AU2012214148A1 - Aptamers to tissue factor pathway inhibitor and their use as bleeding disorder therapeutics - Google Patents

Abstract

The invention relates generally to the field of nucleic acids and more particularly to aptamers that bind to TFPI, which are useful as therapeutics in and diagnostics of bleeding disorders and/or other diseases or disorders in which TFPl has been implicated. In addition, the TFPl aptamers may be used before, during and/or after medical procedures to reduce complications or side effects thereof. The invention further relates to materials and methods for the administration of aptamers that bind to TFPI.

Description

WO 2012/109675 PCT/US2012/024916 APTAMERS TO TISSUE FACTOR PATHWAY INHIBITOR AND THEIR USE AS BLEEDING DISORDER THERAPEUTICS CROSS-REFERENCE TO RELATED APPLICATIONS [00011 This non-provisional patent application is a continuation in part of US Patent Application Serial No. 12/858,369, filed August 17. 2010, which claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Serial Nos.: 61/234,939, filed August 18, Z2009; 61/353,374, filed June 10, 2010; 61/366,362, filed July 21, 2010; and 61/367,766, filed July 26, 2010: the contents of each of which are hereby incorporated by reference in their entirety. FIELD OF THE INVENTION [00021 The invention relates generally to the field of nucleic acids and more particularly to aptamers that bind to tissue factor pathway inhibitor (TFPI), which are useful as therapeutics in and diagnostics of bleeding disorders and/or other pathologies, diseases or disorders in which TFPI has been implicated. The invention further relates to materials and methods for the administration of aptamers that bind to TFPI. BACKGROUND OF THE INVENTION [00031 An aptamer is an isolated or purified nucleic acid that binds with high specificity and affinity to a target through interactions other than Watson-Crick base pairing. An aptamer has a three dimensional structure that provides chemical contacts to specifically bind to a target. Unlike traditional nucleic acid binding, aptamer binding is not dependent upon a conserved linear base sequence, but rather a particular secondary or tertiary structure. That is, the nucleic acid sequences of aptarners are non-coding sequences. Any coding potential that an aptamer may possess is entirely fortuitous and plays no role whatsoever in the binding of an aptamer to a target. A typical minimized aptamer is 5-15 kDa in size (15-45 nucleotides), binds to a target with nanomolar to sub-nanomolar affinity, and discriminates against closely related targets (e.g., aptamers will typically not bind to other proteins from the same gene or functional family).

I

WO 2012/109675 PCT/US2012/024916 [00041 Aptamers have been generated to many targets, such as small molecules, carbohydrates, peptides and proteins, including growth factors, transcription factors, enzymes, immunoglobulins and receptors. [00051 Aptamers are capable of specifically binding to selected targets and modulating the target's activity or binding interactions, e.g.. through binding, aptamers may inhibit or stimulate a target's ability to function. Specific binding to a target is an inherent property of an aptamer. Functional activity, i.e., inhibiting or stimulating a target's function, is not, Often times, an aptamer binds to a target and has little or no effect on the function of the target. Sometimes, an aptamer binds to a target and has an inhibitory or stimulatory effect on a target's function. [00061 Aptamers have a number of desirable characteristics for use as therapeutics and diagnostics, including high specificity arid affinity, biological activity, low immunogenicity, tunable pharnacokirietic properties and stability. Bleeding Disorders 100071 Coagulation is the formation of a stable fibrin/cellular hemostatic plug that is sufficient to stop bleeding. The coagulation process, which is illustrated in Figure 1, involves complex biochemical and cellular interactions that can be divided into three stages. Stage I is the formation of activated Factor X by either the contact (intrinsic) or the tissue factor/VIa (extrinsic) pathway. Stage 2 is the formation of thrombin from prothrombin by Factor Xa. Stage 3 is the formation of fibrin from fibrinogen stabilized by Factor XIIla. 100081 Hemophilia is defined as a congenital or acquired disorder of coagulation that usually, but not always, involves a quantitative and/or functional deficiency of a single coagulation protein. Deficiency of coagulation Factors VIII (hemophilia A) and IX (hemophilia B) are the two most common inherited bleeding disorders. The total overall number of hemophilia A and B patients worldwide is approximately 400,000; however, only about 1/4 (100,000) of these individuals are treated. Hemophilia A and B can be further divided in regard to the extent of factor deficiency. Mild hemophilia is 5-40% of normal factor levels and represents approximately 25% of the total hemophilia population. Moderate hemophilia is 1-5% of normal factor levels and represents approximately 25% of the total hemophilia population. Severe hemophilia is <I% of normal factor levels and represents approximately 50% of the total hemophilia population and the highest users of currently available therapies. 2 WO 2012/109675 PCT/US2012/024916 [00091 Since the discovery of cryoprecipitation by Pool (Pool et al, "High-potency antihaemophilic factor concentrate prepared from cryoglobulin precipitate", Nature, vol. 203, p. 312 (1964)), treatment of these life threatening deficiencies has focused on factor replacement, with a continued effort directed toward improvement in the quality of the Factor VIII and IX concentrates. The most significant improvement has been the availability of recombinant forms of Factors VIII and IX. These highly purified recombinant molecules have a safety and efficacy profile that has made them the primary forni of replacement factors used for the treatment of hemophilia. The majority of mild and moderate patients are treated "on demand", that is when a bleed occurs. Approximately 50-60% of severe patients are treated "on demand", while the remainder of this population uses prophylactic therapy, which involves administering intravenous factor 2-3 times weekly. [0010] Unfortunately, recombinant factors still retain some of the limitations of concentrates and more highly purified plasma derived factors. These limitations include the relatively short half-life of the molecules, which require frequent injection to maintain effective plasma concentration; high cost; and the development of antibody responses, especially to Factor VIII, in a subpopulation of patients called inhibitor patients. [00111 In a majority of patients who develop inhibitory antibodies, the antibody is only transient. In those patients with a sustained antibody response (- 15%), some respond to complex and expensive tolerization protocols. Those who do not respond to tolerization (~5-10%) require the use of non-Factor VIII/Factor IX products to control bleeding. Prothrombin Complex Concentrations (PCC), Factor Eight Inhibitor Bypass Agent (FEIBA) and recombinant Factor VIIa (NovoSeven, FVIIa) are effective Factor VIII/Factor IX bypass treatments for inhibitor patients. [00121 Recombinant Factor Vila (rFVIIa) treatment is the most used of these bypass agents. Factor Vila complexes with endogenous tissue factor to activate the extrinsic pathway. It also can directly activate Factor X. The response to rFVIIa treatment is variable. The variable response, along with the poor pharmacokinetic (PK) profile of rFVIIa, can require multiple injections to control bleeding and significantly limits its utility for prophylactic treatment. [00131 A major effort is currently underway towards development of modified Factor VIII, IX and VIIa molecules with improved potency, stability and circulating half-life. It should be WO 2012/109675 PCT/US2012/024916 noted that in all instances, the products represent incremental improvements to stability, pharmacokinetics and/or formulation of existing replacement factors. [00141 The tissue factor/Vlla (extrinsic) pathway provides for rapid formation of low levels of thrombin that can serve as the initial hemostatic response to initiate and accelerate the Factor VIii, V and IX dependent intrinsic pathway. Tissue factor, Factor Vlla and Factor Xa have a central role in this pathway and it is closely regulated by an endothelial cell associated Kunitz Type proteinase inhibitor, tissue factor pathway inhibitor (TFPI). [00151 Tissue factor pathway inhibitor is a 40 kDa serine protease inhibitor that is synthesized in and found bound to endothelial cell surfaces ("surface TFPI"), in plasma at a concentration of 2-4 nM ("plasma TFPI") and is stored (200 pM/10 8 platelets) and released from activated platelets. Approximately 10% of plasma TFPI is unassociated, while 90% is associated with oxidized LDL particles and is inactive. There are two primary forms of TFPI, TFPIa and TFPI3 (Figures 2 and 3). [0016] TFPIL contains 3 iKunitz decoy domains, K1, K2 and K3. KI and K2 minic protease substrates and inhibit by tight but reversible binding to the target proteases. In the case of TFPIu, K1 binds to and inhibits tissue factor/Vla, while K2 binds to and inhibits Factor Xa. The role for K3 is unknown at this time, but it may have a role in cell-surface binding and enhancing the inhibition of Factor Xa by K2. TFPIu has a basic C-termrinal tail peptide that is the membrane binding site region for the molecule. It is estimated that 80% of the surface TFPI is TFPla. TFlPIu is primarily bound to the endothelial surface associated with the membrane proteoglycans. Heparin has been shown to release TFlPIa from cultured endothelium, isolated veins and following intravenous (IV) heparin (unfractionated and LMWH) injection. The exact nature of the release mechanism is unclear (competition or induced release), but TFPI levels can be increased 3-8 fold following IV heparin administration. Some TFPIla can also be found bound to glycosylated phosphatidylinositol (GPI) via an unidentified co-receptor. [00171 TFPIB3 is an alternatively spliced version of TFPI that is post-translationally modified with a glycosylated phosphatidylinositol (GPI) anchor. It is estimated that it represents about 20% of the surface TFPI in cultured endothelial cells. Although it has in vitro inhibitory activity, the functional in vivo role is less clear. [00181 Surface TFPI may have a more important role in regulation of coagulation based on its localization to the site of vascular injury and thrombus formation. Surface TFPI represents 4 WO 2012/109675 PCT/US2012/024916 the largest proportion of active TFPL Data from several laboratories suggest that TFPI can also have complementary/synergistic effects via interactions with antithrombin III (ATIII) and protein C. [00191 TFPI binds to Factor VIla and Factor Xa via its K1 and, K2 domains and to proteoglycans via its K3 and C-terminal domains. The fact that TFPI has a key role in the inhibition of both tissue factor/VIla and Xa suggests that TFPI inhibition could provide a single treatment or an adjuvant treatment that is given in addition to or combined with recombinant purified factors. An approach to promote a prothrombotic state could be via the upregulation of the tissue factor mediated extrinsic pathway of coagulation. It has been suggested that inhibition of TFPI might improve coagulation in the hemophilia patient. [00201 Studies have demonstrated that TFPI deficiency in mice can increase thrombus formation, and that TFPI antibodies improve bleeding times in Factor VIII deficient rabbits and shorten clotting in plasma from hemophilia patients. In the rabbit, transient hemophilia A was induced by treating rabbits with a Factor VIII antibody. This was followed by treatment with either Factor VIII replacement or an antibody specific to rabbit TFPI. The anti-TFPI treatment produced a reduction in bleeding and a correction of coagulation that was similar to that observed with Factor VIII replacement. Liu et al. (Liu et al, "Improved coagulation in bleeding disorders by Non-Anticoagulant Sulfated Polysaccharides (NASP)", Thromib. Haenost., vol. 95, pp. 68-76 (2006)) reported the effects of a non-anticoagulant polysaccharide isolated from brown algae that inhibits TFPL. A subsequent paper by Prasad et al (Prasad ct al., "Efficacy and safety of a new-class of hemostatic drug candidate, AV513, in dogs with hemophilia A", Blood, vol. 111, pp. 672-679 (2008)) also assessed this polysaccharide in hemophilia A dogs. In both studies, it was found that TFPI inhibition had a positive effect on restoration of a normal coagulation profile and, in the dog model, an improvement in hemostatic profile, including an improved thromboelastogram (TEG) and a reduction in nail bleeding time. These data suggest that inhibition of TFPI could provide an approach to treating hemophilia. [00211 Accordingly, it would be beneficial to identify novel therapies for antagonizing TFPI in the treatment of bleeding disorders, or that are used in conjunction with medical procedures, or that are used in combination with another drug or another therapy to induce a pro-coagulant state. The present invention provides materials and methods to meet these and other needs. 5 WO 2012/109675 PCT/US2012/024916 SUMMARY OF THE INVENTION [00221 The invention provides aptamers that bind to tissue factor pathway inhibitor (TFPI), referred to herein as "TFPI aptamers", and methods for using such aptamers in the treatment of bleeding disorders and other TFPI-mediated pathologies, diseases or disorders, with or without other agents. In addition, the TFPI aptamers may be used before, during and/or after medical procedures, with or without other agents, in order to reduce the complications or side effects thereof [00231 The TFPI aptamers bind to or otherwise interact with TFPI or one or more portions (or regions) thereof. For example, the TFPI aptarners may bind to or otherwise interact with a linear portion or a conformational portion of TFPI. A TFPI aptamer binds to or otherwise interacts with a linear portion of TFPI when the aptamer binds to or otherwise interacts with a contiguous stretch of amino acid residues that are linked by peptide bonds. A TFPI aptamer binds to or otherwise interacts with a conformational portion of TFPI when the aptarner binds to or otherwise interacts with non-contiguous amino acid residues that are brought together by folding or other aspects of the secondary and/or tertiary structure of the polypeptide chain. Preferably, the TFPI is hurnan TFPL Preferably, the TFPI aptamers bind to TFPI and require binding contacts, at least in part, outside of the K1 and K2 regions, such as the K3/C-tenninal region. More preferably, the TFPI aptarners bind at least in part to one or more portions of mature TFPII (for example, Figure 3A) that are selected from the group consisting of: amino acids 148-170. amino acids 150-170, amino acids 155-175, amino acids 160-180, amino acids 165-185, amino acids 170-190, amino acids 175-195, amino acids 180-200, amino acids 185 205, amino acids 190-210, amino acids 195-215, amino acids 200-220, amino acids 205-225, amino acids 210-230, amino acids 215-235, amino acids 220-240, amino acids 225-245, amino acids 230-250. amino acids 235-255, amino acids 240-260, amino acids 245-265, amino acids 250-270, amino acids 255-275, amino acids 260-276, amino acids 148-175, amino acids 150 175. amino acids 150-180, amino acids 150-185, amino acids 150-190, amino acids 150-195, amino acids 150-200, amino acids 150-205, amino acids 150-210, amino acids 150-215, amino acids 150-220, amino acids 150-225, amino acids 150-230, amino acids 150-235, amino acids 150-240, amino acids 150-245, amino acids 150-250, amino acids 150-255, amino acids 150 260, amino acids 150-265, amino acids 150-270., amino acids 150-275, amino acids 150-276, amino acids 190-240, amino acids 190-276, amino acids 240-276, amino acids 242-276, amino 6 WO 2012/109675 PCT/US2012/024916 acids 161-181, amino acids 162-181, amino acids 182-240, amino acids 182-241, and amino acids 182-276. Preferably, the TFPI aptamer has a dissociation constant for TFPI of 100 nM or less. [00241 Examples of TFPI aptamers include, but are not limited to, aptamers that comprise a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, which is referred to herein as ARC26835; SEQ ID NO: 2, which is referred to herein as ARC17480; SEQ ID NO: 3, which is referred to herein as ARC 19498; SEQ ID NO: 4, which is referred to herein as ARC1 9499: SEQ ID NO: 5, which is referred to herein as ARC1 9500; SEQ ID NO: 6, which is referred to herein as ARCl 9501; SEQ ID NO: 7, which is referred to herein as ARC31301; SEQ ID NO: 8, which is referred to herein as ARC18546; SEQ ID NO: 9, which is referred to herein as ARC] 9881: and SEQ ID NO: 10, which is referred to herein as ARC19882. 100251 Preferably, the TFPI aptamer is an aptamer or a salt thereof comprising the following nucleic acid sequence: rnG-rnG-mA-mA-mU-mA-mU-mA-dC-rnU-mU-mG-mG-dC-mU-dC mi-mU-i -mA-mG-mG-mU-rG-dC-mG-mU-mA -mU-mA-rU-mA (SEQ ID NO: 1) (ARC26835), wherein "dN" is a deoxynucleotide and "nN" is a 2-O Methyl containing nucleotide (which is also known in the art as a 2'-OMe. 2'-methoxy or 2'-OC-13 containing nucleotide). In some embodiments, the TFPI aptaner is an aptamer or a salt thereof that consists of the nucleic acid sequence of SEQ ID NO: 1. 100261 More preferably, the TFPI aptamer is an aptamer or a salt thereof comprising the following nucleic acid sequence: mG-mG-mA-mA-mU -mA-nU-mA-dC-mU -mU-mG-mGi-dC mU-dC-mG-mU-mU-mA-mG-mG-mU -mG-dC-mG-nU-mA-mU-mA-mU-mA-3T (SEQ ID NO: 2) (ARC 17480), wherein "31" is an inverted deoxythymidine, "dN" is a deoxynucleotide and "mN" is a 2-0 Methyl containing nucleotide. In some embodiments, the TFPI aptamer is an aptamer or a salt thereof that consists of the nucleic acid sequence of SEQ ID NO: 2. [00271 Even more preferably, the TFPI aptamer is an aptamer or a salt thereof comprising the following nucleic acid sequence: NH--mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU -mG-dC-mG-mU-mA-mU-mA-niU-mA-3T (SEQ ID NO: 3) (ARC19498). wherein "NH2" is from a 5'-hexylamine linker phosphoramidite, "3T" is an inverted deoxythymidine, "dN" is a deoxynucleotide and "mN" is a 2-O Methyl containing nucleotide. In some embodiments, the TFPI aptamer is an aptainer or a salt thereof that consists of the nucleic acid sequence of SEQ ID NO: 3. 7 WO 2012/109675 PCT/US2012/024916 [00281 Most preferably, the TFPI aptamer is an aptamer or a salt thereof comprising the following nucleic acid sequence: PEG4OK-NH-mG-mG-mA-mA-mU-mA-mU-imA-dC-mU-mU mG-mG-dC-mU-dC-mG-mU-mU -mA-mG-mG-mU-mG-dC-mG-miU-mA-mU-mA-mU-mA-3T (SEQ ID NO: 4) (ARC19499), wherein "NH" is from a 5'-hexylamine linker phosphoramidite, "3T" is an inverted deoxythymidine, "dN" is a deoxynucleotide, "inN" is a'-O Methyl containing nucleotide and "PEG" is a polyethylene glycol. In some embodiments, the TFPI aptaner is an aptamer or a salt thereof that consists of the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the PEG40K moiety of SEQ ID NO: 4 is a branched PEG moiety having a total molecular weight of 40 kDa. In other embodiments, the PEG40K moiety of SEQ ID NO: 4 is a linear PEG moiety having a molecular weight of 40 kDa. In further embodiments, the PEG40K moiety of SEQ ID NO: 4 is a methoxypolyethylene glycol (mPEG) moiety having a molecular weight of 40 kDa. In still further embodiments, the PEG40K moiety of SEQ ID NO: 4 is a branched mPEG moiety that contains two mPEG20K moieties, each having a molecular weight of 20 kDa, as shown in Figures 6-9, where "20KPEG" refers to a mPEG moiety having a molecular weight of 20 kDa. In a preferred embodiment, the PEG40K moiety of SEQ ID NO: 4 is the branched PEG40K moiety shown in Figure 6, where "20K-PEG" refers to a nPEG moiety having a molecular weight of 20 kDa, and is connected to the aptamer as shown in Figure 7. In a more preferred embodiment, the PEG40K moiety is connected to the aptamer using a 5'-amine linker phosphoramidite, as shown in Figure 8, where "20KPEG" refers to a mPEG moiety having a molecular weight of 20 kDa. In a most preferred embodiment, the PEG40K moiety is a mPEG moiety having a total molecular weight of 40 kDa and is connected to the aptamer using a 5 hexylamine linker phosphoramidite, as shown in Figure 9A and 9B. [0029] Alternatively, the TFPI aptamer is an aptamer or a salt thereof comprising the following nucleic acid sequence: NH 2 -mG-mG-mA-nmA-mU -mA-mU-mA-dC-mU-mU-mG-mG dC-mU-dC-mG-mU -mU-mA-mG-mG-mU -mG-dC-mG-mU-mA-mU-mA-mU-mA-NH2 (SEQ ID NO: 5) (ARC 19500), wherein "dN" is a deoxynucleotide, "mN" is a 2'-O Methyl containing nucleotide and "NH 2 " is from a hexylamine linker phosphoramidite. In some embodiments, the TFPI aptamer is an aptamer or a salt thereof that consists of the nucleic acid sequence of SEQ ID NO: 5. [00301 Preferable to the TFPI aptamer of paragraph [0029] is an aptamer or a salt thereof comprising the following nucleic acid sequence: PEG20K-NI--mG-mG-mA- nA-mU-mA-mU 8 WO 2012/109675 PCT/US2012/024916 mA-dC-mU-mU-mG-mG-dC-niU-dC-mG-mU-mU-mA-mG-mG-niU-mG-dC-mG-mU-mA-mU mA-mU-mA-NH-PEG20K (SEQ ID NO: 6) (ARC19501), wherein "dN" is a deoxynueleotide, "mN" is a '-0 Methyl containing nucleotide. "NH" is from a hexylamine linker phosphoramidite and "PEG" is a polyethylene glycol. In some embodiments, the TFPI aptamer is an aptamer or a salt thereof that consists of the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the PEG20K moieties of SEQ ID NO: 6 are branched PEG moieties. In other embodiments, the PEG20K moieties of SEQ ID NO: 6 are linear PEG moieties. In further embodiments, the PEG20K moieties of SEQ ID NO: 6 are methoxypolyethylene glycol (mPEG) moieties having a molecular weight of 20 kDa. In still further embodiments, the PEG20K moieties of SEQ ID NO: 6 are branched mPEG moieties that contain two mPEG 1 OK moieties each having a molecular weight of 10 kDa. [0031] Alternatively, the TFPI aptamer is an aptamer or a salt thereof comprising the following nucleic acid sequence: rmG-mG-mA-mA-mU-i-mA-mU-mA-dC-mU-mU n-mG-mG-m mU-dC-mG-mU-mU-mA-mG-rnG-mU-mG-mC-mG-ir-mA-mU-mA -mU -mA (SEQ ID NO: 7) (ARC31301), wherein "dN" is a deoxynucleotide and "nN" is a 2'-( Methyl containing nucleotide. In some embodiments, the TFPIl aptamer is an aptamer or a salt thereof that consists of the nucleic acid sequence of SEQ ID NO: 7. [0032] Preferable to the TFPI aptaner of paragraph [0031] is an aptaner or a salt thereof comprising the following nucleic acid sequence: mG-mG-mA-mA-mU-niA-mU-mA-dC-mU mU-mG-mG-m(C-mL-dC-mG-mU -mU -mA-mG-m(i-mU-mG-mC-mG-mU-mtA-mU -mA-mU mA-3T (SEQ ID NO: 8) (ARC18546), wherein "31" is an inverted deoxythymidine, "dN" is a deoxynucleotide and "nN" is a 2'-) Methyl containing nucleotide. In some embodiments, the TFPI aptamer is an aptamer or a salt thereof that consists of the nucleic acid sequence of SEQ ID NO: 8. [00331 More preferable to the TFPI aptamer of paragraph [0031] is an aptamer or a salt thereof comprising the following nucleic acid sequence: NH 2 mG-mG-mA-mA-mU-mA-nU mA-dC-mU -mU-mG-mG-mC-mU-dC-mG-mU-mU -mA-mG-mG-mU-mG-mC-mG-mU-mA miU-mA-inU-mA-3T (SEQ ID NO: 9) (ARC 19881). wherein "NH2" is from a 5'-hexylamine linker phosphoramidite, "3T" is an inverted deoxythymidine, "dN" is a deoxynucleotide and "muN" is a 2'-O Methyl containing nucleotide. In some embodiments, the TFPI aptaner is an aptaner or a salt thereof that consists of the nucleic acid sequence of SEQ ID NO: 9. 9 WO 2012/109675 PCT/US2012/024916 [00341 Even more preferable to the TFPI aptamer of paragraph [0031] is an aptamer or a salt thereof comprising the following nucleic acid sequence: PEG40K-NH-mG-mG-mA-mA-mU mA-mU -mA-dC-mU-mU-mG-mG-mC-nmU-dC-mG-mU-mU-mA-mG-mG-nmU-mG-mC-mG mU-mA-mU-mA-niU-mA-3T (SEQ ID NO: 10) (ARC19882), wherein "NH" is from a 5 hexylanline linker phosphoramidite, "3T" is an inverted deoxythymidine, "dN" is a deoxynucieotide, "mN" is a 2'-O Methyl containing nucleotide and "PEG" is a polyethylene glycol In some embodiments, the TFPI aptaner is an aptamer or a salt thereof that consists of the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the PEG40K moiety of SEQ ID NO: 10 is a branched PEG moiety having a total molecular weight of 40 kDa, In other embodiments, the PEG40K moiety of SEQ ID NO: 10 is a linear PEG moiety having a molecular weight of 40 kDa, In further embodiments, the PEG40K moiety of SEQ ID NO: 10 is a methoxypolyethylene glycol (mPEG) moiety having a molecular weight of 40 kDa. In still further embodiments, the PEG40K moiety of SEQ ID NO: 10 is a branched mPEG moiety that contains two mPEG20K moieties, each having a molecular weight of 20 kDa, as shown in Figures 6-9, where "20KiPEG" refers to a mPEG moiety having a molecular weight of 20 kDa. In a preferred embodiment, the PEG40K moiety of SEQ ID NO: 10 is the branched PEG40K moiety shown in Figure 6, where "20KPEG" refers to a mPEG moiety having a molecular weight of 20 kDa, and is connected to the aptamer as shown in Figure 7. In a more preferred embodiment, the PEG40K moiety is connected to the aptamer using a 5'-amine linker phosphoramidite, as shown in Figure 8, where "20KPEG" refers to a mPEG moiety having a molecular weight of 20 kDa. In a most preferred embodiment, the PEG40K moiety is a mPEG moiety having a total molecular weight of 40 kDa and is connected to the aptamer using a 5' hexylamine tinker phosphoramidite, as shown in Figure 9A and 9B. [00351 Preferably, the TFPI aptamers are connected to one or more PEG moieties, with or without one or more linkers. The PEG moieties may be any type of PEG moiety. For example, the PEG moiety may be linear, branched, multiple branched, star shaped, comb shaped or a dendrimer. In addition, the PEG moiety may have any molecular weight. Preferably, the PEG moiety has a molecular weight ranging from 5-100 kDa in size. More preferably, the PEG moiety has a molecular weight ranging from 10-80 kDa in size. Even more preferably, the PEG moiety has a molecular weight ranging from 20-60 kDa in size. Yet even more preferably, the PEG moiety has a molecular weight ranging from 30-50 kDa in size. Most preferably, the PEG 1 0 WO 2012/109675 PCT/US2012/024916 moiety has a molecular weight of 40 kDa in size, also referred to herein as "40KPEG". The same or different PEG moieties may be connected to a TFPI aptamer. The same or different linkers or no liners may be used to connect the same or different PEG moieties to a TFPI aptamer. [00361 Alternatively, the TFPI aptamers may be connected to one or more PEG alternatives (rather than to one or more PEG moieties), with or without one or more linkers. Examples of PEG alternatives include, but are not limited to, polyoxazoline (POZ), PolyPEG, hydroxyethylstarch (IHES) and albumin. The PEG alternative may be any type of PEG alternative, but it should function the same as or similar to a PEG moiety, i.e., to reduce renal filtration and increase the half-life of the TFPI aptamer in the circulation. The same or different PEG alternatives may be connected to a TFPI aptamer. The same or different linkers or no linkers may be used to connect the same or different PEG alternatives to a TFPI aptamer. Alternatively, a combination of PEG moieties and PEG alternatives may be connected to a TFPI aptamer, with or without one or more of the same or different linkers. [00371 Preferably, the TFPI aptamers are connected to a PEG moiety or a PEG alternative via one or rnore linkers. However, the TFPI aptamers may be connected to a PEG moiety or PEG alternative directly, without the use of a linker. The linker may be any type of molecule. Examples of linkers include, but are not limited to, amines, thiols and aides. The linkers can include a phosphate group. Preferably, the linker is from a 5'-amine linker phosphoramidite. In some embodiments, the 5'-amine linker phosphoramidite comprises 2-18 consecutive C-12 groups. In more preferred embodiments, the 5'-amine linker phosphoramidit comprises 2-12 consecutive C11 groups. In even more preferred embodiments, the 5-amine linker phosphoramidite comprises 4-8 consecutive CH 2 groups. In most preferred embodiments, the 5' amine linker phosphoramidite comprises 6 consecutive CH 2 groups, i.e., is a 5'-hexylamine linker phosphoramidite. One or more of the same or different linkers or no linkers may be used to connect one or more of the same or different PEG moieties or one or more of the same or different PEG alternatives to a TFPI aptamer. [00381 In preferred embodiments, an aptamer, or a salt thereof, comprising the following structure is provided: 11 WO 2012/109675 PCT/US2012/024916 20KPEG 0 20KPEG' N N- O H-ptarner-3 H P I; 0 wherein HNI PO 3 H is from a 5'-amine linker phosphoramidite, and the aptamer is a TFPI aptamer of the invention. Preferably, the aptamer is selected from the group consisting of SEQ ID NOs: 2 and 8. The 20KPEG moiety can be any PEG moiety having a molecular weight of 20 kDa. Preferably, the 20KPEG moiety is a mPEG moiety having a molecular weight of 20 kDa. [00391 In alternative preferred embodiments, an aptamer, or a salt thereof, comprising the following structure is provided: O 0 20KEGOH ,OH 20KPEG 2OKPEG OH 5'-aptamer-3 - VE H O 00 wherein HN-. P02H is from a 5'-amine linker phosphoramidite, and the aptamer is a TFPI aptamer of the invention. Preferably, the aptamer is selected from the group consisting of SEQ ID NO: 1. The 20KPEG moiety can be any PEG moiety having a molecular weight of 20 kDa. Preferably, the 20KPEG moiety is a mPEG moiety having a molecular weight of 20 kDa 100401 In more preferred embodiments, an aptamer, or a salt thereof, comprising- the following structure is provided: 20KPEG 0 20KPEG' N 'O II wherein the aptamer is a TFPI aptamer of the invention. Preferably, the aptamer is selected from the group consisting of SEQ ID NOs: 2 and 8. The 20KPEG moiety can be any PEG moiety having a molecular weight of 20 kDa. Preferably, the 20KPEG moiety is a mPEG moiety having a molecular weight of 20 kDa 100411 In alternative more preferred embodiments, an aptamer, or a salt thereof, comprising the following structure is provided: 20KPEG H 5'-aptamer-3 O 2KPEG 2OKPEG ,'N20KPEN H 1 0 H wherein the aptaner is a TFPI aptamer of the invention. Preferably, the aptamer is selected from 12 WO 2012/109675 PCT/US2012/024916 the group consisting of SEQ ID NO: 1. The 20KPEG moiety can be any PEG moiety having a molecular weight of 20 kDa. Preferably, the 20KPEG moiety is a mPEG moiety having a molecular weight of20 kDa [00421 In most preferred embodiments, an aptamer, or a salt thereof, comprising the following structure is provided: 0 H n

H

3 CO O 9 0 HO n , wherein ""n" is about 454 ethylene oxide units (PEG 20 kDa), and the aptamer is a TFPI aptamer of the invention. "n" is about 454 ethylene oxide units because the number of n's may vary slightly for a PEG having a particular molecular weight. Preferably, "n" ranges from 400-500 ethylene oxide units. More preferably, "n" ranges from 425-475 ethylene oxide units, Even more preferably, "n" ranges from 440-460 ethylene oxide units. Most preferably, "n" is 454 ethylene oxide units. Preferably, the aptamer is selected from the group consisting of SEQ ID NOs: 2 and 8. [00431 In alternative most preferred embodiments, an aptamer, or a salt thereof, comprising the following structure is provided: MeO0 OH 5-aptamer-3' OH n H 0 0 H wherein "n" is about 454 ethylene oxide units (PEG = 20 kDa), and the aptamer is a TFPI aptamer of the invention. "n" is about 454 ethylene oxide units because the number of n's may vary slightly for a PEG having a particular molecular weight. Preferably. "n" ranges from 400 500 ethylene oxide units. More preferably, "n" ranges from 425-475 ethylene oxide units. Even more preferably, "n" ranges from 440-460 ethylene oxide units, Most preferably, "n" is 454 ethylene oxide units. Preferably, the aptamer is selected from the group consisting of SEQ ID NO: 1. [0044] The invention also provides aptamers that have substantially the same ability to bind to TFPI as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the aptamers have substantially the same structure as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the aptamers have 13 WO 2012/109675 PCT/US2012/024916 substantially the same ability to bind to TFPI and substantially the same structure as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6. 7, 8, 9 or 10. The invention also provides aptamers that have substantially the same ability to bind to TFPI and substantially the same ability to modulate a biological function of TFPI as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The invention further provides aptainers that have substantially the same ability to bind to TFPI and substantially the same ability to modulate blood coagulation as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The invention also provides aptamers that have substantially the same structure and substantially the same ability to modulate a biological function of TFPI as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The invention also provides aptamers that have substantially the same structure and substantially the same ability to modulate blood coagulation as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the aptamers have substantially the same ability to bind to TFPI, substantially the same structure and substantially the same ability to modulate a biological function of TFPI as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 , 9or 10. In some embodiments, the aptamers have substantially the same ability to bind to TFPI, substantially the same structure and substantially the same ability to modulate blood coagulation as any one of the aptarners shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. 100451 The TFPI aptamers may comprise at least one chemical modification. Preferably, the modification is selected from the group consisting of: a chemical substitution at a sugar position, a chemical substitution at an internucleotide linkage and a chemical substitution at a base position. Alternatively, the modification is selected from the group consisting of: incorporation of a modified nucleotide; a 3' cap; a 5' cap; conjugation to a high molecular weight, non immunogenic compound; conjugation to a lipophilic compound; incorporation of a CpG motif; and incorporation of a phosphorothioate or phosphorodithioate into the phosphate backbone. The high molecular weight, non-immunogenic compound is preferably polyethylene glycol. In some embodiments, the polyethylene glycol is methoxypolyethylene glycol (mPEG). The 3' cap is preferably an inverted deoxythymidine cap. [00461 The invention also provides aptamers that bind to TFPI and have one or more of the following characteristics: (i) includes the primary nucleotide sequence of mG-mG-mA-mA-mU mA-mU-mxA-dC-mUI-mUT-mG-mG-dC-m nU-dC-mG-n -n U-mA-m G-mG-m nU-mG-dC-mG-mU 14 WO 2012/109675 PCT/US2012/024916 mA-mU-mA-mU-mA (SEQ ID NO: 1); (ii) includes a primary nucleotide sequence that has at least 70%. 75%, 80%, 85%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the primary nucleotide sequence shown in SEQ ID NO: 1 or 7; (iii) has substantially the same or better ability to bind to TFPI as that of an aptamer that comprises a primary nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and/or (iv) has substantially the same or better ability to modulate or inhibit TFPI as that of an aptamer comprising a primary nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. As used herein, the term primary nucleotide sequence refers to the 5' to 3' linear sequence of nucleotide bases of the nucleic acid sequence that forms an aptamer, without regard to 3' or 5' modifications. For example, ARC26835, ARC 17480, ARC19498, ARC19499, ARCl 9500 and ARC19501 all have the same primary nucleotide sequence. [0047] The invention additionally provides pharmaceutical compositions comprising a therapeutically effective amount of a TFPI aptamer or a salt thereof, and a pharmaceutically acceptable carrier or diluent. [0048] The invention further provides a method for treating, preventing, delaying the progression of, or ameliorating a pathology, disease or disorder mediated by TFPI by administering to a subject the above pharmaceutical composition. Preferably, the subject is a mammal. More preferably, the subject is a human. Preferably, the pathology, disease or disorder is selected from the group consisting of: coagulation factor deficiencies, congenital or acquired, mild or moderate or severe, including hemophilia A (Factor VIII deficiency), hemophilia B (Factor IX deficiency) and hemophilia C (Factor XI deficiency); hemophilia A or B with inhibitors; other factor deficiencies (V, VII, X, XIII, prothrombin. fibrinogen); deficiency of a2-plasmin inhibitor; deficiency of plasminogen activator inhibitor 1; multiple factor deficiency; functional factor abnormalities (e.g.. dvsprothrombinemia); joint hemorrhage (hemarthrosis), including, but not limited to, ankle, elbow and knee; spontaneous bleeding in other locations (muscle, gastrointestinal, mouth, etc.); hemorrhagic stroke; intracranial hemorrhage; lacerations and other hemorrhage associated with trauma; acute traumatic coagulopathy; coagulopathy associated with cancer (e.g., acute promyclocytic leukemia); von Willebrand's Disease: disseminated intravascular coagulation; liver disease; menorrhagia: 15 WO 2012/109675 PCT/US2012/024916 thrombocytopenia and hemorrhage associated with the use of anticoagulants (e.g., vitamin K antagonists, FXa antagonists, etc.). [00491 The pharmaceutical compositions may be administered by numerous routes of administration. Preferably, the compositions are administered intravenously (IV). Most preferably, the compositions are administered subcutaneously (SC or SQ). [00501 The pharmaceutical compositions may be administered using various treatment regimens. For example, the compositions may be administered as a maintenance therapy at a defined dose for a defined period of time, such as when a patient is not suffering from a bleeding episode. Alternatively, the compositions may be administered on demand, i.e., as needed, such as when a patient is suffering from a bleeding episode. In a further alternative embodiment, the compositions may be administered as a combination of maintenance therapy and on demand therapy. In such an embodiment, the compositions may be administered as a maintenance therapy at a defined dose for a defined period of time until a bleed occurs, in which case the dosage of the compositions would be increased on an as needed basis until the bleeding stopped, at which point the dosage of the compositions would be decreased back to the prior maintenance level. In another such embodiment, the compositions may be administered as a maintenance therapy at a defined dose for a defined period of time until a bleed occurs, in which case another bleeding disorder therapy would be administered to the patient (such as Factor VIII) until the bleeding stopped, at which point the other bleeding disorder therapy would be discontinued. During this entire time, the compositions would continue to be administered as a maintenance therapy. In yet another such embodiment, the compositions may be administered as a maintenance therapy at a defined dose for a defined period of time until a bleed occurs, in which case the dosage of the compositions would be decreased and another bleeding disorder therapy would be administered to the patient (such as Factor ViII) until the bleeding stopped, at which point the dosage of the compositions would be increased back to the prior maintenance level and the other bleeding disorder therapy would be discontinued. In another such embodiment, another bleeding disorder therapy (such as Factor VIII) may be administered as a maintenance therapy at a defined dose for a defined period of time until a bleed occurs, in which case the compositions would be administered to the patient until the bleeding stopped, at which point therapy with the compositions would be discontinued, During this entire time, the other bleeding disorder therapy would continue to be administered as a maintenance therapy. In yet another such embodiment, 16 WO 2012/109675 PCT/US2012/024916 another bleeding disorder therapy (such as Factor VIII) may be administered as a maintenance therapy at a defined dose for a defined period of time until a bleed occurs, in which case the dosage of the other bleeding disorder therapy would be decreased and the compositions would be administered to the patient until the bleeding stopped, at which point the dosage of the other bleeding disorder therapy would be increased back to the prior maintenance level and therapy with the compositions would be discontinued. [00511 The pharmaceutical compositions may also be administered prior to, during and/or after a medical procedure. For example, the pharmaceutical compositions may be administered in conjunction (before, during and/or after) with medical procedures, such as: prophylaxis and/or treatment associated with bleeding caused by dental procedures, orthopedic surgery including but not limited to arthroplasty (e.g., hip replacement), surgical or radionuclide synovectomy (RSV), major surgery, venipuncture, transfusion and amputation. [00521 The pharmaceutical compositions may also be administered in combination with another drug, such as: activated prothrombin complex concentrates (APCC), Factor Eight Inhibitor Bypass Agent (FEIBA recombinant Factor VIla (e.g., NovoSeven*), recombinant Factor VIII (Advate Kooenate Recombinate, Lielixate", ReFacto"), plasma-derived Factor VII1 (-1 urnate P [-emofil M"), recombinant Factor IX (BeneFIX"), plasma-derived Factor IX (Bebulin VH*, Konyne*, Mononine*), cryoprecipitate, desmopressin acetate (DDAVP), epsilon aminocaproic acid or tranexamic acid. Alternatively, the pharmaceutical compositions may be administered in combination with another therapy, such as: blood or blood-product transfusion, plasmapheresis, immune tolerance induction therapy with high doses of replacement factor, immune tolerance therapy with immunosuppressive agents (e.g., prednisone, rituximab) or pain therapy. [00531 The TFPI aptamers may be used for identification of the TFPI protein. Specifically, the TFPI aptamers may be used to identify, quantify or otherwise detect the presence of the TFPI protein in a sample, such as a biological sample or other subject-derived sample. For example, the TFPI aptamers may be used in in vitro assays, e.g., ELISA, to detect TFPI levels in a patient sample. [00541 The invention also provides a method for regulating TFPI in which a molecule binds or otherwise interacts with one or more portions of TFPI, wherein at least one portion is outside of the K-1 and K2 domains of TFPI, such as the K3/C terminal region. The molecule can be any 17 WO 2012/109675 PCT/US2012/024916 type of molecule, such as, for example, a small molecule organic compound, an antibody, a protein or peptide, a polysaccharide, a nucleic acid, an siRNA, an aptamer, or any combination thereof. Preferably, the molecule is a small molecule organic compound. More preferably, the molecule is an antibody. Most preferably, the molecule is an aptamer. For example, the molecule may bind to or otherwise interact with a linear portion or a conformational portion of TFPL A molecule binds to or otherwise interacts with a linear portion of TFPI when the molecule binds to or otherwise interacts with a contiguous stretch of amino acid residues that are linked by peptide bonds. A molecule binds to or otherwise interacts with a conformational portion of TFPI when the molecule binds to or otherwise interacts with non-contiguous amino acid residues that are brought together by folding or other aspects of the secondary and/or tertiary structure of the polypeptide chain. Preferably, the molecule binds at least in part to one or more portions of mature TFPI (for example, Figure 3A) that are selected from the group consisting of: amino acids 148-170, amino acids 150-170, amino acids 155-175, amino acids 160-180, amino acids 165-185, amino acids 170-190, amino acids 175-195, amino acids 180 200, amino acids 185-205, amino acids 190-210, amino acids 195-215, amino acids 200-220, amino acids 205-225, amino acids 210-230, amino acids 215-235, amino acids 220-240, amino acids 225-245, amino acids 230-250, amino acids 235-255, amino acids 240-260, amino acids 245-265, amino acids 250-270, amino acids 255-275, amino acids 260-276, amino acids 148 175, amino acids 150-175, amino acids 150-180, amino acids 150-185, amino acids 150-190, amino acids 150-195, amino acids 150-200, amino acids 150-205, amino acids 150-210, amino acids 150-215, amino acids 150-220, amino acids 150-225, amino acids 150-230, amino acids 150-235. amino acids 150-240, amino acids 150-245, amino acids 150-250, amino acids 150 255, amino acids 150-260, amino acids 150-265, amino acids 150-270, amino acids 150-275, amino acids 150-276, amino acids 190-240, amino acids 190-276, amino acids 240-276, amino acids 242-276, amino acids 161-181, amino acids 162-181, amino acids 182-240, amino acids 182-241, and amino acids 182-276. The molecule preferably comprises a dissociation constant for human TFPI, or a variant thereof, of less than 100 pM, less than I PM, less than 500 nM, less than 100 nM, preferably 50 nM or less, preferably 25 nM or less, preferably 10 nM or less, preferably 5 nM or less, more preferably 3 nM or less, even more preferably I nM or less, and most preferably 500 pM or less. 18 WO 2012/109675 PCT/US2012/024916 [00551 The invention further provides for the use of a TFPI aptamer in the manufacture of a medicament in the treatment, prevention, delaying progression, and/or amelioration of a bleeding disorder. For example, ARC2683 5, ARC 17480, ARC 19498, ARC 19499, ARC 19500, ARC19501. ARC31301, ARC18546, ARC19881 and ARC19882 are used in the manufacture of medicament for treating, preventing, delaying progression of or otherwise ameliorating a bleeding disorder. [00561 In one embodiment, the invention provides a TFPI aptamer for use in a method of treatment, prevention, delaying progression and/or amelioration of a bleeding disorder. [00571 In one embodiment, the invention provides for the use of a TFPI aptamer in the manufacture of a diagnostic composition or product for use in a method of diagnosis practiced on the human or animrial body. In some embodiments, the method of diagnosis is for the diagnosis of a bleeding disorder. [00581 In one embodiment, the invention provides a TFPI aptamer for use in a method of diagnosis practiced on the human or animal body. In some embodiments, the method of diagnosis is for the diagnosis of a bleeding disorder. [0059] In one embodiment, the invention provides the use of a TFPI aptamer for diagnosis in vitro. In some embodiments, the in vitro use is for the diagnosis of a bleeding disorder. [0060] The invention further relates to agents that reverse the effects of the TFPI aptamers, referred to herein as "TFPI reversal agents". The TFP1 reversal agent can be any type of molecule, such as a protein, antibody, small molecule organic compound or an oligonucleotide. Preferably, a TFPI reversal agent is an oligonucleotide that is 10-15 nucleotides in length. Preferably, a TFPI reversal agent binds to a TFPI aptamer. Preferably, such binding is via complementary base pairing. Without wishing to be bound by theory, a TFPI reversal agent acts by hybridizing to a TFPI aptamer, thereby disrupting the TFPI aptamer's structure and preventing the binding of the TFPI aptamer to TFPI. [00611 Examples of TFPI reversal agents include, but are not limited to: SEQ ID NO: 15, which is ARC23085; SEQ ID NO: 16, which is ARC23087; SEQ ID NO: 17. which is ARC23088; and SEQ ID NO: 18, which is ARC23089. [00621 Preferably, the TFPI reversal agent is a nucleic acid comprising the structure set forth below: 19 WO 2012/109675 PCT/US2012/024916 ntA-mG-mC-mC-mA-nmA-mG-mU-mA-mU-imA-mU-nmU-mC-mC (SEQ ID NO: 15). wherein "tN" is a 2'-0 Methyl containing residue (which is also known in the art as a 2'-OMe, methox; or '-OCH 3 containing residue). [00631 Alternatively, the TFPI reversal agent is a nucleie acid comprising the structure set forth below: mU -A-U-mA-mU-mA-mC-mG-mC-mA-mC-mC-mU-mA-mA (SEQ ID NO: 16), wherein "iN" is a 2'-O Methyl containing residue. [00641 Alternatively, the TFPI reversal agent is a nucleic acid comprising the structure set forth below: mC-mUt-mA-mnA-mC-nG-nA-mG-mC-mC (SEQ ID NO: 17), wherein "mN" is a 2'-O Methyl containing residue. [00651 Alternatively, the TFPI reversal agent is a nucleic acid comprising the structure set forth below: mC-mA-rnC-mC-mU-mA-mA-rnC-mG-mA-rnG-mC-rnC-mA-mA (SEQ ID NO: 18), wherein "N" is a 2>O Methyl containing residue. 100661 The invention further provides a method for treating, preventing, delaying the progression of and/or ameliorating a bleeding disorder, the method comprising the step of administering a TFPI reversal agent to a patient in need of such treatment. 100671 The invention provides for the use of a TFPI reversal agent in the manufacture of a medicament for the treatment, prevention, delaying progression and/or amelioration of a bleeding disorder. 100681 Accordingly, the invention provides for the use of a TFI reversal agent in the manufacture of a medicament for the treatment, prevention, delaying progression and/or amelioration of a bleeding disorder in a patient wherein the method involves administering the TFPI reversal agent to the patient to control and/or modulate the therapeutic effect of a TFPI aptamer administered to the patient. The TFiPI aptamer may be administered prior to the TFPI reversal agent, simultaneously with the TFPI reversal agent or after the TFPI reversal agent, and may be administered as part of a combination therapy. Preferably, the TFPI aptamer is administered to the patient in order to treat, prevent, delay progression of and/or ameliorate a bleeding disorder in the patient. 20 WO 2012/109675 PCT/US2012/024916 [00691 The invention also provides the use of a TFPI reversal agent in the manufacture of a medicament for use in controlling and/or modulating the treatment of a bleeding disorder, wherein the bleeding disorder is being treated with a TFPI aptamer. [00701 In one embodiment, the invention provides a TFPI reversal agent for use in the treatment, prevention, delaying progression and/or amelioration of a bleeding disorder. [00711 Accordingly, the invention provides a TFPI reversal agent for use in the treatment, prevention, delaying progression and/or amelioration of a bleeding disorder in a patient wherein the method involves adniinistering the TFPI reversal agent to the patient to control and/or modulate the therapeutic effect of a TFPI aptamer administered to the patient. [00721 The invention also provides a TFPI reversal agent for use in the treatment, prevention, delaying progression and/or amelioration of a bleeding disorder, wherein the bleeding disorder is being treated with a TFPI aptamer. [00731 In one embodiment, the invention provides for the use of a TFPI reversal agent in the manufacture of a diagnostic composition or product for use in a method of diagnosis practiced on the human or animal body. In sore embodiments, the method of diagnosis is for the diagnosis of a bleeding disorder. [00741 In one embodiment, the invention provides a TFPI reversal agent for use in a method of diagnosis practiced on the human or animal body. In some embodirnents, the method of diagnosis is for the diagnosis of a bleeding disorder. 100751 In one embodiment, the invention provides the use of a TFPI reversal agent for diagnosis in vitro. In some embodiments, the in vitro use is for the diagnosis of a bleeding disorder. [00761 'The invention also provides a kit comprising at least one container comprising a quantity of one or more TFPI aptamers, along with instructions for using the one or more TFPI aptamers in the treatment, prevention, delaying progression and/or amelioration of a bleeding disorder. For example, the kit includes ARC26835, ARC 17480, ARC 19498, ARC 19499, ARC 19500, ARC 19501, ARC31301, ARC18546, ARC19881 or ARC 19882 and combinations thereof. In some embodiments, the aptamers are formulated as a pharmaceutical composition. The kit may further comprise a TFPI reversal agent, along with instructions regarding administration of the reversal agent. 21 WO 2012/109675 PCT/US2012/024916 [00771 The invention also provides a method for producing an aptamer that binds to TFPI, the method comprising the step of chemically synthesizing a nucleic acid having a nucleic acid sequence of an aptamer that binds to TFPI as described herein. The method may further comprise the step of formulating a pharmaceutical composition by mixing the synthesized nucleic acid sequence, or a salt thereof, with a pharmaceutically acceptable carrier or diluent. [00781 The invention additionally provides a method for producing a reversal agent, the method comprising the step of chemically synthesizing a nucleic acid having a nucleic acid sequence of a TFPI reversal agent as described herein. The method may further comprise the step of formulating a pharmaceutical composition by mixing the synthesized nucleic acid sequence or a salt thereof, with a pharmaceutically acceptable carrier or diluent. [00791 The invention further provides aptamers that have been identified by the SELEX' process, which comprises the steps of (a) contacting a mixture of nucleic acids with TFPI under conditions in which binding occurs; (b) partitioning unbound nucleic acids from those nucleic acids that have bound to TFPI; (c) amplifying the bound nucleic acids to yield a ligand-enriched mixture of nucleic acids; and, optionally, (d) reiterating the steps of binding, partitioning and amplifying through as many cycles as desired to obtain aptanier(s) that bind to TFPI. [00801 The invention further provides methods for identifying aptamers that bind at least in part to or otherwise interact with one or more portions of TFPI, which comprise the steps of (a) contacting a mixture of nucleic acids with one or more portions of TFPI under conditions in which binding occurs; (b) partitioning unbound nucleic acids from those nucleic acids that have bound to TFPI; (c) amplifying the bound nucleic acids to yield a ligand-enriched mixture of nucleic acids; and, optionally, (d) reiterating the steps of contacting, partitioning and amplifying through as many cycles as desired, to obtain aptamer(s) that bind to a portion of TF'PI. This method may also include intervening or additional cycles with binding to full-length TFPI, followed by partitioning and amplification. For example, the TFPI aptamers may bind to or otherwise interact with a linear portion or a conformational portion of TFPI. A TFPI aptamer binds to or otherwise interacts with a linear portion of TFPI when the aptamer binds to or otherwise interacts with a contiguous stretch of amino acid residues that are linked by peptide bonds. A TFPI aptamer binds to or otherwise interacts with a conformational portion of TFPI when the aptamer binds to or otherwise interacts with non-contiguous amino acid residues that are brought together by folding or other aspects of the secondary and/or tertiary structure of the 22 WO 2012/109675 PCT/US2012/024916 polypeptide chain. Preferably, the one or more portions of mature'TFP (for example, Figure 3A) are selected from the group consisting of: amino acids 148-170, amino acids 150-170, amino acids 155-175. amino acids 160-180, amino acids 165-185, amino acids 170-190, amino acids 175-195, amino acids 180-200, amino acids 185-205, amino acids 190-210, amino acids 195 215, amino acids 200-220, amino acids 205-225, amino acids 210-230, amino acids 215-235, amino acids 220-240, amino acids 225-245, amino acids 230-250, amino acids 235-255, amino acids 240-260, amino acids 245-265, amino acids 250-270, amino acids 255-275, amino acids 260-276, amino acids 148-175, amino acids 150-175, amino acids 150-180, amino acids 150 185, amino acids 150-190, amino acids 150-195, amino acids 150-200, amino acids 150-205, amino acids 150-210, amino acids 150-215, amino acids 150-220, amino acids 150-225, amino acids 150-230, amino acids 150-235, amino acids 150-240, amino acids 150-245, amino acids 150-250, amino acids 150-255, amino acids 150-260, amino acids 150-265, amino acids 150 270, amino acids 150-275, amino acids 150-276, amino acids 190-240, amino acids 190-276, amino acids 240-276, amino acids 242-276, amino acids 161-181, amino acids 162-181, amino acids 182-240, amino acids 182-241, and arnino acids 182-276. The aptamer preferably comprises a dissociation constant for human TFPI or a variant or one or more portions thereof, of less than 100 IM, less than I uM, less than 500 nM, less than 100 nM, preferably 50 nM or less, preferably 25 nM or less, preferably 10 nM or less, preferably 5 nM or less, more preferably 3 nM or less, even more preferably I nM or less, and most preferably 500 pM or less. 100811 The invention also provides methods for identifying aptamers that bind at least in part to or otherwise interact with one or more portions of TFPI, which comprise the steps of (a) contacting a mixture of nucleic acids with full-length TFPI or one or more portions of TFPI under conditions in which binding occurs; (b) partitioning unbound nucleic acids from those nucleic acids that have bound to full-length TFPI or one or more portions of TFPI; (c) specifically eluting the bound nucleic acids with a portion of TFPI, or a ligand that binds to full length TFPI or a portion of TFPI; (d) amplifying the bound nucleic acids to yield a ligand enriched mixture of nucleic acids; and, optionally, (c) reiterating the steps of contacting, partitioning, eluting and amplifying through as many cycles as desired to obtain aptamer(s) that bind to one or more portions of TFPI. For example, the TFPI aptamers may bind to or otherwise interact with a linear portion or a conformational portion of TFPI. A TFPI aptamer binds to or otherwise interacts with a linear portion of TFPI when the aptamer binds to or otherwise interacts 23 WO 2012/109675 PCT/US2012/024916 with a contiguous stretch of amino acid residues that are linked by peptide bonds. A TFPI aptamer binds to or otherwise interacts with a conformational portion of TFPI when the aptaner binds to or otherwise interacts with non-contiguous amino acid residues that are brought together by folding or other aspects of the secondary and/or tertiary structure of the polypeptide chain. Preferably, the one or more portions of mature TFPI (for example, Figure 3A) are selected from the group consisting of: amino acids 148-170, amino acids 150-170, amino acids 155-175, amino acids 160-180, amino acids 165-185, amino acids 170-190, amino acids 175-195, amino acids 180-200, amino acids 185-205, amino acids 190-210, amino acids 195-215, amino acids 200 220, amino acids 205-225, amino acids 210-230, amino acids 21 5-235, amino acids 220-240, amino acids 225-245, amino acids 230-250, amino acids 235-255, amino acids 240-260, amino acids 245-265, amino acids 250-270, amino acids 255-275, amino acids 260-276, amino acids 148-175, amino acids 150-175, amino acids 150-180, amino acids 150-185, amino acids 150 190, amino acids 150-195, amino acids 150-200, amino acids 150-205, amino acids 150-210, amino acids 150-215, amino acids 150-220, amino acids 150-225, amino acids 150-230, amino acids 150-235, amino acids 150-240, amino acids 150-245, amino acids 150-250, amino acids 150-255, amino acids 150-260, amino acids 150-265, amino acids 150-270, amino acids 150 275, amino acids 150-276, amino acids 190-240, amino acids 190-276, amino acids 240-276, amino acids 242-276, amino acids 161-181, amino acids 162-181, amino acids 182-240, amino acids 182-241, and amino acids 182-276. The aptamer preferably comprises a dissociation constant for human TFPI or a variant or one or more portions thereof of less than 100 pM, less than I [[M, less than 500 nM, less than 100 nM, preferably 50 nM or less, preferably 25 nM or less, preferably 10 nM or less, preferably 5 nM or less, more preferably 3 nM or less, even more preferably I nM or less, and most preferably 500 pM or less. [00821 The invention further provides methods for identifying aptamers that bind at least in part to or otherwise interact with one or more portions of TFPI, which comprise the steps of (a) contacting a mixture of nucleic acids with full-length TFPI or one or more portions of TFPI under conditions in which binding occurs in the presence of a TFPI ligand (a ligand that binds to TFPI) that blocks one or more epitopes on TFPI from aptamer binding; (b) partitioning unbound nucleic acids from those nucleic acids that have bound to full-length TFPI or one or more portions of TFPI; (c) amplifying the bound nucleic acids to yield a ligand-enriched mixture of nucleic acids; and, optionally, (d) reiterating the steps of contacting, partitioning and amplifying 24 WO 2012/109675 PCT/US2012/024916 through as many cycles as desired to obtain aptamer(s) that bind to one or more portions of TFPL In other embodiments of this method, inclusion of a TFPI ligand that blocks one or more portions on TFPI from aptamer binding can occur during the contacting step, the partitioning step, or both. For example, the TFPI aptamers may bind to or otherwise interact with a linear portion or a conformational portion of TFPI. A TFPI aptamer binds to or otherwise interacts with a linear portion of TFPI when the aptamer binds to or otherwise interacts with a contiguous stretch of amino acid residues that are linked by peptide bonds. A TFPI aptamer binds to or otherwise interacts with a conformational portion of TFPI when the aptamer binds to or other rise interacts with non-contiguous amino acid residues that are brought together by folding or other aspects of the secondary and/or tertiary structure of the polypeptide chain. Preferably, the one or more portions of mature TFPI (for example, Figure 3A) are selected from the group consisting of: amino acids 148-170, amino acids 150-170, amino acids 155-175, amino acids 160-180, amino acids 165-185, amino acids 170-190, amino acids 175-195, amino acids 180 200, arnino acids 185-205, arnino acids 190-210, arnino acids 195-215, arnino acids 200-220, amino acids 205-225, amino acids 210-230, amino acids 215-235, amino acids 220-240, amino acids 225-245, arnino acids 230-250, arnino acids 235-255, arnino acids 240-260, amino acids 245-265, amino acids 250-270, amino acids 255-275, amino acids 260-276, amino acids 148 175, arnino acids 150-175, arnino acids 150-180, arnino acids 150-185, arnino acids 150-190, amino acids 150-195, amino acids 150-200, amino acids 150-205, amino acids 150-210, amino acids 150-215, amino acids 150-220, amino acids 150-225, amino acids 150-230, amino acids 150-235, amino acids 150-240, amino acids 150-245, amino acids 150-250, amino acids 150 255, amino acids 150-260, amino acids 150-265, amino acids 150-270, amino acids 150-275, amino acids 150-276, amino acids 190-240, amino acids 190-276, amino acids 240-276, amino acids 242-276, amino acids 161-181, amino acids 162-181, amino acids 182-240, amino acids 182-241, and amino acids 182-276. The aptamer preferably comprises a dissociation constant for human TFPI or a variant or one or more portions thereof of less than 100 taM, less than 1 pM, less than 500 nM, less than 100 nM, preferably 50 nM or less, preferably 25 nM or less, preferably 10 nM or less, preferably 5 nM or less, more preferably 3 nM or less, even more preferably I nM or less, and most preferably 500 pM or less. [00831 The invention further provides methods for identifying aptamers that bind at least in part to or otherwise interact with one or more portions of TFPI, which comprise the steps of (a) 25 WO 2012/109675 PCT/US2012/024916 contacting a mixture of nucleic acids with full-length TFPI or one or more portions of TFPI under conditions in which binding occurs; (b) partitioning unbound nucleic acids from those nucleic acids that have bound to full-length TFPI or one or more portions of TFPI; (c) partitioning bound nucleic acids that have a desired functional property from bound nucleic acids that do not have a desired functional property; (d) amplifying the bound nucleic acids that have a desired functional property to yield a ligand-enriched mixture of nucleic acids; and, optionally, (e) reiterating the steps of contacting, partitioning, partitioning and amplifying through as many cycles as desired to obtain aptamer(s) that bind to one or more portions of TFPL Steps (b) and (c) can occur sequentially or simultaneously. Preferably, the desired functional property is inhibition of TFPI's interaction with FXa, FVIIa, TFPI receptor or the glycocalyx. For example, the TFPI aptamers may bind to or otherwise interact with a linear portion or a conformational portion of TFPL A TFPI aptamer binds to or otherwise interacts with a linear portion of TFPI when the aptamer binds to or otherwise interacts with a contiguous stretch of amino acid residues that are linked by peptide bonds. A TFPI aptamer binds to or otherwise interacts with a conformational portion of TFPI when the aptamer binds to or otherwise interacts with non contiguous anino acid residues that are brought together by folding or other aspects of the secondary and/or tertiary structure of the polypeptide chain. Preferably, the one or more portions of mature TFPI (for example, Figure 3A) are selected from the group consisting of: amino acids 148-170, amino acids 150-170, amino acids 155-175, amino acids 160-180, amino acids 165 185, amino acids 170-190, amino acids 175-195, amino acids 180-200, amino acids 185-205, amino acids 190-210, amino acids 195-215, amino acids 200-220, amino acids 205-225, amino acids 210-230. amino acids 215-235, amino acids 220-240, amino acids 225-245, amino acids 230-250, amino acids 235-255, amino acids 240-260, amino acids 245-265, amino acids 250 270, amino acids 255-275, amino acids 260-276, amino acids 148-175, amino acids 150-175, amino acids 150-180, amino acids 150-185, amino acids 150-190, amino acids 150-195, amino acids 150-200. amino acids 150-205, amino acids 150-210. amino acids 150-215. amino acids 150-220, amino acids 150-225, amino acids 150-230, amino acids 150-235, amino acids 150 240, amino acids 150-245, amino acids 150-250., amino acids 150-255, amino acids 150-260. amino acids 150-265, amino acids 150-270, amino acids 150-275, amino acids 150-276, amino acids 190-240. amino acids 190-276, amino acids 240-276. amino acids 242-276, amino acids 161-181, amino acids 162-181, amino acids 182-240, amino acids 182-241, and amino acids 26 WO 2012/109675 PCT/US2012/024916 182-276. The aptamer preferably comprises a dissociation constant for human TFPI or a variant or one or more portions thereof of less than 100 pM, less than I PM, less than 500 nM. less than 100 nM. preferably 50 nM or less, preferably 25 nM or less, preferably 10 nM or less, preferably 5 nM or less, more preferably 3 nM or less, even more preferably 1 nM or less, and most preferably 500 pM or less. [0084] The invention also provides an aptamer that binds to a human tissue factor pathway inhibitor (TFPI) polypeptide having the amino acid sequence of SEQ ID NO: 11, wherein the aptamer modulates TFPI-mediated inhibition of blood coagulation, and wherein the aptamer competes for binding to TFPI with a reference aptamer comprising a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 4 (ARC I9499), SEQ ID NO: 1 (ARC26835), SEQ ID NO: 2 (ARC] 7480), SEQ ID NO: 3 (ARCI19498), SEQ ID NO: 5 (ARC19500), SEQ ID NO:6 (ARCI9501), SEQ ID NO: 7 (AR C31l01), SEQ ID NO: 8 (ARC 18546), SEQ ID NO: 9 (ARC19881) and SEQ lI) NO: 10 (ARC19882). Preferably, the reference aptamer comprises the nucleic acid sequence of SEQ ID NO: 4 (ARC19499). [0085] The invention further provides an aptaner that binds to a human tissue factor pathway inhibitor (TFPf) polypeptide having the amino acid sequence of SEQ ID NO: 11, wherein the aptamer binds to a linear portion or a conformational portion of TFPI in which at least a portion of the region recognized by the aptamer is different than the TFPI region bound by Factor VIIa, Factor Xa, or both Factor Vila and Factor Xa. Preferably, the aptamer binds to one or more regions comprising at least a portion of the amino acid sequence of SEQ ID NO: 11 selected from the group consisting of: amino acid residues 148-170, amino acid residues 150 170, amino acid residues 155-175, amino acid residues 160-180, amino acid residues 165-185, amino acid residues 170-190, amino acid residues 175-195, amino acid residues 180-200, amino acid residues 185-205, amino acid residues 190-210, amino acid residues 195-215, amino acid residues 200-220, amino acid residues 205-225. amino acid residues 210-230, amino acid residues 215-235, amino acid residues 220-240, amino acid residues 225-245, amino acid residues 230-250, amino acid residues 235-255, amino acid residues 240-260. amino acid residues 245-265, amino acid residues 250-270, amino acid residues 255-275, amino acid residues 260-276. amino acid residues 148-175. amino acid residues 150-175. amino acid residues 150-180, amino acid residues 150-185, amino acid residues 150-190, amino acid residues 150-195, amino acid residues 150-200, amino acid residues 150-205, amino acid 27 WO 2012/109675 PCT/US2012/024916 residues 150-210, amino acid residues 150-215, amino acid residues 150-220, amino acid residues 150-225, amino acidesisidues 150-230, amino acid residues 150-235. amino acid residues 150-240, amino acid residues 150-245, amino acid residues 150-250, amino acid residues 150-255, amino acid residues 150-260, amino acid residues 150-265, amino acid residues 150-270, amino acid residues 150-275, amino acid residues 150-276, amino acid residues 190-240, amino acid residues 190-276. amino acid residues 240-276. amino acid residues 242-276, amino acid residues 161-181, amino acid residues 162-181, amino acid residues 182-240, amino acid residues 182-241, and amino acid residues 182-276. More preferably, the aptamer competes with a reference aptamer comprising the nucleic acid sequence of SEQ ID NO: 4 (ARC19499) for binding to TFPL [00861 The invention also provides an aptamer that binds to the same region on a human tissue factor pathway inhibitor (TFPI) polypeptide having the amino acid sequence of SEQ ID NO: 11 as the region bound by a TFPI aptamer comprising the nucleic acid sequence of SEQ ID NO: 4 (ARC19499). [0087] The invention further provides an aptarner that binds to a region on a hurnan tissue factor pathway inhibitor (TFPI) polypeptide comprising one or more portions of SEQ ID NO: 11, wherein the one or more portions is selected from the group consisting of: amino acid residues 148-170, amino acid residues 150-170, amino acid residues 155-175, amino acid residues 160 180, amino acid residues 165-185, amino acid residues 170-190, amino acid residues 175-195, amino acid residues 180-200, amino acid residues 185-205, amino acid residues 190-210, amino acid residues 195-215, amino acid residues 200-220, amino acid residues 205-225, amino acid residues 210-230, amino acid residues 215-235, amino acid residues 220-240, amino acid residues 225-245. amino acid residues 230-250, amino acid residues 235-255, amino acid residues 240-260, amino acid residues 245-265, amino acid residues 250-270, amino acid residues 255-275, amino acid residues 260-276, amino acid residues 148-175, amino acid residues 150-175, amino acid residues 150-180, amino acid residues 150-185, amino acid residues 150-190, amino acid residues 150-195, amino acid residues 150-200, amino acid residues 150-90, amino acid residues 150-210, amino acid residues 150-215, amino acid residues 150-220, amino acid residues 150-225, amino acid residues 150-230, amino acid residues 150-235, amino acid residues 150-240, amino acid residues 150-Z45, amino acid residues 150-250, amino acid residues 150-255. amino acid residues 150-260. amino acid 28 WO 2012/109675 PCT/US2012/024916 residues 150-265, amino acid residues 150-270, amino acid residues 150-275, amino acid residues 150-276, amino acid residues 190-240. amino acid residues 190-276, amino acid residues 240-276, amino acid residues 242-276, amino acid residues 161-181, amino acid residues 162-181. amino acid residues 182-240, amino acid residues 182-241. and amino acid residues 182-276. [0088] The invention additionally provides an aptamer that binds to human tissue factor pathway inhibitor (TFPI) and exhibits one or more of the following properties: a) competes for binding to TFPI with any one of SEQ ID NOs: 1-10; b) inhibits TFPI inhibition of Factor Xa; c) increases thrombin generation in hemophilia plasma; d) inhibits TFPI inhibition of the intrinsic tenase complex; e) restores normal hemostasis, as measured by thromboelastography (TEG*) in whole blood and plasma; f) restores normal clotting, as indicated by shorter clot time, more rapid clot formation or more stable clot development, as measured by thromboelastography (TEG*) or rotational thromboelastometry (ROTEM) in whole blood and plasma; or g) decreases the clot time, as measured by dilute prothrombin time (dPT), tissue factor activated clotting time (TF-ACT) or any other TFPI-sensitive clot-time measurement. [0089] The invention also provides an aptarner that binds to human tissue factor pathway inhibitor wherein the aptamer competes for binding to TFPI with a reference aptamer selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. 100901 The invention further provides an aptamer that binds to tissue factor pathway inhibitor (TFPi) wherein the aptamer competes, either directly or indirectly, for binding to TFPI with a reference antibody selected from the group consisting of: AD4903. [00911 The invention also provides an aptamer that binds to human tissue factor pathway inhibitor (TFPI) and comprises a stem and loop motif having the nucleotide sequence of SEQ ID NO: 4, wherein: a) any one or more of nucleotides 1, 2, 3, 4, 6, 8, 11, 12, 13, 17, 20, 21, 22. 24, 28 30 and 32 may be modified from a 2'-OMe substitution to a 2'-deoxy substitution; b) any one or more of nucleotides 5, 7, 15, 19, 23, 27, 29 and 31 may be modified from a 2'-OMe uracil to either a 2'-deoxy uracil or a 2'-deoxy thymine; c) nucleotide 18 may be modified from a 2'-OMe uracil to a 2'-deoxy uracil; and/or d) any one or more of nucleotides 14, 16 and 25 may be modified from a 2'-deoxy cytosine to either a 2'-OMe cytosine or a 2'-luoro cytosine. 29 WO 2012/109675 PCT/US2012/024916 [00921 The invention additionally provides an aptamer that binds to human tissue factor pathway inhibitor (TFPI) and comprises nucleotides 7-28 of SEQ ID NO: 2. [00931 The invention further provides a method for treating a bleeding disorder comprising administering any one of the above aptamers. [00941 The invention further provides an aptamer that binds to tissue factor pathway inhibitor (TFPI), wherein the aptamer comprises a primary nucleic acid sequence selected from the group consisting of SEQ ID NOs.: 4, 1, 2, 3, 5, 6, 7, 8, 9 and 10. BRIEF DESCRIPTION OF THE DRAWINGS [00951 Figure 1 is a schematic representation of the coagulation cascade. [00961 Figure 2 is an illustration of the forms of TFPI, which are associated with the vascular endothelium or in the plasma pool, [00971 Figure 3 is a schematic representation of the two forms of TFPI found on the endothelium, (Figure 3A.) TFPlu and (Figure 3B) TFPI. [00981 Figure 4 is a schematic representation of the in vitro aptamer selection (SELEXTIMI) process fro n pools of random sequence oligonucleotides. 100991 Figure 5 is an illustration of the amino acid sequence of the mature hunan TFPI protein. 1001001 Figure 6 is an illustration of a 40 kDa branched PEG. [001011 Figure 7 is an illustration of a 40 kDa branched PEG that is attached to the 5' terminus of an amine aptamer. 1001021 Figure 8 is an illustration of a 40 kDa branched PEG that is attached to the 5' terminus of an aptamer using a 5'-amine linker phosphoramidite. 1001031 Figure 9A is an illustration of a 40 kDa branched PEG that is attached to the 5' terminus of an aptamer using a 5'-hexylamine liner phosphoramidite. Figure 9B is an alternative illustration of a 40 kDa branched PEG that is attached to the 5' terminus of an aptamer using a 5'-hexylamine linker phosphoramidite. 1001041 Figure 10 A is an illustration of a TFPI aptamer, which is comprised of 2 '-O Methyl (circles) and 2'-deoxy (squares) nucleotides and is modified at its 5'-terminus with a 40 kDa PEG moiety and at its 3 'terminus with an inverted deoxythymidine residue (3T, which is also known in the art as idT). Figure 10B is an illustration of a TFPI aptamer, which is comprised of 2-O Methyl (circles) and 2'-deoxy (squares) nucleotides and is modified at its 5'-terminus with 30 WO 2012/109675 PCT/US2012/024916 a 40 kDa PEG moiety and linker, and at its 3'-terminus with an inverted deoxythymidine residue (3T). Figure lOC is an illustration of the putative structure of ARC19499. which is comprised of 2'-O Methyl (circles) and 2'-deoxy (squares) nucleotides and is modified at its 5'-terminus with a 40 kDa branched PEG moiety and a hexylainine phosphate-containing linker, and at its 3' terminus with an inverted deoxythymidine residue (3T). [00105] Figure II is an illustration depicting various PEGylation strategies, such as standard mono-PEGylation, multiple PEGylation and oligomerization via PEGylation. [00106] Figure 12A is a graph showing that ARC 17480 binds tightly to full-length TFPI. The data are fit to both nonophasic and biphasic models to determine a KD for binding. Figure 12B is a graph showing that tRNA shifts the affinity of ARC17480 for TFPI. The aptamer still binds tightly to TFPI in the presence of tRNA, indicating that the binding of ARC17480 to TFPI is specific. [00107] Figure 13 depicts the results of binding-competition experiments with radiolabeled ARC17480, full-length TFPI and various unlabeled aptamers. Unlabeled ARCI 7480 and ARC19499 (Figure 13A); ARC1 9498 (Figure 1313), ARC18546 (Figure 13C); ARC26835 and ARC31301 (Figure 13D); ARC19500, ARCI 9501, ARC19881 and ARC19882 (Figure 13E) all compete for binding with radiolabeled ARC 17480. [00108] Figure 14 is a set of graphs showing binding experiments with ARC17480 and various proteins, including coagulation factors, protease inhibitors and coagulation zymogens. Figure 14A is a graph of a binding experiment with ARC 17480 and various proteins. Figure 14B is a graph of a binding experiment with ARC17480 and TFPI or various activated coagulation factors. Figure 14C is a graph of a binding experiment with ARC 17480 and TFPI or various protease inhibitors. Figure14D is a graph of a binding experiment with ARC 17480 and TFPI or various coagulation zymogens. ARC17480 showed significant binding to TFPI, but not to any of the other proteins tested, [001091 Figure 15 is a graph showing data from a plate-based assay demonstrating binding of ARC 19499 to recombinant TFPI. [001101 Figure 16 is a graph showing data from a plate-based competition assay demonstrating binding of ARC19498 to TFPI in competition with ARC19499. [001111 Figure 17A depicts the results of a binding assay with radiolabeled ARC17480, full length TFPI and TFPI-His. Figure 17B depicts the results of a binding assay with radiolabeled 31 WO 2012/109675 PCT/US2012/024916 ARC17480, full-length TFPI, truncated TFPI-K1K2, TFPI K3-C-terminal domain protein, and the peptide that contains the C-terminal region of TFPI in the presence of neutravidin, [001121 Figure 18A depicts the results of a binding assay with radiolabeled ARC 17-480 and full-length TFPI in the absence or presence of 0.1 mg/mL heparin. Figure 18B depicts the results of a binding-competition assay with radiolabeled ARC] 7480, 12.5 nM full-length TFPI, and different concentrations of heparin and low molecular weight heparin (LMWH) as competitors. [001131 Figure 19, A and B, illustrates competition of various anti-TFPi antibodies with ARC1 9499 in a plate-based binding assay. [001141 Figure 20, A, B and C, illustrates competition of various anti-TFPi antibodies with ARC1 9499 in a nitrocellulose filtration (dot-blot) assay. [001151 Figure 21 is a series of graphs showing the activity of ARC19499 in the extrinsic Xase inhibition assay. In Figure 2 1A, the rate (mOD/min) was plotted vs time (minutes). In the absence of TFPI, the rate was linear. I nM TFPI decreased the rate dramatically. Increasing concentrations of ARC19499 from 0.01 to 1000 nM increased the rate in a dose-dependent manner until nearly the level of no TFPI. In Figure 21B, the rates at the 4 minute time point were normalized to the rate in the absence of TFPI at 4 minutes. ARC] 9499 showed a dose dependent improvement on the rate of the assay, reaching levels close to that of no TFPI by 10 niM aptamer. Data for Figure 21 A were representative from three experiments. Data for Figure 21B represent mean i standard error, n:=3. 1001161 Figure 22, A-C, depicts the results of a Factor Xa (FXa) activity assay with full length TFPI and ARC 17480, ARC18546, ARC26835, ARC3 1301, ARC19498, ARC19499, ARC19500, ARC19501, ARC19881 or ARC19882. The adjusted rate of FXa substrate degradation is plotted as a function of aptamer concentration. The rates are adjusted by subtraction of the rate observed with FXa and TFPI in the absence of aptamer. All of the aptamers inhibit TFPI, which results in a concentration-dependent increase in FXa activity in this assay. [001171 Figure 23 is a graph that shows protection of Factor Xa (FXa) activity by ARC19499 from TFPI inhibition in a chromogenic FXa activity assay. [001181 Figure 24 is a graph that shows protection of the extrinsic FXase by ARC 19499 from TFPI inhibition in a chromogenic assay of Factor X (FX) activation. 32 WO 2012/109675 PCT/US2012/024916 [001191 Figure Z25 is a graph that shows protection of the TF:FVIIa complex by ARC 19499 from TFPI inhibition in a fluorogenic assay of TF:FVIIa activity. [001201 Figure 26 is a graph that shows the effect of ARC 19499 on tissue factor (TF)-initiated thrombin generation in a Normal Synthetic Coagulation Proteome (SCP). [001211 Figure 27 is a graph that shows the effect of ARC 19499 on tissue factor (TF)-irnitiated thrombin generation in a hemophilia A Synthetic Coagulation Proteome. [001221 Figure 28 is a graph that shows the effect of ARC 19499 on tissue factor (TF)-irnitiated thrombin generation in a hemophilia B Synthetic Coagulation Proteome. [001231 Figure 29 is a graph that shows the effect of increasing Factor VIII (FVIII) concentrations on tissue factor (TF)-initiated thrombin generation in the absence of ARC19499. [001241 Figure 30 is a graph that shows the effect of increasing Factor VIII (FVIII) concentrations on tissue factor (TF)-initiated thronbin generation in the presence of 1.0 nM ARC19499. 1001251 Figure 31 is a graph that shows the effect of increasing Factor VIII (FVII I) concentrations on tissue factor (TF)-initiated thrombin generation in the presence of 2.5 nM ARC 19499. [001261 Figure 32 is a graph that shows the effect of increasing ARC19499 concentrations on tissue factor (TF)-initiated thrombin generation in the absence of Factor VIII (FVIII). 1001271 Figure 33 is a graph that shows the effect of increasing ARC 19499 concentrations on tissue factor (TF)-initiated thrombin generation in the presence of 100% Factor VIII (FVIII). 1001281 Figure 34 is a graph that shows the effect of increasing ARC 19499 concentrations on tissue factor (TF)-initiated thrombin generation in the presence of 2% Factor VIII (F VIII). [001291 Figure 35 is a graph that shows the effect of increasing ARC 19499 concentrations on tissue factor (TF)-initiated thrombin generation in the presence of 5% Factor VIII (FVIII). [001301 Figure 36 is a graph that shows the effect of increasing ARC 19499 concentrations on tissue factor (TF)-initiated thrombin generation in the presence of 40% Factor VIII (FVIII). [001311 Figure 37 is a series of graphs showing the activity of ARC 19499 in the calibrated automated thrombogram (CAT) assay in pooled normal plasma (PNiP) initiated with 0.1 pM tissue factor (TF; Figure 37A) or 1.0 pM TF (Figure 37B). The endogenous thrombin potential (ETP; Figure 37C) and peak thrombin (Figure 37D) both showed a dose-dependent increase with increasing concentrations of ARC]19499 at both TF concentrations. The lag time (Figure 37E) 33 WO 2012/109675 PCT/US2012/024916 showed a dose-dependent decrease with increasing concentrations of ARC 19499 at both TF concentrations. [001321 Figure 38 is a series of graphs showing the activity of ARC 19499 in the calibrated automated thrombogram (CAT) assay in TFPI-depleted plasma initiated with 0.01, 0.1 or 1.0 pM tissue factor (TF). Figure 38A shows thrombin generation curves at increasing ARC19499 concentrations with three different TF concentrations. The endogenous thrombin potential (ETP; Figure 38B), peak thrombin (Figure 38C) and lag time (Figure 38D) showed little or no change over all tested ARC19499 concentrations at all tested TF concentrations. [001331 Figure 39 is a series of graphs showing the activity of ARC19499 in the calibrated automated thrombogram (CAT) assay in pooled normal plasma (PNP) previously treated with a neutralizing, polyclonal anti-TFPI antibody. The assay was initiated with 0.01 pM tissue factor (TF; Figure 39A), 0.1 pM TF (Figure 3913) or 1.0 pM TF (Figure 39C). The endogenous thrombin potential (ETP; Figure 39D), peak thrombin (Figure 39E) and lag time (Figure 39F) remained largely unchanged at all ARCI 9499 concentrations independent of the TF concentration. [00134] Figure 40 is a series of graphs showing a calibrated automated thrombogram (CAT) assay with ARC1480 (Figure 40A), ARC1 9498 (Figure 40B) and ARC19499 (Figure 40C) at various concentrations. The endogenous thrombin potential (ETP; Figure 40D) and peak thrombin (Figure 40E) measured with various concentrations of ARC 17480, ARC 19498 and ARC 19499 in hemophilia A plasma were similar to one another, with ARCl 9499 having slightly greater activity, reaching an ETP plateau close to normal plasma levels by 30 nM aptamer. The thrombin generation curves (Figure 40A-C) are representative data. The ETP (Figure 40D) and peak thrombin (Figure 40E) data represent the mean ± standard error, n=3. [001351 Figure 41 is a graph of thrombin generation in platelet-poor normal plasma from a single, healthy volunteer. The plasma was treated with an anti-FVIII antibody to generate a hemophilia A-like state. ARC19499 showed a dose-dependent increase in thrombin generation in the antibody-treated plasma. [001361 Figure 42 is a series of graphs showing a calibrated automated thrombogram (CAT) assay with ARC19499 (Figure 42A) and ARCI7480 (Figure 42B) at various concentrations in hemophilia B plasma. 34 WO 2012/109675 PCT/US2012/024916 [001371 Figure 43 is a series of graphs showing the effect of ARC19499 (diamonds) and ARC 17480 ( riangles) on endogenous thrombin potential (FTP), peak thrombin and lag time in hemophilia B plasma. The solid line designates the level of each parameter in the absence of any drug. The hatched line designates the level of each parameter in pooled normal plasma (PNP) without any additional drug. Data represent mean ± standard error, n=3. Both aptainers behaved very similarly to each other in hemophilia B plasma. [001381 Figure 44 is a series of graphs showing the effects of ARC 19499 compared to a negative control aptamer on thrombin generation as measured by the calibrated automated thrombogram (CAT) assay in plasmas from patients with hemophilia A (Figure 44A), hemophilia A with inhibitors (Figure 44B) or hemophilia B (Figure 44C. The results are given in terms of the lag time (left), endogenous thrombin potential (ETP) (middle) and peak thrombin concentration (right). In all graphs, lines represent activity of normal plasma (solid) and factor deficient plasma (dashed) in the absence of aptamer, and shading around the lines represents the standard error of the rnean. [00139] Figure 45 depicts the results of thrombin generation experiments with ARCI 7480, ARCI18546, ARC26835 and ARC31301 in hemophilia A plasma. Adjusted endogenous thrombin potential (ETP; Figure 45A and C) and adjusted peak thrombin (Figure 4513 and D) values are plotted as a function of aptamer concentration. The ETP and peak thrombin values for hemophilia plasma were subtracted from each value to give the adjusted values. ARC17480, ARC18546, ARC26835 and ARC31301 increase thrombin generation in a concentration dependent manner in hemophilia A plasma. 1001401 Figure 46 depicts the results of thrombin generation experiments with ARC17480, ARC19500. ARC19501, ARC19881 and ARC19882 in hemophilia A plasma. Adjusted endogenous thrombin potential (ETP; Figure 46A) and adjusted peak thrombin (Figure 46B) values are plotted as a function of aptamer concentration. The ETP and peak thrombin values for hemophilia plasma were subtracted from each value to give the adjusted values. ARC! 7480, ARC19500. ARC19501, ARC19881 and ARC19882 increase thrombin generation in a concentration-dependent manner in hemophilia A plasma. [001411 Figure 47 is a series of graphs from thrombin generation experiments showing the effect of NovoSeven" (empty triangles) and ARCI 9499 (filled diamonds) on endogenous thrombin potential (ETP; Figure 47A), peak thrombin (Figure 47B) and lag time (Figure 47C) in 35 WO 2012/109675 PCT/US2012/024916 normal plasma. The solid black line designates the level of each parameter in the absence of any drug. Data represent mean ± standard error, n=3. [001421 Figure 48 is a series of graphs from thrombin generation experiments showing the effect of NovoSeven* (empty triangles) and ARC 19499 (filled diamonds) on endogenous thrombin potential (ETP; Figure 48A), peak thrombin (Figure 48B) and lag time (Figure 48C) in hemophilia A plasma. The solid black line designates the level of each parameter in the absence of any drug. The dashed line designates the level of each parameter in pooled normal plasma (PNP) without anv additional drug. Data represent mean ± standard error, n=3. [001431 Figure 49 is a series of graphs from thrombin generation experiments showing the effect of NovoSeven* (empty triangles) and ARC19499 (filled diamonds) on endogenous thrombin potential (ETP; Figure 49A), peak thrombin (Figure 49B) and lag time (Figure 49C) in hemophilia A inhibitor plasma. The solid black line designates the level of each parameter in the absence of any drug. The dashed line designates the level of each parameter in pooled normal plasma (PNP) without any additional drug. Data represent mean + standard error, n=:::. [001441 Figure 50 is a series of graphs from experiments showing the effect of NovoSeven* (empty triangles) and ARC 19499 (filled diamonds) on R-value (Figure 50A), angle (Figure 5013) and maximum amplitude (MA; Figure 50C) in a thromboelastography (TEG*) assay in citrated whole blood frorn healthy volunteers. The solid black line designates the level of each parameter in the absence of any drug. Data represent mean ± standard error, n::::3. 1001451 Figure 51 is a series of graphs from experiments showing the effect of NovoSevenI (empty triangles) and ARC1 9499 (filled diamonds) on R-value (Figure 51A), angle (Figure 51B) and maximum amplitude (MA; Figure 51C) in a thromboelastography (TEG") assay in citrated whole blood from healthy volunteers treated with an anti-FVIll antibody. 'The solid black line designates the level of each parameter in the absence of any drug. 'The dashed line designates the level of each parameter in whole blood not treated with antibody. Data represent mean i standard error, n=3. [001461 Figure 52 is a series of graphs from thromboelastography experiments showing the lag time (Figure 52A). peak thrombin (Figure 52B) and endogenous thrombin potential (ETP; Figure 52C). Each line represents the dose response of ARC19499 in the presence of a different percent of Factor VIII (filled diamonds, 0%; empty triangles, 1.4%; filled squares, 2.5%; filled triangles, 5%; empty squares, 14%; and filled circles, 140%). The dashed line designates level 36 WO 2012/109675 PCT/US2012/024916 of each parameter in the presence of pooled normal plasma (PNP) alone. The solid line designates the level of each parameter in hemophilia A plasma without any additions. Data represent mean ± standard error, n=3. [001471 Figure 53 is a series of graphs from thrombin generation experiments demonstrating ARC19499 activity in plasma with various concentrations of Factor VIII (FVIII). In Figure 53A, the endogenous thrombin potential (ETP) is plotted as a function of ARC19499 concentration. The dashed lines represent the ETP after addition of different amounts of FVIII to hemophilia A plasma. The solid lines show that ARC1 9499 increases ETP in hemophilia A plasma (line with triangles) and hemophilia A plasma with 5% FVIII added (line with diamonds), In Figure 53B, the ETP is plotted versus FVIII concentration. ETP data is shown with and without the addition of '00 nM ARC 19499. [00148] Figure 54 illustrates the experimental design of the spatial clotting model. Figure 54A is a diagram of the spatial clotting chamber. Figure 54B is a schematic illustration of the components of the system for measuring clot progression in the chamber. [00149] Figure 55 shows two graphs illustrating clot propagation in the spatial clotting model, as measured by light scattering, plotted as a function of distance from the activating surface. Clotting was activated by low density tissue factor in normal pooled plasma in the absence (Figure 55A) and in the presence (Figure 55B) of 300 nM ARC1 9499. 1001501 Figure 56 is a graph of clot size versus time, in the absence (thick black line) and the presence (thin grey line) of 300 nM ARCi 9499 in normal pooled plasma. The parameters that can be derived from this graph include the lag time (time until beginning of clot growth), initial velocity (co or Vitia,; mean slope during the first 10 minutes of growth), stationary velocity (P or Vstationarv; mean slope during the next 30 minutes of growth) and clot size after 60 minutes (an integral parameter of clot formation efficiency). 100151] Figure 57 is a series of graphs showing the lag time (Figure 57A), Viitiai (Figure 5713), Vstationary (Figure 57C) and clot size after 60 minutes (Figure 571D) in normal pooled plasma, each plotted as a function of tissue factor density in the presence (circles) and absence (squares) of ARC] 9499. 100152] Figure 58 is a series of graphs showing the lag time (Figure 58A), Viiti 1 (Figure 58B), Vstationary (Figure 58C) and clot size after 60 minutes (Ficure 58D) in normal pooled 37 WO 2012/109675 PCT/US2012/024916 plasma, each plotted as a function of ARC 19499 concentration under conditions of low surface tissue factor density. [001531 Figure 59 is a series of graphs illustrating the effect of ARC 19499 on the lag time (Figure 59A), Vtia (Figure 59B), Vstajonary (Figure 59C) and clot size after 60 minutes (Figure 59D) in normal pooled plasma under conditions of low surface tissue factor density. An asterisk indicates a statistically significant difference ± ARC 19499 (P<0,05). [001541 Figure 60 is a series of graphs showing the lag time (Figure 60A), Vinitiai (Figure 60B), Vtationary (Figure 60C) and clot size after 60 minutes (Figure 60D) in normal pooled plasma, each plotted as a function of ARC1 9499 concentration under conditions of mediun surface tissue factor density. [001551 Figure 61 is a series of graphs illustrating the effect of ARC19499 on the lag time (Figure 61 A), Vi;itial (Figure 6113), Vsiationary (Figure 61 C) and clot size after 60 minutes (Figure 61 D) in normal pooled plasma under conditions of medium surface tissue factor density. An asterisk indicates a statistically significant difference A- ARCi 9499 (P<0.05). [001561 Figure 62 compares clot propagation in normal pooled plasma (Figure 62A) to normal pooled plasma containing 100 nM ARC19499 (Figure 62B) or 100 nM recombinant factor VIlla (rVI I a or Novoseven" Figure 62C) under conditions of low surface tissue factor density. [00157] Figure 63 is an illustration showing a series of light scattering images from the spatial clotting model. Each row depicts clot propagation from a surface (bottom) over time 0, 10, 20, 30, 40, 50 and 60 minutes. The top row shows clot propagation in severe hemophilia A plasma, followed by severe hemophilia A plasma containing 100 nM ARC 19499 in the second row and severe hemophilia A plasma containing 100 nM recombinant factor VlIa (rVIla) in the third row. [00158] Figure 64 is a graph of clot size versus time, in normal plasma (dark grey, dashed line), severe hemophilia A plasma (black, solid line), severe hemophilia A plasma containing 100 nM ARC 19499 (light grey, solid line) or 100 nM recombinant factor VIIa (rVIIa) (light grey, dashed line). [001591 Figure 65 is a table summarizing the demographics of hemophilia A patients from which plasma samples were drawn for spatial clot formation experiments. [001601 Figure 66 shows the effects of ARC 19499 or recombinant factor VIla (rVIla), titrated into severe hemophilia A plasma from Patient 1, on spatial clot formation activated with low surface tissue factor density. The effects of ARC19499 and rVIla on lag time are depicted in 38 WO 2012/109675 PCT/US2012/024916 Figure 66A and B, respectively, while the effects of ARC19499 and rVIIa on Vina are depicted in Figure 66C and D, respectively. [001611 Figure 67 shows the effects of ARC 19499 or recombinant factor VIIa (rVIIa), titrated into severe hemophilia A plasma from Patient 2, on spatial clot formation activated with low surface tissue factor density. The effects of ARC19499 and rVIIa on lag time are depicted in Figure 67A and B, respectively, while the effects of ARC19499 arid rVIIa on Vinitia are depicted in Figure 67C and D, respectively. [001621 Figure 68 shows the effects of ARC 19499 or recombinant factor VIla (rVIla), titrated into severe hemophilia A plasma from Patient 3, on spatial clot formation activated with low surface tissue factor density. The effects of ARC19499 and rVIIa on lag time are depicted in Figure 68A and B, respectively, while the effects of ARCI 9499 and rVIia on Vinmi are depicted in Figure 68C and D, respectively. [00163] Figure 69 shows the effects of ARCi 9499 (black symbols) or recombinant factor Vlla (rVIa; grey symbols) on Vstationary in hemophilia A plasma samples from Patients 1-3, activated with low surface tissue factor density. [00164] Figure 70 shows the effects of ARC 19499 (Figure 70A) or recombinant factor VIla (rVI a; Figure 70B) on clot size at 60 minutes in hemophilia A plasma samples from Patients I 3, activated with low surface tissue factor density. 1001651 Figure 71 is a series of graphs illustrating the effect of 300 nM ARC19499 on the average lag time (Figure 71A), V 'nia (Figure 71 B), Vstaionary (Figure 71C) and clot size after 60 minutes (Figure 71D) in hemophilia A plasma activated with low surface tissue factor density (n:=3). An asterisk indicates a statistically significant difference ± ARCi 9499 (P<0.05). [00166] Figure 72 is a series of graphs showing the lag time (Figure 72A), V,iia 3 (Figure 72B), Vstationary (Figure 72C) and clot size after 60 minutes (Figure 72D) in hemophilia A plasma from Patient 4 activated with medium surface tissue factor density. Each parameter is plotted as a function of ARC 19499 (squares) or recombinant factor Vlla (rVIIa; circles). [001671 Figure 73 is a series of graphs showing the lag time (Figure 73A), Vnjiai (Figure 73B) Vstationary (Figure 73C) and clot size after 60 minutes (Figure 73D) in hemophilia A plasma from Patient 5 activated with medium surface tissue factor density. Each parameter is plotted as a function of ARC] 9499 (squares) or recombinant factor VIla (rVIIa; circles). 39 WO 2012/109675 PCT/US2012/024916 [001681 Figure 74 is a series of graphs showing the lag time (Figure 74A), Vinita, (Figure 74B), Vstaionary (Figure 74C) and clot size after 60 minutes (Figure 74D) in hemophilia A plasma from Patient 6 activated with medium surface tissue factor density. Each parameter is plotted as a function of ARC19499 (squares) or recombinant factor Vlla (rVIIa; circles). [001691 Figure 75 is a series of graphs illustrating the effect of 300 nM ARC19499 on the average lag time (Figure 75A), Viiij (Figure 75B), Vstationary (Figure 75C) and clot size after 60 minutes (Figure 75D) in hemophilia A plasma activated with medium surface tissue factor density (n=3). [001701 Figure 76 is a series of graphs illustrating the lag time (Figure 76A), Vinitial (Figure 76B), Vstationary (Figure 76C) and clot size after 60 minutes (Figure 76D) in normal plasma compared to hemophilia A plasma or hemophilia A plasma containing 300 nM ARC19499, activated with low surface tissue factor density. [00171] Figure 77 is a bar graph illustrating the efficiency of AR-C19499 in promoting clot propagation in normal plasma (solid bars) versus hemophilia A plasma (hatched bars) as reflected in the lag time (white), Vinitija (light grey), Vtationary (medium grey) and clot size after 60 minutes (black). Efficiency is defined as the ratio of the parameter determined in the presence of 300 nM ARC] 9499 to the parameter in the absence of ARCI 9499. [00172] Figure 78 illustrates the concentration dependence of the lag time (Figure 78A) and clot size at 60 minutes (Figure 78B) on ARC 19499 in hemophilia A plasma activated with low surface tissue factor density. These data were used to calculate the IC.

5 values shown in the table below the graphs. 100173] Figure 79 is a series of graphs illustrating the lag time (Figure 79A), Vini ial (Figure 79B), Vstationary (Figure 79C) and clot size after 60 minutes (Figure 79D) in hemophilia A plasma alone compared to hemophilia A plasma containing 300 nM ARC19499 or 300 nM recombinant factor Vita (rVIIa), activated with low surface tissue factor density. [001741 Figure 80 compares the lag time (Figure 80A) and Vinitia (Figure 80B) in TFPI depleted plasma activated with low surface tissue factor density. Each graph shows parameters measured in TFPI depleted plasma alone ("PBS"), TFPI depleted plasma supplemented with +10 nM recombinant TFPI ("TFPI"), TFPI depleted plasma containing 300 nM ARC 19499 ("ARC"). and TFPI depleted plasma supplemented with 10 nM recombinant TFPI and 300 nM ARC19499 ("ARC-TFPI"'). 40 WO 2012/109675 PCT/US2012/024916 [001751 Figure 81 is a series of tables showing the effects of ARC19499 on the TF-activated clotting time (TF-ACT) assay in whole blood samples from normal, severe hemophilia B and severe hemophilia A individuals. [001761 Figure 82 is a series of tables showing the effects of ARC 19499 on the dilute prothrombin time (dPT) assay in plasma samples from normal, severe hemophilia B and severe hemophilia A individuals, [001771 Figure 83 shows the effect of different ARCI 9499 concentrations on ROTEM parameters in whole blood samples (without corn trypsin inhibitor (CTI)) from hemophilia patients (filled squares) and healthy controls (empty circles). The following parameters were analyzed: the clotting time (CT), the clot formation time (CFT), the maximum clot firmness (MCF) and the alpha angle (alpha), [00178] Figure 84 shows the effect of different ARC19499 concentrations on the clotting time (CT) in blood samples from healthy controls (empty circles) compared to hemophilia A patients stratified according to FVIII level: <1% (filled squares), 1-5% (filled, inverted triangles), >5% (filled triangles). The hatched region indicates the range in healthy controls. [00179] Figure 85 shows the effect of different ARC19499 concentrations on ROTEM parameters in whole blood samples (with corn trypsin inhibitor (CTI)) from hemophilia patients (filled squares) and healthy controls (empty circles). The following parameters were analyzed: the clotting time (CT), the clot formation time (CFT), the maximum clot firmness (MCF) and the alpha angle (alpha). 1001801 Figure 86 shows the effect of different ARC19499 concentrations on ROTEM parameters in whole blood samples from a single patient with acquired hemophilia A. The following parameters were analyzed: the clotting time (CT), the clot formation time (CFT), the maximum clot firmness (MCF) and the alpha angle (alpha). [001811 Figure 87 shows ROTEM parameters for healthy control blood pre-incubated with a neutralizing FVIII antibody. Graphs show clotting time (CT) (left panel) and clot formation time (CFT) (right panel) in the same controls; on the left side of each graph, values after inhibition by an FYIII antibody are depicted. [001821 Figure 88 shows thrombin generation curves from the calibrated automated thrombogram (CAT) assay in plasma from a representative severe hemophilia A patient (left 41 WO 2012/109675 PCT/US2012/024916 panel) and a healthy control (right panel). Both graphs show results in the presence (empty circles) and absence (filled squares) of 200 nM ARC 19499. [001831 Figure 89 shows plots of calibrated automated thrombogram (CAT) parameters versus ARC 19499 concentration, including the endogenous thrombin potential (ETP). time to peak, peak thrombin concentration and start tail. In each graph, the response to ARC 19499 in plasma from hemophilia patients (filled squares) is compared to healthy controls (empty circles). [001841 Figure 90 is a plot of calibrated automated thrombogram (CAT) lag time versus ARC1 9499 concentration, comparing the response in hemophilia patients (filled squares) to healthy controls (empty circles). [001851 Figure 91 shows the effect of different ARC19499 concentrations on peak thrombin in plasma samples from healthy controls (empty circles) compared to hemophilia A patients stratified according to FVIII level: <1% (filled squares), 1-5% (filled, inverted triangles), >5% (filled triangles). The hatched region indicates the range observed in healthy controls. 100186] Figure 92 shows thrombin generation curves in plasma fiom a single patient with acquired hemophilia A containing 0 nM (filled squares), 2 nM (asterisks), 20 nM (empty circles) or 200 nM (filled stars) of ARCI 9499. [001871 Figure 93 shows calibrated automated thrombogram (CAT) parameters for healthy control plasma pre-incubated with a neutralizing FVIII antibody. Graphs show endogenous thrombin potential (ETP) (left panel) and peak thrombin (right panel) in the same controls: on the left side of each graph, values after inhibition by an FVIII antibody are depicted. 1001881 Figure 94 shows representative calibrated automated thrombogram (CAT) data from a healthy volunteer (ARC IIV 01). [00189] Figure 95 shows representative calibrated automated thrombogram (CAT) data from a patient with severe hemophilia A (ARC SHA 05). [001901 Figure 96 shows representative calibrated automated thrombogram (CAT) data from a patient with moderate hemophilia A (ARC MoHA 01). [001911 Figure 97 shows representative calibrated automated thrombogram (CAT) data from a patient with mild hemophilia A (ARC MiHA 03). [001921 Figure 98 is a series of graphs depicting median calibrated automated thrombogram (CAT) parameters (endogenous thrombin potential (ETP), peak thrombin. lag time and time to peak) measured in fresh plasma samples from patients with severe hemophilia A (empty 42 WO 2012/109675 PCT/US2012/024916 diamonds), moderate hemophilia A (empty squares), mild hemophilia A (empty triangles) or severe hemophilia B (filled triangles) compared to healthy controls (filled circles). [001931 Figure 99 is a series of graphs depicting median calibrated automated thrombogram (CAT) parameters (endogenous thrombin potential (ETP), peak thrombin, lag time and time to peak) measured in frozen/thawed plasma samples from patients with severe hemophilia A (empty diamonds), moderate hemophilia A (empty squares), mild hemophilia A (empty triangles) or severe hemophilia B (filled triangles) compared to healthy controls (filled circles). [001941 Figure 100 shows representative whole blood thromboelastography (TEG*) data from a healthy volunteer (ARC HV 01). [001951 Figure 101 shows representative whole blood thromboelastography (TEG) data from a patient with severe hemophilia A (ARC SHA 02). 100196] Figure 102 shows representative whole blood thromboelastography (TEG*) data from a patient with moderate hemophilia A (ARC MoIHA 01). 100197] Figure 103 shows representative whole blood thromboelastography (TEG*) data from a patient with mild hemophilia A (ARC MiHA 01). [00198] Figure 104 is a series of graphs depicting median thromboelastography (TEG*) parameters (R-tine, K and angle) measured in whole blood samples from patients with severe hemophilia A (empty diamonds), moderate hemophilia A (empty squares), mild hemophilia A (empty triangles) or severe hemophilia B (filled triangles) compared to healthy controls (filled circles). 1001991 Figure 105 shows representative plasma thromboelastography (TEG*) data from a patient with severe hemophilia A (ARC SHA 02). [00200] Figure 106 shows representative plasma thromboelastography (TEG*) data from a patient with moderate hemophilia A (ARC MoHA 01). [002011 Figure 107 shows representative plasma thromboelastography (TEG*) data from a patient with mild hemophilia A (ARC MiHA 03). [002021 Figure 108 is a series of graphs depicting median thromboelastography (TEG*) parameters (R-time, K and angle) measured in plasma samples from patients with severe hemophilia A (empty diamonds), moderate hemophilia A (empty squares), mild hemophilia A (empty triangles) or severe hemophilia B (filled triangles) compared to healthy controls (filled circles). 43 WO 2012/109675 PCT/US2012/024916 [002031 Figure 109 is a series of graphs showing that ARC 19499 activity can be reversed. ARC 19499 (dashed line) improved thrombin generation in the calibrated automated thrombogram (CAT) assay compared to hemophilia A plasma alone (solid line), as measured by endogenous thrombin potential (ETP; Figure 109A) and peak thrombin (Figure 109B). Addition of ARC23085 (filled diamonds), ARC23087 (empty triangles), ARC23088 (filled squares) and ARC23089 (filled triangles) can reverse this improvement at concentrations .I 00 nM, reaching similar levels to the absence of ARC19499. In Figiure 109C, R-values from the thromboelastography (TEG*) assay showed that 500 nM ARC 19499 shortens the R-value that is prolonged in hemophilia A plasma. I gM ARC23085 partially reversed this improvement with and without a 5 minute preincubation at 37 'C. ARC23087 reversed the improvement with the addition of a 5 minute preincubation at 37 'C. ARC23088 showed little reversal at either condition. ARC23089 also reversed the ARCI 9499 improvement with a 5 minute preincubation at 37 C. 1002041 Figure 110 is a series of thrombin generation curves from the calibrated automated thrombogram (CAT) assay showing the activity of ARCI 9499 in hemophilia A plasma in the presence of 0.00 (Figure 1 10A), 0.156 (Figure 1101B), 0.312 (Figure 1 10C), 0.625 (Figure 110D), 1.25 (Figure II OE), 2.50 (Figure II OF) or 5.00 IU/mL (Figure 110G) low molecular weight heparin (LMWH). 1002051 Figure III is a series of graphs showing the endogenous thrombin potential (ETP; (Figure IIIA) and peak thrombin (Figure 111 B) from calibrated automated thrombogram (CAT) assays performed in hemophilia A plasma with increasing concentrations of both ARCi 9499 and LMWI. The concentration of LMWI is denoted on the x-axis in units of IU/mL. At therapeutic doses of LMWH (>1.25 IU/mL), the procoagulant activity of ARC19499 was reversed. [002061 Figure 112 is a series of graphs showing the endogenous thrombin potential (ETP; (Figure 112A) and peak thrombin (Figure 112B) from calibrated automated thrombogram (CAT) assays performed in hemophilia A plasma with increasing concentrations of both ARC19499 and LMWH. The concentration of LMWI is denoted on the x-axis in units of pM. The data in these graphs were analyzed by curve-fitting to generate estimates of LMWi-I IC 5 0 in the presence of various ARCi 9499 concentrations. The IC 5 values may be found in the table below the graphs. 44 WO 2012/109675 PCT/US2012/024916 [002071 Figure 113 is a series of graphs showing the in vitro stability of several TFPI aptamers in serum. The stability of ARC19498 (Figure 113A), ARC19499 (Figure 113B), ARC19500 (Figure 113C), ARC19501 (Figure 113D), ARC19881 (Figure 113E) and ARC19882 (Figure 113F) in human, monkey and rat serum were measured over the course of 72 hours. [002081 Figure 114 is a graph of a thromboelastography (TEG") assay where plasma from cynomolgus monkeys that were treated previously with an anti-human FVIII antibody was mixed with increasing concentrations of ARC 19499 and assayed for activity. The solid line represents plasma from untreated monkeys and the dashed line represents plasma from antibody treated monkeys, both in the absence of aptamer. The data represents mean ± standard error, with the shaded areas representing the standard error of the non-aptamer samples. [002091 Figure 115 is a graph showing that regardless of treatment following Factor VIII antibody injection in cynomoigus monkeys, Factor VIII activity decreased to <1% and remained there for the duration of the study (5.5 hours). Data represent mean + standard error, n:::3-6. [00210] Figure 116 is a series of graphs showing prothrombin (PT) and activated partial thromboplastin (aPTT) times before and after ARC19499 treatment in cynomolgus monkeys. [00211] Figure 117 is a series of graphs frorn thromboelastography (TEG*) analysis showing that R-values (Figure 117A), a measure of clot time; angles (Figure 11713), a measure of rate of clot formation; and maximum amplitudes (MA; Figure 117C), a measure of clot strength, determined in monkeys treated with saline (filled triangles), NovoSeven" (x), 600 ptg/kg ARCi 9499 (empty squares), 300 qg/kg ARC19499 (empty triangles) or 100 gg/kg ARC19499 (empty diamonds). The time course of the study is denoted on the x-axis. Data represent mean i standard error, n:=3-6. [00212] Figure 118 is a series of graphs from thromboelastography (TEG*) analysis showing that R-values (Figure I I8A), a measure of clot time; angles (Figure I 18B), a measure of rate of clot formation: and maximum amplitude (MA; Figure 118C), a measure of clot strength, were determined in additional monkeys treated with NovoSeven* (x) or 300 pg/kg ARC19499 (triangles) for a longer time course than in Figure 117. The time course of the study is denoted on the x-axis. Data represent mean ± standard error, n=5 for NovoSeven* treatment and n=6 for ARC 19499 treatment. [002131 Figure 119 is a graph showing TFPI levels in cynomolgus monkeys following a 20 mg/kg intravenous (IV, solid) or subcutaneous (SC, hatched) dose of ARC 19499 in nM on the y 45 WO 2012/109675 PCT/US2012/024916 axis. The time course is denoted on the x-axis. The pattern of TFPI release was very similar for both IV and SC dosing. Data represent mean 1 standard error, n=3. [002141 Figure 120 shows the schedule for bleeding time assessment and related FVIII antibody and ARC 19499 dosing and blood sampling in the non-human primate (NHP) bleeding model. [00215] Figure 121 is a series of graphs showing FVIii activity levels in plasma samples from various dosing groups of cynomoigus monkeys treated with FVIII antibody and ARC 19499: Group 1, monkeys whose bleeding times were corrected with one dose of 1 ng/kg ARC19499 (Figure 121 A); Group 2, monkeys whose bleeding times were corrected with two doses of I mg/kg ARC1 9499 (Figure 121B); Group 3, monkey whose bleeding time was corrected with three doses of 1 mg/kg ARC 19499 (Figure 12 1C); and Group 4, monkey whose bleeding time was not corrected with three doses of I mg/kg ARC 19499 (Figure 121 D). [002161 Figure 122 shows mean group bleeding times for Group I monkeys in seconds (Figure 1 22A) and in terms of % of baseline bleeding time (Figure 12213). [00217] Figure 123 shows individual bleeding times for Group 1 monkeys in seconds (Figure 123A) and in terms of% of baseline bleeding time (Figure 12313). [00218] Figure 124 shows mean group bleeding times for Group 2 monkeys in seconds (Figure 124A) and in terms of % of baseline bleeding time (Figure 12413). 1002191 Figure 125 shows individual bleeding times for Group 2 monkeys in seconds (Figure 125 A) and in terms of % of baseline bleeding time (Figure 125B). 1002201 Figure 126 shows bleeding times for the Group 3 monkey in seconds (Figure 126A) and in terms of % of baseline bleeding time (Figure 126B). [00221] Figure 127 shows bleeding times for the Group 4 monkey in seconds (Figure 127A) and in terms of % of baseline bleeding time (Figure 127B). [002221 Figure 128 is a graph of mean group whole blood thromboelastography (TEG") R values plotted against sampling timepoint for Group I monkeys. The time of anti-Factor VIII antibody dosing is indicated by a plus-sign (+) and the time of ARC 19499 dosing is indicated by an asterisk (*) [002231 Figure 129 is a graph of individual whole blood thromboelastography (TEG*) R values plotted against sampling timepoint for Group I1 monkeys. The time of anti-Factor VIII 46 WO 2012/109675 PCT/US2012/024916 antibody dosing is indicated by a plus-sign (+) and the time of ARC 19499 dosing is indicated by an asterisk (*). [002241 Figure 130 is a graph of mean group whole blood thromboelastography (TEG") R values plotted against sampling timepoint for Group 2 monkeys. The time of anti-Factor VIII antibody dosing is indicated by a plus-sign (+) and the times of ARC19499 dosing are indicated by asterisks (*) [002251 Figure 131 is a graph of individual whole blood throinboelastography (TEG") R values plotted against sampling timnepoint for Group 2 monkeys. The time of anti-Factor VIii antibody dosing is indicated by a plus-sign (+) and the times of ARC19499 dosing are indicated by asterisks (*) [002261 Figure 132 is a graph of individual whole blood throinboelastography (TEGt) R values plotted against sampling timepoint for the Group 3 monkey. The time of anti-Factor VIII antibody dosing is indicated by a plus-sign (+) and the times of ARC19499 dosing are indicated by asterisks (*) [002271 Figure 133 is a graph of individual whole blood thromboelastography (TEG*) R values plotted against sampling timepoint for the Group 4 monkey. The time of anti-Factor VIII antibody dosing is indicated by a plus-sign (+) and the times of ARC19499 dosing are indicated by asterisks (*). 1002281 Figure 134 is a graph of mean group plasma thromboelastography (TEG*) R-values plotted against sampling timepoint for Group 1 monkeys. The time of anti-Factor VIII antibody dosing is indicated by a plus-sign (+) and the time of ARCI9499 dosing is indicated by an asterisk (* [002291 Figure 135 is a graph of individual plasma thromboelastography (TEG*) R-values plotted against sampling timepoint for Group 1 monkeys. The time of anti-Factor VIII antibody dosing is indicated by a plus-sign (+) and the time of ARC 19499 dosing is indicated by an asterisk (*) [002301 Figure 136 is a graph of mean group plasma thromboelastography (TEG") R-values plotted against sampling timepoint for Group 2 monkeys, The time of anti-Factor VIII antibody dosing is indicated by a plus-sign (+) and the times of ARC 19499 dosing are indicated by asterisks (*). 47 WO 2012/109675 PCT/US2012/024916 [002311 Figure 137 is a graph of individual plasma thromboelastography (TEG*) R-valiues plotted against sampling timepoint for Group 2 monkeys. The time of anti-Factor VIII antibody dosing is indicated by a plus-sign (+) and the times of ARC 19499 dosing are indicated by asterisks (* [002321 Figure 138 is a graph of individual plasma thromboelastography (TEG*) R-vaiues plotted against sampling timepoint for the Group 3 monkey. The time of anti-Factor VIII antibody dosing is indicated by a plus-sign (+) and the times of ARC19499 dosing are indicated by asterisks (*) [002331 Figure 139 is a graph of individual plasma thromboelastography (TEG*) R-vaiues plotted against sampling timepoint for the Group 4 monkey. The time of anti-Factor VIII antibody dosing is indicated by a plus-sign (+) and the times of ARC19499 dosing are indicated by asterisks (*). [002341 Figure 140 depicts derivatives of A RC17480 that contain single and multiple 2' substitutions in the ARC17480 sequence. Differences relative to ARC17480 are shaded. [002351 Figure 141 depicts derivatives of AR-C17480 that contain a single phosphorothioate substitution between each pair of residues in the AR C17480 sequence. Each phosphorothioate is indicated by an "s" between the pairs of residues in the sequence. Differences relative to ARCI 7480 are shaded. 1002361 Figure 142A depicts tolerated and non-tolerated 2'-substitutions mapped onto the putative secondary structure of ARC 17480. Figure 142B depicts active ARC 1 7480 derivatives with multiple 2'-deoxy to 2'-( Methyl and/or 2 t fluoro substitutions at the four deoxycytidine residues of ARC 17480 (residues 9, 14, 16 and 25). [00237] Figure 143 depicts derivatives of ARC17480 that contain single or multiple deletions in the ARC 17480 sequence. Differences relative to ARC 17480 are highlighted in black. [002381 Figure 144A depicts tolerated and non-tolerated single residue deletions mapped onto the putative secondary structure of ARCI7480. ARC17480 is comprised of 2'-O Methyl (circles) and 2'-deoxy (squares) nueleotides and is modified at its 3'-terminus with an inverted deoxythymidine residue (3T). The corresponding double residue deletion is also depicted in cases where two adjacent nucleotides were identical. Tolerated deletions are highlighted in gray and non-tolerated deletions are highlighted in black. Tolerated and non-tolerated double deletions are indicated. Figure 144B depicts active ARC] 7480 derivatives ARC33889 and 48 WO 2012/109675 PCT/US2012/024916 ARC33895. These molecules each have seven of the ARC17480 residues deleted, which are represented by black circles. [002391 Figure 145 depicts the results of a thrombin generation experiment with 3 %truncated ARC 19499 derivatives. ARC19499, ARC21383. ARC21385, ARC2 1387 and ARC21389 all increase thrombin generation in a concentration-dependent manner in hemophilia A plasma, as measured by endogenous thrombin potential (ETP; Figure 145A) and peak thrombin (Figure 145B). [002401 Figures 146A and 146B depict derivatives of ARC 17480 that contain single nucleotide mutations in the ARC 17480 sequence. Differences relative to ARC 7480 are shaded. [002411 Figure 147 depicts the single tolerated nucleotide mutations mapped onto the putative secondary structure of ARC17480. The letters at each position indicate the nucleotides that are tolerated at that position when substituted individually in the context of the ARCI 7480 sequence. [002421 Figure 148 depicts derivatives of ARC17480 that contain multiple mutations and/or deletions in the ARC 17480 sequence. Deletions relative to ARC17480 are highlighted in black and mutations relative to ARC 17480 are shaded. 1002431 Figure 149 depicts derivatives of ARCI 7480 that contain multiple mutations relative to the ARCi 17480 sequence that retain Watson-Crick base-pairing at residues 6 and 29, 7 and 28, and 8 and 27. Mutations relative to ARC17480 are shaded. Sore of these molecules also contain deletions relative to ARC 17480, which are highlighted in black. 1002441 Figure 150 depicts derivatives of ARCI17480 that contain multiple mutations in the ARC 17480 sequence, each of which is tolerated individually (see Figure 147 and Table 40), Mutations relative to ARC 17480 are shaded. [00245] Figure 151 is a graph showing different concentrations of FXa and ARC19499 (2000 0.10 nM) competing with trace amounts of radiolabeled ARC17480 for binding to TFPI (10 nM). Also shown is a graph of a control experiment in which different concentrations of FXa (2000 -- 0.10 nM) are bound to trace amounts of radiolabeled ARC17480. [002461 Figure 152 is a series of graphs depicting computer simulations of spatial clotting illustrating the effects of factor VIII and TFPI depletion. Panels A and B show clot size versus time plots for clotting activation with TF at 5 or 100 pmole/m, respectively. Panel C shows the lag time in hemophilia A plasma with and without TFPI as a function of TF surface density. 49 WO 2012/109675 PCT/US2012/024916 [002471 Figure 153 is a series of graphs showing the predicted effect based on computer simulation of the combination of TFPI and factor VIII concentration variations on spatial clot growth in hemophilia A. Individual panels show the dependence on concentrations of TFPI and factor VIII for the following clotting parameters: lag time (a), initial velocity (b), stationary velocity (c) and clot size (d.) [00248] Figure 154 illustrates the experimental design of the spatial clotting model. Panel A is a diagram of the spatial clotting chamber. Panel B is a schematic illustration of the components of the system for measuring clot progression in the chamber. [002491 Figure 155 is a table showing characteristics of hemophilia A patients used in the spatial clotting experiments, including factor VIII level on the day of the experiment and the activated partial thromboplastin time (APTT). [00250] Figure 156 is a set of graphs showing the factor VIII level (Panel A) and the APTT (Panel B) measured prior to and at various time points after administration of replacement factor VIII. Individual measurements are shown for samples from all 9 patients used in the study. [00251] Figure 157 illustrates the effects of ARC19499 and factor VII on spatial clotting in hemophilia A. Panel A shows typical light-scattering time-lapse irnages of clot growth in plasma initiated by immobilized TF at surface density of 2 pmole/m2: hemophilia A before (0 hours) and at I and 24 hours after factor VIII administration, with and without addition of 100 nM ARC 19499. TF-coated activator is seen as a vertical black strip on the left side of each image. White bar shows the scale of 0.5 mm. Panels B through D show clot size versus time plots for each of the timepoints shown in Panel A: (b) 0 hours; (c) 1 hour post factor VIII administration; (d) 24 hours post factor VIII administration. Panel C also illustrates parameters used for experiment analysis throughout the study: lag time (time of clot growth initiation); a, initial velocity (mean slope over the first 10 min); p, stationary velocity (mean slope over the following 30 min); clot size after 60 min of the experiment. [002521 Figure 158 is a bar graph illustrating the efficiency of ARC19499 in hemophilia A plasmas prepared with different methodologies. Ratios of clotting parameter with or without 300 nM ARC 19499 are shown for freshly prepared plasma collected into CTI and the same frozen/thawed plasma. The error bars were calculated based on standard errors. [002531 Figure 159 is a set of graphs showing the spatial clotting parameter dependence on ARC1 9499 concentration at 0, 1, and 24 h post factor VIII administration for patient 1. 50 WO 2012/109675 PCT/US2012/024916 [002541 Figure 160 is a set of graphs showing the spatial clotting parameter dependence on ARC 19499 concentration at 0, 1, and 24 h post factor VIII administration for patient 2. [002551 Figure 161 is a set of graphs showing the spatial clotting parameter dependence on ARC 19499 concentration at 0, 1, and 24 h post factor VIII administration for patient 3. [002561 Figure 162 is a set of graphs showing the spatial clotting parameter dependence on ARC1 9499 concentration at 0, 1, and 24 h post factor VIII administration for patient 4. [002571 Figure 163 is a set of graphs showing the spatial clotting parameter dependence on ARC1 9499 concentration at 0, 1, and 24 h post factor VIII administration for patient 5. [002581 Figure 164 is a set of graphs showing the spatial clotting parameter dependence on ARC1 9499 concentration at 0, 1, and 24 h post factor VIII administration for patient 6. [002591 Figure 165 is a set of graphs showing the spatial clotting parameter dependence on ARC] 9499 concentration at 0, 1, and 24 h post factor VIII administration for patient 7. [00260] Figure 166 is a set of graphs showing the spatial clotting parameter dependence on ARC] 9499 concentration at 0, 1, and 24 h post factor VIII administration for patient 8. [00261] Figure 167 is a set of graphs showing the spatial clotting parameter dependence on ARC] 9499 concentration at 0, 1, and 24 h post factor VIII administration for patient 9. [00262] Figure 168 is a set of graphs showing the effect of 100 nM ARC19499 on spatial clotting parameters for three different ranges of factor VIII, <5%, 5-30% and >30%. Spatial clotting parameters are shown as the average values (+ S.E.M.) for samples from 9 patients, as follows: lag time (a), initial velocity (b), stationary velocity (c) and clot size (d). 1002631 Figure 169 is a graph showing the concentration dependence of clotting parameters on ARC 19499 in plasma samples taken prior to factor VI administration (squares), and at 1 hr (circles) and 24 hr (triangles) after factor VIII administration: (a) lag time, (b) initial velocity, (c) stationary velocity, (d) clot size. Each data point is the average ( S.E.M.) for 9 patient samples. [002641 Figure 170 is a series of pictures illustrating the role of TFPI in spatial clot formation and consequences of its inactivation. (a) Under normal conditions, TFPI rapidly inhibits extrinsic tenase in a fXa-dependent manner; this does not stop fibrin clot spatial propagation because additional fXa is activated by intrinsic tenase. (b) Clot size in hemophilia A plasma is significantly impaired because fVIIIa absence prevents spatial propagation. (c) Addition of ARC 19499 to hemophilia A plasma inactivates TFPL This does not restore spatial propagation 51 WO 2012/109675 PCT/US2012/024916 velocity but initiates coagulation even earlier than in normal plasma. As a result, overall fibrin clot size is normalized. [002651 Figure 171 is a computer simulation illustrating the effects of TFPI and factor VIII depletion on coagulation. (a) Effects of TFPI and factor VIII depletion on fibrin clot propagation as shown in a clot size versus time plot. (b) Factor Xa produced by intrinsic tenase (right panels) or extrinsic tenase (left panels) illustrating the effects of TFPI and factor VIII depletion on the generation of factor Xa. Factor Xa generation is plotted as a function of time from activation of the cascade and distance from activator. [002661 Figure 1 '72 is a series of graphs depicting FXa activity under various conditions. In Panels A and B, TFPI, FXa and substrate were mixed in the absence of ARC 19499. In Panel A, FXa was added last, while in Panel B, substrate was added last. The lower panels show the Same assays in the presence of ARCI19499. Panel C shows results with TFPJ, substrate and ARC19499 mixed prior to FXa addition, and Panel D depicts the results with ARC19499, TFPI and FXa mixed prior to substrate addition. [00267] Figure 173 shows the relationship between ARC19499 and TFPI in FXa activity after a 30 mmi incubation of TFPI, FXa, and ARC] 9499 prior to substrate addition. In panel A, the results are plotted with the ARC19499 concentration on the x-axis. In panel B, the same results are plotted with the TFPf concentration on the x-axis. 1002681 Figure 174 illustrates competition of various anti=TFPI antibodies with ARC 19499 in a plate-based binding assay. 1002691 Figure 175, A and B, illustrates competition of various anti-TFPI antibodies with ARC 17480 in a nitrocellulose filtration (dot-blot) assay. [00270] Figure 176 is a graph illustrating the effect of ARC19499 on TFPI inhibition of FXa, shown as the relative FXa activity plotted against ARC 19499 concentration. Either I nM (triangles) or 2 nM (closed circles) TFPI was incubated with 0.Z2 nM of factor Xa and various of the ARC 19499 before chromogenic substrate was added to measure residual factor Xa activity, [002711 Figure 177 is a set of graphs illustrating TFPI inhibition of FXa under different conditions in the absence (Panel A) or presence (Panel B) of 4 jpM ARC19499. In Panel A, curve #1 (circles) shows the factor Xa (0.2 nM) cleavage of a chromogenic substrate in the absence of TFPI, curve #2 (triangles) shows the factor Xa cleavage of substrate when TFPI (2 nM) and the chromogenic substrate were added to factor Xa at the same time, and curve #3 (plus signs) shows 52 WO 2012/109675 PCT/US2012/024916 factor Xa cleavage of substrate when TFPI (2 nM) was pre-incubated with factor Xa (0.2 nM) before adding the substrate. In Panel B. curves 44 (circles), #5 (triangles), and #6 (plus signs) represent the factor Xa cleavage of substrate under the same conditions as #1, #2, and #3, respectively except that 4 pM of ARC19499 was included. Both graphs in figures 177A and 1 77B are representative from four repeats with almost identical results. [002721 Figure 178 is a series of graphs illustrating the effects of ARCI19499 on TFPI inhibition of the extrinsic Xase, Panel A shows progress curves of factor Xa cleavage of substrate where FXa was activated from the reactions of 1 pM tissue factor, I nM factor VIla, 150 niM FX, mM CaCl 2 , and various amount of ARC1 9499, Line I represents FXa cleavage of substrate when TFPI is absent; lines 2-6 represent the FXa cleavage of substrate in the present of 2 nM TFPI and 4000, 1000, 100, 10 and 0 nM of ARCI19499. Panel B shows the data from Panel A transformed into the active factor Xa concentrations plotted versus time. In Panel C, the data are expressed in terms of the extrinsic Xase turn-off time due to TFPI plotted as a function of increasing ARC 19499 concentration. [00273] Figure 179 is a set of graphs illustrating the effects of ARC19499 on TFPI inhibition of prothrombinase. In Panel A, curve #1 (circles) represents the thrombin generation by 0.1 pM prothroibinase in the absence of TFPI, curve #2 (triangles) represents the thrombin generation when I nM of TFPI was included with 0.1 pM prothrombinase, and curve #3 (plus signs) represents the thrombin generation when FVa was absent. In Panel B, curves #4 (circles), #5 (triangles) and #6 (plus signs) are from assays performed under the same conditions as #1, #2 and #3 from Panel A, respectively, except that 4 pM ARC 19499 was included. These graphs are representative from four repeats with almost identical results. [00274] Figure 180 is a set of graphs showing the effects of ARC19499 on the dilute prothrombin time (dPT) measured in FViii-deficient human plasma. In Panel A, either 0.25 pM (closed circles) or 0.5 pM (triangles) recombinant tissue factor was used to initiate the dPT in plasma samples prepared with increasing concentrations of FVIII. FVIII content was varied in these samples by mixing FViii-deficient plasma with normal plasma at different rations. In Panel B, the ARC 19499 shortened the dPT in the factor VIII-deficient plasma when initiated by either 0.25 pM (closed circles) or 0.5 pM (triangles) recombinant tissue factor. In Panel B, clot times measured in FViii-deficient plasma mixed with 0.25 pM TF in the absence of ARC19499 failed to clot within the pre-set tipper limit of 360 seconds; for the purposes of graphing, a value of 360 53 WO 2012/109675 PCT/US2012/024916 seconds was assigned to this data point. these data. Results are averaged from duplicated measurements with the less than 4-second difference. [002751 Figure 181 is a graph showing the effects of ARC19499 on the whole blood clotting time measured in FVIII-deficient whole blood samples. Recombinant tissue factor (0.5 pM Innovin") and ARC 19499 (at indicated concentrations) were added into the freshly drawn blood (containing 5 mM EDTA as anti-coagulant). The treated blood samples were incubated at 37 'C for 3 minutes before adding CaC 2 to initiate clotting reactions. [002761 Figure 182 is a drawing illustrating the hydrogen-deuterium exchange (HIDX) strategy used to examine the interaction between TFPI and ARC] 9498. [002771 Figure 183 is a drawing illustrating the level of deuteration achieved for segments of TFPI for different pi~s and exchange times at 23 'C. The location of each segment is indicated by juxtaposition with the TFPI arnino acid sequence and the level of deuteration is indicated by color code according to the color key at the bottorn of the figure. 1002781 Figure 184 is a table showing the % deuteration for individual TFPI protein segments at different pHs and exchange times at 23 0 C. This data was used to generate the drawing in Figure 2. [002791 Figure 185 is a series of graphs showing the % deuteration plotted as a function of exchange time for TFPI 23 C. Each graph represents a peptide segment. Within each graph, squares represent data collected at pH 5 and diamonds represent data collected at pi 7. For the purposes of this figure, the pH 5 timepoints were converted to p1- 7 equivalents (e.g. 30 seconds at pH 5 is equal to 0.3 seconds at p1H 7). 1002801 Figure 186 is a set of tables showing the buffers used in HDX with TFI and ARC 19498. [002811 Figure 187 is a series of graphs showing the % deuteration plotted as a function of exchange time for TFPI at 3 'C Each graph represents a peptide segment. Within each graph, blue triangles represent data from on-solution/off-column experiments and purple diamonds represent data from on-column/off-column control experiments. For the purposes of this figure, the pH 5 and pH 6 timepoints were converted to pH 7 equivalents. [002821 Figure 188 is a table showing the differences in deuteration levels in each segment of TFPI after on/off exchange experiments at pH 5, 6 and pH 7 at 3 C. Values in this table were calculated by subtracting the % deuteration from on-column/off-colunmn experiments from the % 54 WO 2012/109675 PCT/US2012/024916 deuteration from on-solution/off-column experiments. TFPI segments showing differences '> 5% are regions protected in the presence of ARC 19498. [002831 Figure 189 is a drawing illustrating the differences in deuteration levels in each segment of TFPI after on/off exchange experiments at pH 5, 6 and pH 7 at 3 'C. Dark blue indicates no protection by ARC1 9498. Lighter colors indicate differences >5% and are regions showing protection in the presence of ARC1 9498. Panel A shows the data for individual p1is and exchange times. Panel B reflects data averaged across all p1-s and exchange times. DETAILED DESCRIPTION OF THE INVENTION [002841 The details of one or more embodiments of the invention are set forth in the accompanying description below, Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described. Other features, objects and advantages of the invention will be apparent froi the description. In the description, the singular form also includes the plural unless the context clearly dictates otherwise, Unless defined otherwise, all technical and scientific terms used herein have the sarne meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present description will control. [002851 The invention provides aptarners that bind to TFPI, which are described herein as "TFPI aptarners", and methods for using such aptamers in the treatment of bleeding disorders and other TFPI-mediated pathologies, diseases and disorders, with or without other agents. In addition, the TFP1 aptamers may be used before, during and/or after medical procedures, with or without other agents, in order to reduce or otherwise delay the progression of the complications or side effects thereof. IDENTIFICATION OF APTAMERS 1002861 The aptamers described herein are preferably identified through a method known in the art as Systematic Evolution of Ligands by EXponential Enrichment, or SELEXTM, which is shown generally in Figure 4. More specifically, starting with a mixture containing a starting pool of nucleic acids, the SELEX T method includes steps of: (a) contacting the mixture with a target under conditions favorable for binding; (b) partitioning unbound nucleic acids from those nucleic acids that have bound to the target; (c) amplifying the bound nucleic acids to yield a 55 WO 2012/109675 PCT/US2012/024916 ligand-enriched mixture of nucleic acids; and. optionally, (d) reiterating the steps of contacting, partitioning and amplifying through as many cycles as desired to yield highly specific, high affinity aptamers to the target. In those instances where transcribed aptamers, such as RNA aptamers, are being selected, the amplification step of the SELEX' method includes the steps of: (i) reverse transcribing the nucleic acids dissociated from the nucleic acid-target complexes or otherwise transmitting the sequence information into a corresponding DNA sequence; (ii) PCR amplification; and (iii) transcribing the PCR amplified nucleic acids or otherwise transmitting the sequence information into a corresponding RNA sequence before restarting the process. The starting pool of nucleic acids can be modified or unmodified DNA, RNA or DNA/RNA hybrids, and acceptable modifications include modifications at a base, sugar and/or internucleotide linkage. The composition of the starting pool is dependent upon the desired properties of the final aptamer. Selections can be performed with nucleic acid sequences incorporating modified nucleotides to, e.g., stabilize the aptamers against degradation in vivo. For example, resistance to nuclease degradation can be greatly increased by the incorporation of modifying groups at the 2' position. [00287] In one embodiment, the invention provides aptamers including single 2' substitutions at all bases or combinations of 2'-01-1, 2'-F, 2 '-deoxy, 2 -N and 2'-OMe modifications of the adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymidine triphosphate (TTP) and uridine triphosphate (UTP) nucleotides. In another embodiment, the invention provides aptamers including combinations of 2'-0H, 2'-F, 2'-deoxy, 2'-OMe, 2'-NI-1 2 and 2'-methoxyethyl modifications of the ATP, GTP1, CTP, TTP and UTP nucleotides. In a further embodiment, the invention provides aptamers including all or substantially all 2'-OMe modified ATP. GTP, CTP, TTP and/or UTP nucleotides. [002881 In some embodiments, 2'-modified aptamers of the invention are created using modified polymerases, e.g.. a modified RNA polymerase having a rate of incorporation of modified nucleotides having bulky substituents at the furanose 2' position that is higher than that of wild-type polymerases. In one embodiment, the modified RNA polymerase is a mutant T7 polymerase in which the tyrosine at position 639 has been changed to phenylalanine (Y639F). In another embodiment, the modified RNA polymerase is a mutant T7 polymerase in which the tyrosine at position 639 has been changed to phenylalanine and the lysine at position 378 has been changed to arginine (Y639F/K378R). In yet another embodiment, the modified RNA 56 WO 2012/109675 PCT/US2012/024916 polymerase is a mutant T7 polymerase in which the tyrosine at position 639 has been changed to phenylalanine, the histidine at position 784 has been changed to an alanine, and the lysine at position 378 has been changed to arginine (Y639F/H784A/K378R), and the transcription reaction mixture requires a spike of 2tOH GTP for transcription. In a further embodiment, the modified RINA polymerase is a mutant T7 polymerase in which the tyrosine at position 639 has been changed to phenylalanine and the histidine at position 784 has been changed to an alanine (Y639F/H784A). [002891 In one embodiment, the modified RNA polymerase is a mutant T7 polymerase in which the tyrosine at position 639 has been changed to leucine (Y639L). In another embodiment, the modified R-NA polymerase is a mutant T7 polymerase in which the tyrosine at position 639 has been changed to leucine and the histidine at position 784 has been changed to an alanine (Y639L/H784A). In yet another embodiment, the modified RNA polymerase is a mutant T7 polymerase in which the tyrosine at position 639 has been changed to leucine, the histidine at position 784 has been changed to alanine, and the lysine at position 378 has been changed to arginine (Y639L/Fl784A1/K378R). 1002901 Another suitable RNA polymerase having a rate of incorporation of modified nucleotides having bulky substituents at the furanose 2' position that is higher than that of wild type polymerases is, for example, a mutant T3 RNA polymerase. In one embodiment, the mutant T3 RNA polymerase has a mutation at position 640, wherein the tyrosine at position 640 is replaced with a phenylalanine (Y640F). In another embodiment, the mutant T3 RNA polymerase has mutations at positions 640 and 785, wherein the tyrosine at position 640 is replaced with a leucine and the histidine at position 785 is replaced with an alanine (Y640L/H7 85A). [002911 2 %modified oligonucleotides may be synthesized entirely of modified nucleotides or with a subset of modified nucleotides. The modifications can be the same or different. Some or all nucleotides may be modified, and those that are modified may contain the same modification. For example, all nucleotides containing the same base may have one type of modification, while nucleotides containing other bases may have different types of modification. All purine nucleotides may have one type of modification (or are unmodified), while all pyrimidine nucleotides have another, different type of modification (or are unmodified). In this way, transcripts, or pools of transcripts, are generated using any combination of modifications, 57 WO 2012/109675 PCT/US2012/024916 including for example, ribonucleotides (2'-OH), deoxyribonucleotides (2'-deoxy), 2-amino nucleotides (2'-NH2), '-fluoro nucleotides (2'-F) and 2'-0--methyl (2'-OMe) nucleotides. [002921 As used herein, a transcription mixture containing only 2'-OMe A, G, C and U and/or T triphosphates (2'-OMe ATP, 2'-OMe UTP and/or 2'-OMe TTP. 2'-OMe CTP and 2'-OMe GTP) is referred to as an MNA or mRmY mixture, and aptamers selected therefrom are referred to as MNA aptamers or mRmY aptamers and contain only 2' -O-methyl nucleotides. A transcription mixture containing 2'-OMe C and U and/or T, and 2'-OH A and G is referred to as an "rRmY" mixture, and aptamers selected therefrom are referred to as "rRmY" aptamers. A transcription mixture containing deoxy A and G, and 2'-OMe U and/or T, and C is referred to as a "dRmY" mixture, and aptamers selected therefrom are referred to as "dRmY" aptamers. A transcription mixture containing 2'-OMe A, C and U and/or T, and 2'-OH G is referred to as a "rGmHl" mixture, and aptaiers selected therefrom are referred to as "rGmH-" aptamers. A transcription mixture alternately containing 2'-OMe A, C, U and/or T and G, and 2'-OMe A, U and/or T, and C, and 2 -F G'3 is referred to as an "alternating mixturee, and aptarners selected therefrom are referred to as "alternating mixture" aptarners. A transcription mixture containing 2'-01H A and G, and 2 '-F C and U and/or T is referred to as an "rRfY" mixture, and aptamers selected therefrom are referred to as "rRfY" aptainers. A transcription mixture containing 2 OMe A and G, and 2'-F C and U and/or T is referred to as an "miRtY" mixture, and aptarners selected therefrom are referred to as "mRfY" aptamers. A transcription mixture containing 2' OMe A, U and/or T, and C, and 2'-F G is referred to as a "fKmHl" mixture, and aptamers selected therefrom are referred to as "fGmiHJ" aptamers. A transcription mixture containing 2 OMe A, U and/or I, C and G, where up to 10% of the G's are ribonucleotides is referred to as a "r/mGmH" mixture, and aptamers selected therefrom are referred to as "r/mGmH" aptamers. A transcription mixture containing 2'-OMe A. U and/or T, and C, and deoxy G is referred to as a "dGmH" mixture, and aptamers selected therefrom are referred to as "dGmHfl" aptamers. A transcription mixture containing deoxy A, and 2'-OMe C, G and U and/or T is referred to as a "dAmB" mixture, and aptamers selected therefrom are referred to as "dAmB" aptamers. A transcription mixture containing 2'-OH A, and 2'-OMe C, G and U and/or T is referred to as a "rAmB" mixture, and aptamers selected therefrom are referred to as "rAmB" aptamers. A transcription mixture containing 2'-OH A and 2 '-OH G, and 2 '-deoxy C and 2'-deoxy T is referred to as an rRdY mixture, and aptamers selected therefrom are referred to as "rRdY' 58 WO 2012/109675 PCT/US2012/024916 aptamers. A transcription mixture containing 2' t OMe A, U and/or T, and G. and deoxy C is referred to as a "dCrmD" mixture, and aptamers selected there from are referred to as "dCmD" aptamers. A transcription mixture containing 2 tOMe A, G and C, and deoxy T is referred to as a "dTniV" mixture, and aptamers selected there from are referred to as "dTrnV" aptamers. A transcription mixture containing 2'-OMe A, C and G, and 2'-OH U is referred to as a "rLmV" mixture, and aptamers selected there from are referred to as "rUmV" aptamers. A transcription mixture containing 2'-OMe A, C and G, and 2'- deoxy U is referred to as a "dUmV" mixture, and aptamers selected therefrom are referred to as "dUmV" aptamers. A transcription mixture containing all 2'-011 nucleotides is referred to as a "rN" mixture, and aptamers selected therefrom are referred to as "rN", "rRrY" or RNA aptamers. A transcription mixture containing all deoxy nucleotides is referred to as a "dN" mixture, and aptamers selected therefrom are referred to as "dN", "dRdY" or DNA aptamers. A transcription mixture containing 2'-F C and 2'-OMe A, G and U and/or T is referred to as a "fCmD" rmixture, and aptamers selected therefrom are referred to as "fCrnD" aptamers. A transcription mixture containing 2'F U and 2'-OMe A, G and C is referred to as a "fUmV mixture, and aptamers selected therefrom are referred to as "fUmiV" aptamers. A transcription mixture containing 2'-F A and (G, and 2'-OMe C and U arid/or T is referred to as a "fRmY" mixture, and aptamers selected therefrom are referred to as "fRmY" aptamers. A transcription mixture containing 2'-F A and 2'-OMe C, G and U and/or T is referred to as a "fAimB" mixture, and aptamers selected therefrom are referred to as "fAmB" aptamers. 1002931 A number of factors have been determined to be useful for optimizing the transcription conditions used to produce the aptarners disclosed herein. For example, a leader sequence can be incorporated into the fixed sequence at the 5' end of a DNA transcription template. The leader sequence is typically 6-15 nucleotides long, eg., 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides long, and may be composed of all purines. or a mixture of purine and pyrimidine nucleotides. [002941 For compositions that contain Z'-OMe GTP. another useful factor can be the presence or concentration of '-OH guanosine or guanosine monophosphate (GMP). Transcription can be divided into two phases: the first phase is initiation, during which the RNA is extended by about 10-12 nucleotides; the second phase is elongation, during which transcription proceeds beyond the addition of the first about 10-12 nucleotides. It has been found that 2'-OH GMP or 59 WO 2012/109675 PCT/US2012/024916 guanosine added to a transcription mixture containing an excess of 2'-OMe GTP is sufficient to enable the polymerase to initiate transcription. Priming transcription with 2'-OH guanosine e.g. or GM1 is useful due to the specificity of the polymerase for the initiating nucleotide. The preferred concentration of GMP is 0.5 mM and even more preferably 1 mM. [002951 Another useful factor for optimizing the incorporation of 2'-OMe substituted nucleotides into transcripts is the use of both divalent magnesium and manganese in the transcription mixture. Different combinations of concentrations of magnesium chloride and manganese chloride have been found to affect yields of 2'-O modified transcripts, the optimum concentration of the magnesium and manganese chloride being dependent upon the concentration of NTPs in the transcription reaction mixture that complex divalent metal ions. [002961 Other reagents that can be included in the transcription reaction include buffers such as N-2-hydroxyethylpiperazine-N '-2-ethanesulfonic acid ( HEPES) buffer, a redox reagent such as dithiothreitol (DTT), a polycation such as spermidine, spermine, a surfactant such as Triton XI 00, and any combinations thereof. [00297] In one embodiment, the IEPES buffer concentration can range from 0 to 1 M. The invention also contemplates the use of other buffering agents having including, for example, Tris-hvdroxymethyl-aminorethan e. In some embodiments, the DTT concentration can range from 0 to 400 mM. The methods of the invention also provide for the use of other reducing agents, including, for example, mercaptoethanol. In some embodiments, the spermidine and/or spermine concentration can range from 0 to 20 mM. In some embodiments, the PEG-8000 concentration can range from 0 to 50 % (w/v). The methods of the invention also provide for the use of other hydrophilic polymers, including, for example, other molecular weight PEGs or other polyalkylene glycols. In some embodiments, the Triton X-100 concentration can range from 0 to 0.1% (w/v). The methods of the invention also provide for the use of other non-ionic detergents, including, for example, other detergents, including other Triton-X detergents. In some embodiments, the MgCl 2 concentration can range from 0.5 nM to 50 mM. The MnCl2 concentration can range from 0.15 mM to 15 nM. In some embodiments, the 2'-OMe NTP concentration (each NTP) can range from 5 gM to 5 mM. In some embodiments, the 2'-O1H1 GTP concentration can range from 0 pIM to 300 pM. In some embodiments, the 2'-OH GNIP concentration can range from 0 to 5 mM. The pH1 can range from pH 6 to pH 9. 60 WO 2012/109675 PCT/US2012/024916 [002981 Variations of the SELEX process may also be used to identify aptamers. For example, one may use agonist SELEX, toggle SELEX, 2'-Modified SELEX or Counter SELEX. Each of these variations of the SELEX process is known in the art. TFPI APTAMERS [002991 The invention includes nucleic acid aptamers, preferably of 20-55 nucleotides in length, that bind to tissue factor pathway inhibitor (TFPI) and which, in some embodiments, functionally modulate, e.g., stimulate, block or otherwise inhibit or stimulate, the activity of TFPL [003001 The TFPI aptamers bind at least in part to TFPI or a variant or one or more portions (or regions) thereof. For example, the TFPI aptamers may bind to or otherwise interact with a linear portion or a conformational portion of TFPL A TFPI aptarner binds to or otherwise interacts with a linear portion of TFPI when the aptamer binds to or otherwise interacts with a contiguous stretch of amino acid residues that are linked by peptide bonds. A TFPI aptarner binds to or otherwise interacts with a confornational portion of TFPI when the aptamer binds to or otherwise interacts with non-contiguous amino acid residues that are brought together by folding or other aspects of the secondary and/or tertiary structure of the polypeptide chain. [00301] A TFPI variant, as used herein, encompasses variants that perform essentially the same fiction as TFPI functions, preferably includes substantially the same structure and in some embodiments includes at least 70% sequence identity, preferably at least 80% sequence identity, more preferably at least 90% sequence identity, and more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of human TFPI, which is shown in Figure 5 as SEQ ID NO: 11. [00302] Preferably, the TFPI aptamers bind to full length TFPL If an aptamer binds to one or more portions of TFPI, it is preferable that the aptamer require binding contacts or other interaction with a portion of TFPI, at least in part, outside of the Ki and, K2 regions, such as the K13/C-terminal region. For example, the TFPI aptamers may bind to or otherwise interact with a linear portion or a conformational portion of TFPL A TFPI aptamer binds to or otherwise interacts with a linear portion of TFPI when the aptamer binds to or otherwise interacts with a contiguous stretch of amino acid residues that are linked by peptide bonds. A TFPI aptamer binds to or otherwise interacts with a conformational portion of TFPI when the aptamer binds to or otherwise interacts with non-contiguous amino acid residues that are brought together by 61 WO 2012/109675 PCT/US2012/024916 folding or other aspects of the secondary and/or tertiary structure of the polypeptide chain. More preferably, the TFPI aptamers bind at least in part to one or more portions of mature TFPI (for example, Figure 3A) that are selected from the group consisting of: amino acids 148-170, amino acids 150-170, amino acids 155-175, amino acids 160-180, amino acids 165-185, amino acids 170-190, amino acids 175-195, amino acids 180-200, amino acids 185-205, amino acids 190 210, amino acids 195-215, amino acids 200-220, amino acids 205-225, amino acids 210-230, amio acids '15 '235, amino acids 220-240, amino acids 225-245, amino acids '230-250, amino acids 235-255, amino acids 240-260, amino acids 245-265, amino acids 250-270, amino acids 255-275, amino acids 260-276, amino acids 148-175, amino acids 150-175, amino acids 150 180, amino acids 150-185, amino acids 150-190, amino acids 150-195, amino acids 150-200, amino acids 150-205, amino acids 150-2 10, amino acids 150-215, amino acids 150-220, amino acids 150-225, arnino acids 150-230, amino acids 150-235, arnino acids 150-240, amino acids 150-245, amino acids 150-250, amino acids 150-255, amino acids 150-260, amino acids 150 265, arnino acids 150-270, arnino acids 150-275, arnino acids 150-276, arnino acids 190-240, amino acids 190-276, amino acids 240-'276, amino acids 242 276, amino acids 161-181, amino acids 162-181, arnino acids 182-240, arnino acids 182-241, and amino acids 182-276. [00303] The TFPI rnay be from any species, but is preferably human. [00304] The TFPI aptamers preferably comprise a dissociation constant for human TFPI, or a variant thereof, of less than 100 pM, less than I tiM, less than 500 nM, less than 100 nM, preferably 50 nM or less, preferably 25 nM or less, preferably 10 nM or less, preferably 5 nM or less, more preferably 3 nM or less, even more preferably I nM or less, and most preferably 500 pM or less. In some embodiments, the dissociation constant is determined by dot blot titration. [003051 The TFPI aptamers may be ribonucleic acid, deoxyribonucleic acid, modified nucleic acids (for example, 2'-modified) or mixed ribonucleic acid, deoxyribonucleic acid and modified nucleic acids, or any combination thereof. The aptamers may be single stranded ribonucleic acid, deoxyribonucleic acid, modified nucleic acids (for example, 2'-modified), ribonucleic acid and modified nucleic acid, deoxyribonucleic acid and modified nucleic acid, or mixed ribonucleic acid, deoxyribonucleic acid and modified nucleic acids, or any combination thereof. [003061 In some embodiments, the TFPI aptamers comprise at least one chemical modification. In some embodiments, the chemical modification is selected from the group consisting of: a chemical substitution at a sugar position, a chemical substitution at an 62 WO 2012/109675 PCT/US2012/024916 internucleotide linkage and a chemical substitution at a base position. In other embodiments, the chemical modification is selected from the group consisting of: incorporation of a modified nucleotide; a 3' cap; a 5' cap; conjug'ation to a high molecular weight, non-immunogenic compound; conjugation to a lipophilic compound; incorporation of a CpG motif; and incorporation of a phosphorothioate or phosphorodithioate into the phosphate backbone. In a preferred embodiment, the non-immunogenic, high molecular weight compound is polyalkylene glycol, and more preferably is polyethylene glycol (PEG). In some embodiments, the polyethylene glycol is methoxypolyethylene glycol (mPEG). In another preferred embodiment, the 3' cap is an inverted deoxythymidine cap. [003071 The modifications described herein may affect aptamer stability, e.g., incorporation of capping moiety may stabilize the aptamer against endonuclease degradation. Additionally, the modifications described herein may affect the binding affinity of an aptamier to its target, e.g., site specific incorporation of a modified nucleotide or conjugation to a PEG may affect binding affinity. The effect of such modifications on binding affinity can be determined using a variety of art-recognized techniques, such as, e.g., functional assays, such as an ELISA, or binding assays in which labeled trace aptamer is incubated with varying target concentrations and complexes are captured on nitrocellulose and quantitated, to compare the binding affinities pre and post-incorporation of a modification. 1003081 Preferably, the TFPI aptamers bind at least in part to TFPI or a variant or one or more portions thereof and act as an antagonist to inhibit the ffinction of TFPI. 1003091 The TFII aptamers completely or partially inhibit, reduce, block or otherwise modulate TEPI-mediated inhibition of blood coagulation. The TFPI aptamers are considered to completely modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with TFPI biological activity, such as TFPI-mediated inhibition of blood coagulation, when the level of TFPI-mediated inhibition in the presence of the TFPI aptamer is decreased by at least 95%. e.g., by 96%, 97%, 98%, 99% or 100% as compared to the level TFPI-mediated inhibition in the absence of the TFPI aptamer. The TFPI aptamers are considered to partially modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with TFPI biological activity, such as TFPI-mediated inhibition, when the level of TFPI-mediated inhibition in the presence of the TFPI aptamer is decreased by less than 95%, e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 63 WO 2012/109675 PCT/US2012/024916 50%. 55%, 60%, 65%, 70%, 75%, 80%. 85% or 90% as compared to the level of TFPI activity in the absence of the TFPI aptamer. [003101 Examples of aptamers that bind to and modulate the function of TFPI for use as therapeutics and/or diagnostics include, but are not limited to. ARC26835, ARC 17480, ARC19498, AR C19499, ARCI 9500, ARCI9501, ARC31301, ARC18546, ARC19881 and ARC19882. [003111 Preferably, the TFPI aptamers comprise one of the following nucleic acid sequences: (ARC26835) mG-niG-mA-mA-mU-mA- nU-mA-dC-mU-mU-mG-muG-dC-mU-dC-mG -mU-mU-mA-nG mG-mU-mG-dC-mG-mU-mA-m-A-mU-mA (SEQ ID NO: 1), wherein "dN" is a deoxynucleotide and "inN" is a 2'-O Methyl containing nucleotide (which is also known in the art as a 2'-OMe, 2'-methoxy or 2'-OC1 3 containing nucleotide); and (ARC17480) mG-m(i-mA-miA-mU-mA -mU-mA-dC-mrdjU-mU-mG-mG-dC-rnj-dC-mG-mU-mU-mA-mG mG-m -mG-dC-mG-mU-mA-mUdJ-mA-mU-mA-3T (SEQ ID NO: 2), wherein "31T" is an inverted deoxythymidine, "dN" is a deoxynucleotide and "mN" is a 2'-O Methyl containing nucleotide; and (ARC 19498) NI-1 2 -mG-mG-iA-mA-mU-mA-niu-mA-dC-mU -mU-mG-mG -dC-mLU-dC-mG-mU-mU-mA mG -mG-mU-mG-dC-mG-mU-mA-mU-nmA-mLU-mA-3T {SEQ ID NO: 3), wherein "Nil 2 " is from a 5'-hexylamine linker phosphoramidite, "3T" is an inverted deoxythymidine, "dN" is a deoxynucleotide and "mN" is a 2'-) Methyl containing nucleotide; and (ARC19499) PEG4OK-NH-mG-mG-mnA-mA-mU-mA-mU-mA-dC-niU -mU-mG-mG-dC-mU-dC-mG-niU mU-mA-mG-mG-niU-mG-dC-mG-mU-mA-mU -mA-niU-mA-3T (SEQ ID NO: 4), wherein "NH" is from a 5'-hexylamine linker phosphoramidite, "3T" is an inverted deoxythymidine, "dN" is a deoxynucleotide, "mN" is a 2'-O Methyl containing nucleotide and "PEG" is a polyethylene glycol; and (ARC19500) NH-nG-mG -nmA -mA -mU-mA-mU-nA-dC-nmU-niU-mG -mG-dC-nU-dC-mG-n mIU-mU-mA mG-mG-inU-mG-dC-mG -mU-mA-i mU- nA-mU-mA-NH2 (SEQ ID NO: 5), wherein "dN" is a 64 WO 2012/109675 PCT/US2012/024916 deoxynicleotide, "nmN" is a 2-0 Methyl containing nucleotide and "NH2" is from a hexylamine linker phosphoramidite; and (ARC19501) PEG20K-NH-mG-mG-mA-mA-mU -mA-mU-nA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU mU-niA-mG-mG-mU-mG-dC-mnG-mnU-mA-niU-mA-mU-mA-NH-PEG20K (SEQ ID NO: 6), wherein "dN" is a deoxynucleotide, "nN" is a'-O Methyl containing nucleotide, "NH" is from a hexylamine linker phosphoramidite and "PEG" is a polyethylene glycol; and (ARC31301) mG-mG-mA -mA-mU-mA-nU-mA-dC-iU-mU-mG-mG- nC-mU-dC-mnG-mU-m-inU-mA-mG mG-mU-mG-mC-mG-mU-mA-mU-mA-mtU-mA (SEQ ID NO: 7), wherein "dN" is a deoxynucleotide and "inN" is a '-0 Methyl containing nucleotide; and (ARC18546) mC-mG-mA-mA-mU- nA-mriU-mA-dC-mU -mU-mG-mG-inC-mU-dC-mG-mU-mU-mA-rG nG-mnU-i(i-m(--i-mU-mA-mj-mA-mU-mA-3T (SEQ ID NO: 8), wherein "3T" is an inverted deoxythymidine, "dN" is a deoxvnucleotide and "irN" is a 2'-O) Methyl containing nucleotide; and (ARC19881) NH2-mG-mG-mA-mA-mJ-mA-mU -mA-dC-mU-mnlU-mG-G-mC U-dC-mG-mU-rnU-mA miG-mG-mU-mG-nC-mG-mU-mA-mU-mA-mU -mA-3T (SEQ ID NO: 9), wherein "NT" is from a 5'-hexylamine liner phosphoramidite, "31"' is an inverted deoxythymidine, "dN" is a deoxynucleotide and "MN" is a 24O Methyl containing nucleotide; and (ARC19882) PEG40K-NH-mG-mG-mA-mA-mU -mA-mU -mA-dC-mU-mU-mG-mG-mC-mU-dC-mG-mU niU-mA-mG-mG-mU-mG-mC-mG-mU -mA-ni-mA-mU-mA-3 T (SEQ ID NO: 10), wherein "NH" is from a 5'-hexylamine linker phosphoramidite, "3T" is an inverted deoxythymidine, "dN" is a deoxynucleotide. "niN" is a 2'-0 Methyl containing nucleotide and "PEG" is a polyethylene glycol. [003121 The chemical name of ARC26835 is 2'-OMe-guanylyi-(3'-+5')-2'-OMe-guanyiyl (3'-45')-2'-OMe-adenyiyil-(3'-+5')-2 '-OMe-adenylyl-(3'--5')-2.)'-OMe-uracylyl-(3'-+5')-2' OMe-adenyiyl-(3 '-+5 ')-2 '-OMe-uracylyi-(3 '-+5 ')-2'-OMe-adenylyl-(3 '-+5 ')-2' deoxycytidylyl-(3' ->5')-2'-OMe-uracylyl-(3'-+5')-2'-OMe-uracylyi-(3'->5')-2 '-OMe 65 WO 2012/109675 PCT/US2012/024916 guanyiyl4(3 '->)5 ')>2 '-OMe-guanyiyl(3 '->)5 '>2Z '-deoxyeyvtidyiyi41-3 '--±,5 ')'-OM-'-uracylyi (3 '-± 5 ')-2 '-deoxycytidyiyl4(3 '-5'2'-OMe-guanyiyi4(3 '-> 5 '>2 '-OMe-uraeyiyi4(3 '->-)5 '2 OMe-uracyiyl4(3 '-±-5 '>2Z '-.OMe-adeii4-3 '-±-,5 '>2'-OMe1-guanyiyl4(3 '-±- 5 '>2 '-OMe g uan yy-(3 '-±5 '5)-2 '-OMe-uracylyi(3 ' -±-5 '-2 '-OMe-oruanvlyi(3 '-5 '>2'-deo~xycytidylyl (3 '-±)5 '>)2 '-OMe-guainyly-43 '--5 '>)2 '-OMe--uraeyiyi(3 '-±5 ),'>2 '-OMe-adenyivi-(3 '-5 '>2' OMe-uiraeyiyi(3 '-±5 ),'>2 '-OMe-adenyiyi-(3' ')-'>2'-OMe-uiracyiyi43 "'-5')-2 l'-OMe-adenNylt [003131 The chemical name of AR-C17480 is 2 Oegayy-3'± > -~-unll (io '-± ')2 -O e' nl-(3-- '-'-±5 2 -Oc -adeny i-'-±5 '>2' l-3e-5ey-( '-±5 2 O 5e-adeiycy]d- -- ± ' 2--->5e-urcy-( -±5 '>2ily- '-O e-ady'y-(3 '-u>5 y- '>2 ---- > OMe-uracvilyl-3 -(-- >5')-2'-OMe-adenylyl (3 ---- >' )-2 '-GM e-guarivylJ-(i) ->5 '>2'-OMe guanykl- (3' ---- )5 '>-2'-OMe-uracylvi-(3 ' ---- >5'>2 '-OMe-gnanylvi-(3 '--6 5' '-deoxycytidylyl ('--5'-2'O eguanylyl-(3 '---->5 ')2 '-OMe-uracylyl-(3 '----> 5')-2 '-OMe--adenylvi-(3 -6 '>2- ') GM e-uracy Iyl-(3 '----->5')-2 '-OMe--adenylvi-(.3 ' --- 5)'>2 '-OMe-uracyl-(3 '--6 >2'-OMe-adenylyJ (3' ---- 5 '1)4-(3'---- 0')-2' -deoxythymidi ne. 1003141 The chemical name of ARC] 9498 is 6-aminiohexyk4l-(i-6 ... >2'-OMe-gnUanvlvl (3 5)-'-OMe-guanylyl(3 -'---> 5' )-2 '-0 'e-adenyiyl-(3 '--'2 '-GMe-adenylv-(3 )-----tS')-2 o e-uracyiyl-(5 --- 5)-2'-OMe-adenylvl-(3 '----tS5)-2 '-GMe-uiracvl1yl-(3 ''-±-,5 '>2'- OMe-adeny lyl (3' ->5 '>2 '-deoxycvtidylyl-(3'---> '> -)-rayy-3----tS5'>2 '-GMe-uracylyl-3 '----5' o e-guanylyi-(3 -'--- >5')-2 '-OMe-g-uanylyl-(3'---->5')-2'-deoxyc-ytidylyl-'3 -'--- >5')-2 '-OMe uracvyy-(3 '-±,5 '>2 '-deoxy)cyfidyiyi-('3'-±-,5 '2'-OMe-guanyiyl-(3 '-±- 5 '>-2'-OMe-uracylyl (3' -±-5 ')>2 '-OMe-uiracyiyl-(3 '-± 5 ')-2 '-OMe-adeny lli-43' -±-5 )-2 '-OMe-oruanvliN-(3 '-5 '>2' OMe-guiaii7-(3 '-±)5')-'-'-OM-uracylyil-(3 '-±-)5 '>2'-OMe-guiaii-(3 '-±)51) '>3 deo~xycytidyyi-43 '-± 5 ')-2 '-OMe-guanylyi-(3 '-±- 5 ')2' -OMe-uracyiyi-(3 '-± 5 '.)2'-OMe adenviyl-(3 '--)5 ')-2'Z.-OMe-uiracyiyi1-('3'-±-5 '- -Me-adenyiyl-3'-5)2 '-OMe-uracylyl (3 -±5'>2'-O e-aeniyi(3 -±5')-3 '±3'2'-deoxythymidine, [003151 The chemical name of ARC 19499 is N-(metho~xy-poiyethylenegilycoil)-6 aminohexyiyl-( 1-±)5' )-' -OMe-guianylyi-(3'-±-5' )-2 '-OMe-gruanyliN-(3 '-5 ')2'-OMe-ade-nylyl (3 '-±-5 ')-2 '-OMe-adeniIyi-(3 '-±-5 )')2'-OMe-uiracylyi-(3 '-±-5 ')-2 '-OMe-adenyiyi-(3 '-±)5 '>2 ' 66 WO 2012/109675 PCT/US2012/024916 OMe-uracyiyl-(3 '-+- 5 ')-2 '-OMe-adeniyiy-(3 '--+,5 ')-2 '-deoxycy-tidylyil-(3--,5 ')-2 -OMe-uiracylyl (3 1 -- +5 ')-2'-OMe-uracyIyi-(3 -+,-5 ')-2~ %OM,-guanmyIy-(3 1 -- +5 ')-2 '-OMe-cguanyl vi-(3 '--+5 ')-2 ' decoxvyyidyiyi4(3 '-- 5 ')-2 '-OMe-uracyI37i-(3 '-+,5 ')-2 '-deoxycytidylyil-(3 -- +)5 ')-2%'OMe guainyiyi-(3 '-- 5 ')-2 '-OMe-uracylyi4(3 '--+5 ')-2 '-OMe-uracyiyl-(3 '-+)5 ')-2 '-OM,--adenyIyi (3 '-+)5 '>2 '-OMe-guainylyi-(3 '-+-5 ')-2 '-OMe-guaanii -(3 '-+-5')-2 '-OMe-uiracyiyi-(3 '-+-5 ')-2 ' OMe-guiaaniyy-(3 '--+>5')-2~'-deoxyeytidyiyil-(3 '-+-5 ')2 '-OMe-guaanii -(3 '-+-5 )-2 '-OMe uraeyiyi-(3 '-+-)5 ')2 '-OMe-ade-nylyi-(3' 5'-'2'-OMe-ujracyiyil-(3 '-5')-2 '-OMe-adenylyl ('-5'-2 '-OMe-nraeyiyi -(3 '-+-5 ')-2 '-OMe-adeny, yi-(3 '-+-5 ')-(3 '-+-3'>? '-deoxyiltymidine, [003161 The chemical name of AR-C19500 is 6-amrinohexyiyi -(1 -+-5 ')2' -OMe-guianyivi (3 '-+-5 ')-2 '-OMe-gitanyiyi1-(3 '-5 '>2'-OMe-aidenyiyil-(3 '-+-5 ')-2 '-OMe-adeniyl-(3 '-+-5 '>2' OMe-uiraiyl-(3 '-->5 ')-2 '-OMe-adeniyl-(3 '-+-5 ')-2 '-OMe-uiraeylyi-(3 '-+-5 '>2 '-OMe-aidenylyl (3' ---- > 2'-deoxycytidylyl -(3 -'--- >5 ')-2 '-OMe-uracylyl -(3 -'--->5 ')-2 '-GM e-uraey Iyl-(3' '---5 '>)2 ' GMe-guanylyi-(3 ' ---- +5')-2 '4)Me-guaniy yl-(31 --- + 5)-2'-deoxyevti dyk'l-(3 ' -- +)5 ')-2'4) Me uraevyi-(3' >5') 1 -deoxyeytidyiyl-(3' ->' T2 OMe-guanyiyl-(3 '-+5 ')-2 '-OMe-uracylyI (31 ---- > 2 )Me-uraeylvl-(3 ' -- +)5 ')-2 '-O\L adetiylyi-(3 ' --- >5')-2 '-OMe-guanyli-(3 ' -- 5 ')-2 ' OMe-guany lyl (3 ---- > ')-2 '-OMe-uraeyly 1 (3'- ---- >2'-OMe-guanyiyI-G --- 5 )-2 deoxycytidylyl I ('->5')-2 '-OMe-guanyli(3 -- >5)-2 '41)Ne-uraevyI-(3 '-+ >-2'-OMe adeniyl-(3 -'--- >5 '-2 '-OMe-uracyly 1(3' ---- 5 T~2 '-GM e-adeny Iyl-(Y --- +>5')-2 '-OMe-uraevli (3' ---- 5 ')-2'OMe-adenylyl-6-amiinohexylyl. 1003171 The chemical name of ARC] 19501 is N -(miethoxy-polyetlwyleneglyeol)-6 aminohexylyl-(I ->5' )-2 '-OMe-guiaiwylyl-(3---+5 '>2 '4Me-guianylyl-(35-+,5 '2 '-OMe,-adenylyl (3'- -- >5' -2'-OMe-adenylyI-(3 - -- >5' )-2 '-OMe-uracylyl-(3 -'----+ 5 ')-2 '-GM e-adenyIy1-(3 -+5 ' )-2 ' O)Ne-uraeyiyl-(3 '-45 ')-2 '-OMe-adenylyi-(3 '-5' )-2 '-decoxycyitidylyi4(3 '-->5 ')-2 '-OMe-uracylyl (3 '->5 ')2 '-OMe-uracylyi-(3 '-5')-2 '-OMe-guainyiyl-(3 '->5 ')-2 '-OMe-guianiyiy-(3 '--)5 ')-2 deoxycytidyiyl-(3 '->5 ')-2 '-OMe-uiraeyiyi-(3 '-5')-2 '-deoxycyitidylyi(3' -->5 ')-2 '-OMe guanyiyi-(3 '--+5')-2 '-OMe--uraeylyi-(3-'-,5 ')-2 '-OMe-uiraeyiyl-(3 '-->5 ')-2 '-OMe-ade-nylyl1 (3 '--> 5 ')-2 '-OMe-guanyliy-(3 '-->5 ')-2 '-OMe-guanyiyl-(3 '-->5 ')-2 '-OMe-uracylyi-(3 '-->5 ')-2 ' OMe-guanylyi-(3 '-- 5' )-2 '-deoxyeyiidylyi4(3 '-+-5' )-2 '-GMe-guanyiyi-(3 '-+5 '5)-2 '-ON'i uracyiyi-(3 '-+- 5 ')-2 '-OMe-adeniyiy-(3 '-+ ,5 '-2 '-OM-uraeyiyl-(3'--+ 5 ')-2 '-OMe-adenylyl (3' ---5 ')2 '-OMe-uiraeyiyl-(3 '-> 5 ')-2 '-OMe-ade-nylyi-6-aminochexylyi-N-(methoxy polyethylenegiyeoi> 67 WO 2012/109675 PCT/US2012/024916 [003181 The chemical name of ARC 31301 is 2' -OMe-cguanviN-(3'-5')-2 '-OMe-guanylyvl (3 '-> 5 ')-2 '-OMe-adenii4-3 -> r'-.OMe-adenyvlyl-(3-')-' >-OMe-uiracyiyl-"3 '-->5 -'>2 Odex dyilyl43 '->5 ')-2 '-OMe-uracyiyl-(3 '-5 '>2'-OMe-aeylyl-(3 '->-5 '>2'-Oe deoxycytdyiy-('->5 '>- ' '-~e-guayyi-(3'->5' )-2 '-OMe-uracdyly3 '-5 ')-2 '-OMe- cl (3 '>' '--- exetiyy-3>5 5 '>2 '-O~le-guanyiy]i43 '-->5 ')-2'-OMe-turacyiyi-(3 '-*-5 )'>2 OMe-uracyiy]-('3'-->5 ')2'-OMe-adenyiyi-(3-'->5 ')>2 '-OMe-guanyiy]-(3 '-->5 ')-2 '-OMe guany iyi-(3 '->5 ')"-2' -OMe-ur-aevIyi(3 '-5')-2 '-OMe-gii-y ivi-(3' ')-2'-OMe-cytidylyl (3'->-)5')-2 '-OMe- uainylyi-(3-'->5 '>2 '-OMe-uiraeyyi--(3--'->5 '>2 '-OMe-adenyliy-(3'-5 '>2'- OMe-uiraeyyi--(3 '->5 ),')2' -OMe-adenyiyi-(3' ')-'2'-OMe-.uracyiyi43 "'-5')-2 l'-OMe-adenNyll [003191 The chemical name of AR-CI18546 is 2 Oegayy-3'> > -~-unll (3' >5' >2'-OMe-adenyiyI-(3 ----- *5 ')-2 '-ON/e-adeniyLyI(3 -'---- >5 ')2' -OMe-uracylyI3 1- 3---- 5')-2' OMe-adenyvlyl -(3' ---- *5 ')-2 '-OMe--uracyly I-(3 -'---- >5')>2 '-OMe-ade-rylyI-(3 -'---- >5 '>2' deoxyevtidylyl3 - (3'- >5 ')-2'-OMe-utac1yivl(3'---- 5'>2'4)Me-uracvlyi-.- (K-->)-''Oe guanyiyk-( 3 ---- > *' )-2-ON/e-guanyl"---- *'5 )- -OMe-cytidy Iy (31 --- ')2 '-OMe-uracyl ('>5'>2 -dcoxycytidyiyJ43 ----- >5)-2 - \e guaniviyi-('->5)- 'OMe-uracyIyl--(3 ----- *5')-2 OMe-uracvyl-) -(-- >5')-'-OMe-adenylylI --- >"5 )-2'-OMe-guaniviyJ () >5'-_ -~ guanylyi-(3 '-----)5 '>-2'-OMc-uraeylvi-(3 ' ---- 6 >2 '4Me-guanyl-(3 '-5 '>2- '--0 M-cytidyly I (3 5)-'-OMe-guanylyI-(3 -'----> 5 '>)2 '-0Me-uracvIyl-(3 -- >,5 '>-2'-OMe-adenvyl-(3 - '>2- 5) 0Me-uracvIy1-(3 -- ,5 '>2'-OMe-adenvyl-(5- '>2 '-0Me-uiracevlyl-(' >--,5')-'- OMe-adeny lyl 1003201 The chemical name of ARC 19881 is 6-aminohexylyl-( I ---- '>-2' -O~le-guanylyl (3 '-->5')-2'-OMe-gruanylyi-(3 '-->5' -2'-OMe-adeniyiy-(3 '->- 5 ')2'-OMe-adeny-('3'->-,5 )'>2' OMe-uracyiyl-(3 '->- 5 '>'-OMe-adenyiyi1-('3'->-,5 '>2 '-OMe-uiraey7lyl-(3 '->45 '>2'- OMe-adenylyl (3 -> 5' '-2 '-deoxyeytidyiyi1-(3 '->)5 '>2 -'-OMe-uiraeylyl-('3 '->5 '>2 '-OMe-uiraeyiyl-(3 '->5 '>-2' OMe-gruanvyl-(3 '-5 '>2'-OMe-g~uanyiyl-(3 '->5,*')>2 '-OMe-eytidyiyl-(3'->- 5')-'-OMe uracvyy-(3' ->-5 '>' -deoxy7ctidviyi-('3'->-,5 '>2 '-OMe-guianyiyl-(3 '->- 5 '>2 '-OMe-uiracylyl (3' ->5 ')>2 '-OMe-uiraeyiyl-(3 '-> 5 ')-2 '-OMe-adeny lvi-(3' ->-5 '-2 '-OMe-cruanvliN-(3 '-5 ')2' Oldvlvliy-( -> ) -Oereyi-3'5''-OMe-uanyyi-(3 '-5 )-'-OMe-n~ cytidy- (3 '-5)-2 )'-OMe-guani~iyiv-(3' '-5'2'Oeureii(-5' )-2'--~-dni] 68 WO 2012/109675 PCT/US2012/024916 (3'-+5')-2 '-OMe-uracylyl-(3'-+5')-2'-OMe-adenylyl-(3' ->5')-2'-OMe-uracyiyl-3 '->5 ')2' OMe-adenyiyl-(3'-+5')-(3'-3 ')-2 '-deoxythymidine. [003211 The chemical name of ARC19882 is N-(methoxy-polyethyleneglycol)-6 aminohexyiyl-( 1-+5')-2 '-OMe-guanylyl-(3' -+5 ')-2'-OMe-guanyiyl-(3'-+5')-2'-OMe-adenylyl (3 '-+5')-2'-OMe-adenyiyl-(3'-+5')-2 '-OMe-uracyy-(3 '->4'-2'-OMe-adenyy-(3'-+5 ')-2 ' OMe-uracylyl-(3'-+5 ')-2'-OMe-ade-nylyl-(3' -5')-2'-deoxycytidylyi-(3'-45')-2'-OMe-uracylyl (3 '-+5')-2'-OMe-uracyiyi-(3 '-+5')-2'-OMe-guanyiyi-(3 '-+5')-2 '-OMe-guanyiyi-(3'-+5')2 2' OMe-cytidyiyl-(3'-+5'-2'-OMe-uracy-(3'-+5')-2'-deoxycytidyiy-(3'-+5 ')2'-OMe guanylyl -(3'->5 ')2'-OMe-uracylyl-(3' ->5')-2'-OMe-uracylyl-(3'-+5')-2'-OMe-adenyiyi (3 '-+5')-2 '-OMe-guanyiyi-(3'-4'-2 '-OMe-guanyliyi-(3 '-+5')-2'-OMe-.uracyiyl-(3 '->4'-2' OMe-guanyliyi-(3 '-+5')-2'-OMe-.cytidyiyi-(3 '-+5 ')>2'-OMe-guanyiyi-(3 '-+5 ')-2 '-OMe uracylyl-(3 '->5 ')-2'-OMe-adenylyl-(13'- >5')-2'-OMe-uracylyl-(3'- >5')-2'-OMe-adenylyl (3' ->5')-2'-OMe-uracyyl-(3 '-+ 5')-2'-OMe-adenylyi-(3 '->5')-(3 '-+3')-2'-deoxythymidine. [00322] The TFPI aptamers of the invention nay have any secondary structure. Preferably, the TFPI aptamers conupise a stem and a loop motif, such as in Figure IA and B. The putative secondary structure of ARC19499 is depicted in Figure 10C, which comprises a stem and a loop motif. [00323] Preferably, the TFPI aptamers are connected to one or more PEG moieties, with (Figure lOB) or without (Figure IA) one or more linkers. The PEG moieties may be any type of PEG moiety. For example, the PEG moiety may be linear, branched, multiple branched, star shaped, comb shaped or a dendrimer. In addition, the PEG moiety may have any molecular weight. Preferably, the PEG moiety has a molecular weight ranging from 5-100 kDa in size. More preferably, the PEG moiety has a molecular weight ranging from 10-80 kDa in size. Even more preferably, the PEG moiety has a molecular weight ranging from 20-60 kDa in size. Yet even more preferably, the PEG moiety has a molecular weight ranging from 30-50 kDa in size. Most preferably, the PEG moiety has a molecular weight of 40 kDa in size. The same or different PEG moieties may be connected to a TFPI aptamer. The same or different linkers or no linkers may be used to connect the same or different PEG moieties to a TFPI aptamer [003241 Alternatively, the TFPI aptamers may be connected to one or more PEG alternatives (rather than to one or more PEG moieties), with or without one or more liners. Examples of PEG alternatives include, but are not limited to, polyoxazoline (POZ), PolyPEG, 69 WO 2012/109675 PCT/US2012/024916 hydroxyethylstarch (HES) and albumin. The PEG alternative may be any type of PEG alternative, but it should function the same as or similar to a PEG moiety, i.e., to reduce renal filtration and increase the half-life of the TFPI aptamer in the circulation. The same or different PEG alternatives may be connected to a TF'P1 aptamer. The same or different linkers or no linkers may be used to connect the same or different PEG alternatives to a TFPI aptamer. Alternatively, a combination of PEG moieties and PEG alternatives may be connected to a TFPI aptamer, with or without one or more of the same or different linkers. [003251 Preferably, the TFPI aptamers are connected to a PEG moiety via a linker (Figure 10B). However, the TFPI aptamers may be connected to a PEG moiety directly, without the use of a linker (Figure 10A). The linker may be any type of molecule. Examples of linkers include, but are not limited to, amines, thiols and azides. For example, amines (RN-i 2 ) and activated esters (R'C(=O)OR") or anhydrides (R'C(=O)OC(=O)R") can be used as linkers to yield an amide (R'(C=O)NR). Activated esters include, without limitation, NIS (N hydroxysuccinimide) and sulfo derivatives of NIHS, nitrophenyl esters and other substituted aromatic derivatives. Anhydrides can by cyclic, such as succinic acid anhydride derivatives. Amines (RNiH2) and activated carbonates (R'OC(:::O)OR") can be used to yield carbamates (ROC(0:::O)NR). Activated carbamates include, without liimitation, N-IS (N-hydroxysuccinimide) and sulfo derivatives of N-IS, nitrophenyl carbamates. Amines (RNH2) and isothiocyanates (R 'N=C=S) can be used as linkers to yield isothioureas (RNHC(=S)NHIR'). Amines (RNH 2 ) and isocyanates (R'N:=C=:O) can be used as linkers to yield isoureas (RNH (C=:O)NHR'). Amines (RN H 2 ) and acyl azides (R'(C:=O)N2) can be used as linkers to yield amides (RN H(C:0)R'). Amines (RN12) and aldehydes or glyoxals ('(C=0)ii) can be used as linkers to yield imines (R'CH=NR) or amines via reduction (R'CH-I=NHR). Amines (RNH2) and sulfonyl chlorides (R'SO2Ci) can be used as linkers to yield suifoamides (R'SO 2 NHR). Amines (RNH2) and epoxides and oxiranes can be used as linkers to give a-hydroxyamines. Thiols (RSH) and iodoacetyls (R'(C:::)C1 2 1) can be used as linkers to yield thioethers (RSClH2(:::C)R'). Thiols (RS) and maleimides or maleimide derivatives can be used as linkers to give thioethers. Thiols (RSH) and aziridines can be used as linkers to give a-amine thioethers. Thiols (RSH) and acryloyl derivatives (R'CH=C1H2) can be used as linkers to give thioethers (R'CHI 2 CH2SR). Thiols (RSH) and disulfides (R'SSR") can be used as linkers to give disulfides (RSSR' or R"). Thiols (RSI) and vinylsulfones (C l=:CJH1SO 2 R') can be used as linkers to yield thiol ethers 70 WO 2012/109675 PCT/US2012/024916

(RSCH

2

CH

2

SO

2 R'). Azides (RN3) and alkynes (R'C=H) can be used as linkers to yield triazolines. Preferably, the linker contains a phosphate group. Preferably, the linker is from a 5' amine linker phosphoramidite. In some embodiments, the 5'-amine linker phosphoramidite comprises 2-18 consecutive CH 2 groups. In more preferred embodiments, the 5'-amine linker phosphoramidite comprises 2-1 consecutive CH2 groups. In even more preferred embodiments, the 5'-amine linker phosphoramidite comprises 4-8 consecutive Ci 2 groups. In most preferred embodiments, the 5'-amine linker phosphoramidite comprises 6 consecutive Cl- 2 groups, iC., is a 5'-hexylamine linker phosphoramidite. One or more of the same or different linkers or no linkers may be used to connect one or more of the same or different PEG moieties or one or more of the same or different PEG alternatives to a TFPI aptamer. [003261 In preferred embodiments, an aptamer, or a salt thereof, comprising the following structure is provided: 20KPEG 0 20KPEG' NN~/5-aptamer-3' H P || 0 wherein IN " PO-3 is from a 5'-amine linker phosphoramidite, and the aptamer is a TFPI aptamer of the invention. The 20KPEG moiety can be any PEG rnoiety having a molecular weight of 20 kDa. Preferably, the 20KPEG moiety is a mPEG moiety having a molecular weight of 20 kDa. [00327] In a particular embodiment, the aptamer, or a salt thereof, comprises the following structure: 20KPEG 0 20KPEGN N-- O -5-aptamer-3 H P wherein HN P0 3 H is from a 5'-amine linker phosphoramidite, and the aptamer has the nucleic acid sequence of rnG-mG-mA-mA-mU-mA-mU-mA-dC mU-mU -mG-mG-dC-mU-dC-mG-mU -mU-mA-mG-mG-mUL-mG-dC-mG-mU -mA-mU-mA mU-mA-3T (SEQ ID NO: 2), wherein "3T" is an inverted deoxythymidine, "dN" is a deoxynucleotide and "mN" is a 2-0 Methyl containing nucleotide. In some embodiments, the 5'-amine linker phosphoramidite comprises 2-18 consecutive CH 2 groups. In more preferred embodiments, the 5'-amine linker phosphoramidite comprises 2-12 consecutive -12 groups. In 71 WO 2012/109675 PCT/US2012/024916 even more preferred embodiments, the 5 -amine linker phosphoramidite comprises 4-8 consecutive CH 2 groups. In most preferred embodiments, the 5'-amine linker phosphoramidite comprises 6 consecutive CH 2 groups. The 20KPEG moiety can be any PEG moiety having a molecular weight of 20 kDa. Preferably, the 20KPEG moiety is a mPEG moiety having a molecular weight of 20 kDa. [003281 In a particular embodiment, the aptamer, or a salt thereof, comprises the following structure: 20KPEG 0 20KPEG' '- N''OOH-5'aptamer' H P 1; 0 wherein UN" P0 3 11 is from a 5'-amine linker phosphoramidite, and the aptamer has the nucleic acid sequence of mG-mG- nA- nA-mU-mA-mU-mA-dC mU-mU-mG-mG-nC-mU-dC-mG-mU-mU-mA-mG-iG -mU-mG-mC-mG-mU-mA-mnU-mA mU-mA-3T (SEQ ID NO: 8), wherein "3T" is an inverted deoxythymidine, "dN" is a deoxynucleotide and "irN" is a 2 -O Methyl containing nucleotide. In some embodiments, the 5'-amine linker phosphoramidite comprises 2-18 consecutive CH2 groups. In more preferred embodiments, the 5'-arnine linker phosphoramidite comprises 2-12 consecutive Cl-i 2 groups. In even more preferred embodiments, the 5'-amine linker phosphorarnidite comprises 4-8 consecutive CH 2 groups. In most preferred embodiments, the 5'-amine linker phosphoramidite comprises 6 consecutive CH21 groups. The 20KPEG moiety can be any PEG moiety having a molecular weight of 20 kDa. Preferably, the 20KPEG moiety is a mPEG moiety having a molecular weight of 20 kDa. 1003291 In alternative preferred embodiments, an aptamer, or a salt thereof, comprising the following structure is provided: 0 0 20KPEGOH 5,,aptarner , OH ,20KPEG H p P H H P I: 0 0 wherein UN" P021 is from a 5'-amine linker phosphoramidite, and the aptamer is a TFPI aptamer of the invention. The 20KPEG moiety can be any PEG moiety having a molecular weight of 20 kDa. Preferably, the 20KPEG moiety is a mPEG moiety having a molecular weight of 20 kDa. 72 WO 2012/109675 PCT/US2012/024916 [003301 In a particular alternative embodiment, the aptamer, or a salt thereof, comprises the following structure: 0 0 20KPEG O NH 5-aptaer-3 OH .20KPEG H PPH I I! 0 0 wherein I-N" P04-I is from a 5'-amine linker phosphoramidite, and the aptarner has the nucleic acid sequence of mG-mG-mA-mA-rnU-mA-riU-mA-dC mL nU-mG-mG-dC-nj-dC-mG-mU-riU-mA-mG-mG -mU-rnG-dC-mG-IriU-mA-mU-mA mU-mA (SEQ ID NO: 1), wherein "dN" is a deoxynucleotide and "m1N" is a 2'- Methyl containing nucleotide. In some embodiments, the 5'-amine linker phosphorami dite comprises 2 18 consecutive C2 groups. In more preferred embodiments, the 5'-amine linker phosphoramidite comprises 2-12 consecutive Ci groups. In even more preferred embodiments, the 5'-amine linker phosphoramidite comprises 4-8 consecutive (112 groups. In most preferred embodiments, the 5'-amine linker phosphoramidite comprises 6 consecutive CH2 groups. The 20KPEG moiety can be any PEG moiety having a molecular weight of 20 kDa. Preferably, the 20KPEG moiety is a mPEG moiety having a molecular weight of 20 kDa. 1003311 In more preferred embodiments, an aptamer, or a salt thereof, comprising the following structure is provided: 20KPEG 0 O 5'-aptarer-3' 2OKPEG' N'A " '' 0pl H , wherein the aptamer is a TFPI aptamer of the invention. The 20KPEG moiety can be any PEG moiety having a molecular weight of 20 kDa. Preferably, the 20KPEG moiety is a mPE G moiety having a molecular weight of 20 kDa. [003321 In a particular embodiment, the aptamer, or a salt thereof, comprises the following structure: 20KPEG 0 20KPE' N H,.5'-aptamrer-3' H r wherein the aptamer has the nucleic acid sequence of mG-mG-mA-mA-mU-mA-mU mA-dC-mU -mU-mG-mG-dC-mU-dC-mG-mU-iU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU 73 WO 2012/109675 PCT/US2012/024916 mA-mU-niA-3T (SEQ ID NO: 2), wherein "3T" is an inverted deoxythymidine. "dIN" is a deoxynucleotide and "mN" is a 2'-O Methyl containing nucleotide, The 20KPEG moiety can be any PEG moiety having a molecular weight of 20 kDa. Preferably, the 20KPEG moiety is a mPEG moiety having a molecular weight of 20 kda. [003331 In a particular embodiment, the aptarner, or a salt thereof, comprises the following structure: 20KPEG 0 OH5'-aptamr.er-3' 20KPEG'N N..-OH H 11 0 wherein the aptamer has the nucleic acid sequence of mG-mG-iA-mA-mU-mA-mU mA-dC-mU -mU-mG-mG-mC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-mC-mG-mU-mA niU-mA-mU-rnA-3T (SEQ ID NO: 8), wherein "31" is an inverted deoxythymidine, "dN" is a deoxynucleotide and "mN" is a 2'-O Methyl containing nucleotide. The 20KPEG moiety can be any PEG moiety having a molecular weight of 20 kDa. Preferably, the 20K-PEG moiety is a mPEG moiety having a molecular weight of 20 kDa. [003341 In alternative more preferred embodiments, an aptamer, or a salt thereof, comprising the following structure is provided: O H OH0 20KPEG NH5'-aptamer-3 N' 2KE N2-KPEGPE H 0 0 H wherein the aptamer is a TFPI aptamer of the invention. The 20KPEG moiety can be any PEG moiety having a molecular weight of 20 kDa. Preferably, the 20KPEG moiety is a mPEG moiety having a molecular weight of 20 kDa. [003351 In a particular alternative embodiment, the aptamer, or a salt thereof, comprises the following structure: 0 OH -'-aptamer-3 OH 2OKPEG p''"-"N 2KE H o 0 H wherein the aptamer has the nucleic acid sequence of mG-mG-mA-mA-mU-mA-mU mA-dC-mL-mU-mG-mG-dC-mU-dC-mG-mU-mnU-mA-mG-mG-mU -mG-dC-mG-mU -mA-mU mA-mU-mA (SEQ ID NO: 1), wherein "dN" is a deoxynucleotide and "mN" is a 2'-0 Methyl containing nucleotide. The 20KPEG moiety can be any PEG moiety having a molecular weight 74 WO 2012/109675 PCT/US2012/024916 of 20 kDa, Preferably, the 20KiPEG moiety is a mPEG moiety having a molecular weight of 20 kDa. [003361 In most preferred embodiments, an aptamer, or a salt thereof, comprising the following structure is provided: 0 H H3ON N 5t~N..~ aptamer-3' n 0 0 HO wherein "In" is about 454 ethylene oxide units (PEG = 20 kDa), and the aptamer is a TFPI aptamer of the invention. "n" is about 454 ethylene oxide units because the number of n's may vary slightly for a PEG having a particular molecular weight. Preferably, "n" ranges from 400-500 ethylene oxide units. More preferably, "n" ranges from 425-475 ethylene oxide units. Even more preferably, "n" ranges from 440-460 ethylene oxide units. Most preferably, "n" is 454 ethylene oxide units. [003371 In a particular embodiment, the aptamer, or a salt thereof, comprises the following structure: 0 H u H3CO Nor~N 0 O' O5'-aptamer-3'

H

3 CO OO HO wherein the aptamer has the nucleic acid sequence of mG-mG-mA-mA-mU-mA-mU mA-dC-mU-mU-i mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU mA-mU-mA-3T (SEQ ID NO: 2), wherein "n" is approximately 450, "3T" is an inverted deoxythymidine, "dN" is a deoxynucleotide and "mN" is a 2'-0 Methyl containing nucleotide. This aptamer is also known as ARC19499. [003381 In a particular embodiment, the aptamer, or a salt thereof, comprises the following structure: 0 H u1 H3jCO ' N 'N PO' O'5'-aptamner-3' _" 011 010 03COO HO wherein the aptamer has the nucleic acid sequence of mn(i-nG-mA-mA-mU-mA-mU mA-dC-mU-mU-mG-mG-mC -MU-dC-mG-m-U-mA-mnG-mG-mL-mG-m(-mG-mU-mA 75 WO 2012/109675 PCT/US2012/024916 niUi-mA-mU-mA-3T (SEQ ID NO: 8), wherein "n" is approximately 450, "3T" is an inverted deoxythymidine, "dN" is a deoxynucleotide and "mN" is a 2-O Methyl containing nucleotide. This aptamer is also known as ARC19882. [003391 In alternative most preferred embodiments, an aptamer, or a salt thereof, comprising the following structure is provided: 0 OH 5tataamer-3' OH n H O H n wherein "n" is about 454 ethylene oxide units (PEG = 20 kDa), and the aptamer is a TFPI aptamer of the invention. "n" is about 454 ethylene oxide units because the number of n's may vary slightly for a PEG having a particular molecular weight. Preferably, "n" ranges from 400 500 ethylene oxide units. More preferably, "n" ranges from 425-475 ethylene oxide units. Even more preferably, "n" ranges from 440-460 ethylene oxide units, Most preferably, "n" is 454 ethylene oxide units. [00340] In a particular alternative embodiment, the aptamer, or a salt thereof, comprises the following structure: MeOH 5O-aDtamer-3 OH 0 -- P -', -,) 0

NX

4 -- OMe n H H n H H wherein the aptamer has the nucleic acid sequence of m(i-mG-mA-minA-mU-mA-nU mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-rnU-rnU-mA-mG-mG-mU-mG -dC-rnG-mU-mA-ndJ mA-mU-nA (SEQ ID NO: 1), wherein "n" is approximately 450, "dN" is a deoxynucleotide and "imN" is a -O Methyl containing nucleotide. This aptamer is also known as ARC19501. 1003411 The invention also provides aptamers that have substantially the same ability to bind to TFIPI as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5. 6, 7, 8, 9 or 10. In some embodiments, the aptamers have substantially the same structure as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the aptamers have substantially the same ability to bind to TFPI and sub stantially the same structure as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The invention also provides aptamers that have substantially the same ability to bind to TFPI and substantially the same ability to modulate a biological function of TFPI as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The invention further provides aptamers that have 76 WO 2012/109675 PCT/US2012/024916 substantially the same ability to bind to TFPI and substantially the same ability to modulate blood coagulation as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5. 6. 7. 8, 9 or 10. The invention also provides aptamers that have substantially the same structure and substantially the same ability to modulate a biological function of TFPI as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The invention also provides aptamers that have substantially the same structure and substantially the same ability to modulate blood coagulation as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the aptamers have substantially the same ability to bind to TFPI, substantially the same structure and substantially the same ability to modulate a biological function of TFPI as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the aptamers have substantially the same ability to bind to TFPI, substantially the same structure and substantially the same ability to modulate blood coagulation as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. As used herein, substantially the same ability to bind to TFPI rneans that the affinity is within one or two orders of magnitude of the affinity of the nucleic acid sequences and/or aptamers described herein. It is well within the skill of those of ordinary skill in the art to determine whether a given sequence has substantially the same ability to bind to TFPL In some embodiments, the aptarner that binds to TFPI has a nucleic acid sequence at least 70%, 80%, 90% or 95% identical to SEQ ID NOs: 1, 2,3, 4, 5, 6, 7, 8, 9 or 10. 1003421 The ability of an aptamer to bind to TFPI may be assessed in a binding-competition assay, e.g., as described in Example 34, in which one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 may be selected as the competitor acting as a control aptamer. For example, a suitable assay may involve incubating 10 nM human TFPI (American Diagnostica, Stamford, CT, catalog #4500PC) with trace amounts of radiolabeled control aptamer and 5000 nM, 16667 nM, 556 nM, 185 nM, 61.7 nM, 20.6 nM, 6.86 nM, 2.29 nM, 0.76 nM or 0.25 nM of unlabeled competitor aptamer. A control aptamer is included in each experiment. For each molecule, the percentage of radiolabeled control aptamer bound at each competitor aptamer concentration is used for analysis. The percentage of radiolabeled control aptamer bound is plotted as a function of aptamer concentration and fitted to the equation y=(max/(1 + x/ICso)) + int where y=the percentage of radiolabeled control aptamer bound, x=the concentration of aptamer, max=the maximum radiolabeled control aptamer bound, and int=the y-intercept, to 77 WO 2012/109675 PCT/US2012/024916 generate an IC 50 value for binding-competition. The IC 50 of each aptamer is compared to the

IC

50 of the control aptamer evaluated in the same experiment, An aptamer having substantially the same ability to bind may include an aptamer having an IC 50 that is within one or two orders of magnitude of the IC 5 O of the control aptamer, and/or an aptamer having an IC 0 that is not more than 5-fold greater than that of the control aptamer evaluated in the same experiment. [003431 The ability of an aptamer to modulate a biological function and/or to modulate blood coagulation may be assessed in a calibrated automated thrombogram (CAT) assay, e.g.. as described in Example 34, in which one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 may be selected as a control aptamer. For example, a suitable assay may involve evaluation in a CAT assay in pooled hemophilia A plasma at 500 nM, 167 nM, 55.6 nM, 18.5 nM, 6.17 nM and 2.08 nM aptamer concentration. A control aptamer is included in each experiment. For each molecule, the endogenous thrombin potential (ETP) and peak thrombin values at each aptamer concentration are used for analysis. The ETP or peak thrombin value for hemophilia A plasma alone is subtracted from the corresponding value in the presence of aptamer for each molecule at each concentration, Then, the corrected ETP and peak values are plotted as a function of aptamer concentration and fitted to the equation y=(max/(i + ICso/x)) int, where y:::ETP or peak thrombin, x=:concentration of aptamer, max:::the maximurn ETP or peak thrombin, and int:::the y-intercept, to generate an IC;o value for both the ETP and the peak thrombin. The IC 5 o of each aptamer is compared to the IC 5 0 of the control aptamer that is evaluated in the same experiment. An aptamer having substantially the same ability to modulate a biological function and/or to modulate blood coagulation may include an aptamer having an

IC

5 0 that is within one or two orders of magnitude of the IC 50 of the control aptamer, and/or an aptamer for which one or both of the ETP and peak thrombin ICo of that molecule are not more than 5-fold greater than that of the control aptamer evaluated in the same experiment. [003441 The ability of an aptamer to modulate a biological function and/or to modulate blood coagulation may be assessed by evaluating inhibition of TFPI in a Factor Xa (FXa) activity assay, e.g., as described in Example 34, in which one of the aptamers shown in SEQ ID NOs: 1, 2, 3. 4. 5. 6. 7, 8, 9 or 10 may be selected as a control aptamer. A suitable assay may involve measuring the ability of FXa to cleave a chromogenic substrate in the presence and absence of TFPI, with or without the addition of aptamer. For example, 2 nM human FXa is incubated with 8 nM human TFPI. Then, 500 pM chromogenic substrate and aptamers are added and FXa 78 WO 2012/109675 PCT/US2012/024916 cleavage of the substrate is measured by absorbance at 405 nm (A 405 ) as a function of time. Aptamers are tested at 500 nM, 125 nM, 31.25 nM. 7.81 nM, 1.95 nM and 0.49 nM concentrations. A control aptamer is included in each experiment. For each aptamer concentration, the A 40 5 is plotted as a function of time and the linear region of each curve is fitted to the equation y=mx + b, where y=A 4 0 5 , x=the aptaner concentration, m=the rate of substrate cleavage, and b=the y-intercept, to generate a rate of FXa substrate cleavage. The rate of FXa substrate cleavage in the presence of TFPI and the absence of aptamer is subtracted from the corresponding value in the presence of both TFPI and aptamer for each molecule at each concentration. Then, the corrected rates are plotted as a function of aptamer concentration and fitted to the equation y=(Vmax/(i + IC/x)i), where y=the rate of substrate cleavage, x=concentration of aptamer, and Vmax=the maximum rate of substrate cleavage, to generate an

IC

50 and maximum (Vmax) value. The ICo and Vmrax values of each aptarner are compared to the ICIsc and Vmax values of the control aptamer evaluated in the same experiment. An aptamer having substantially the same ability to modulate a biological function and/or to modulate blood coagulation may include an aptamer having an IC 5 o that is within one or two orders of magnitude of the IC 50 of the control aptamer, and/or an aptamer having an ICso that is not more than 5-fold greater than that of the control aptarner evaluated in the same experiment, and/or an aptamer having a Vmax value not less than 80% of the Vm, value of the control aptarner evaluated in the same experiment. [00345] 'The terms "sequence identity" or"% identity", in the context of two or more nucleic acid or protein sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. Optimal alignment of sequences for comparison can be conducted. e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981); by the homology alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48: 443 (1970); by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988); by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.); or by 79 WO 2012/109675 PCT/US2012/024916 visual inspection (see generally, Ausubel, F. M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987)). [003461 One example of an algorithm that is suitable for determining percent sequence identity is the algorithm used in the basic local alignment search tool (hereinafter "BLAST"), see, e.g. Altschul et al., J Mol. Biol. 215: 403-410 (1990) and Altschul et al., Nucleic Acids Res., 15: 3389-3402 (1997), which is publicly available through the National Center for Biotechnology Information (hereinafter "NCBI"). [003471 Aptamers of the invention including, but not limited to, aptamers identified by the SELEX" method, 2'-Modified SELEX, minimized aptamers, optimized aptamers and chemically substituted aptamers, can be manufactured using any oligonucleotide synthesis technique that is well known in the art, such as solid phase oligonucleotide synthesis techniques (see, e.g., Gualtiere, F. Ed., New Trends in Synthetic Medicinal Chemistry, Ch. 9, Che mistrv of Antisense Oligonucleoticles, p. 261-335, 2000, Wiley-VCH, New York). The manufacturing of aptamiers using solid phase oligonucleotide synthesis techniques can also be done at commercial scale. Solution phase methods, such as triester synthesis methods (see, e.g., Sood et al., Nucl. Acid Res. 4:2557 (1977) and Hirose et al., Tet. Lett., 28:2449 (1978)), may also be used to manufacture aptamers of the invention, as well as recombinant means. [00348] In addition, a variety of functional groups can be introduced during the solid phase synthesis. The functionality can be a simple linker that results in a functional group, such as an amine or thiol, or may be a more complex construct, such as a biotin or a fluorescent dye. Typically, functional group linkers or more complex moieties are introduced using a phosphoramidite, or they can be introduced post-synthetically (i.e., after solid phase synthesis). Alternatively, by utilizing a modified solid support, a variety of functionalities can be introduced at the 3'-end of the oligonucleotide, thereby enabling a wider variety of conjugation techniques. [003491 The invention further provides aptamers that have been identified by the SELEXmI process, which comprises the steps of (a) contacting a mixture of nucleic acids with TFPI under conditions in which binding occurs; (b) partitioning unbound nucleic acids from those nucleic acids that have bound to TFPI; (c) amplifying the bound nucleic acids to yield a ligand-enriched mixture of nucleic acids; and, optionally, (d) reiterating the steps of binding, partitioning and amplifying through as many cycles as desired to obtain aptamer(s) that bind to TFPI. 80 WO 2012/109675 PCT/US2012/024916 [003501 The invention further provides methods for identifying aptamers that bind at least in part to or otherwise interact with one or more portions of TFPI, which comprise the steps of (a) contacting a mixture of nucleie acids with one or more portions of TFPI under conditions in which binding occurs; (b) partitioning unbound nucleic acids from those nucleic acids that have bound to TFPi; (c) amplifying the bound nucleic acids to yield a ligand-enriched mixture of nucleic acids; and, optionally, (d) reiterating the steps of contacting, partitioning and amplifying through as many cycles as desired, to obtain aptamer(s) that bind to a portion of TFPl. This method may also include intervening or additional cycles with binding to full-length TFPI, followed by partitioning and amplification. For example, the TFPI aptamers may bind to or otherwise interact with a linear portion or a conformational portion of TFPl. A TFPI aptamer binds to or otherwise interacts with a linear portion of TFPI when the aptamer binds to or otherwise interacts with a contiguous stretch of amino acid residues that are linked by peptide bonds. A TFPI aptamer binds to or otherwise interacts with a conformational portion of TFPI when the aptamer binds to or otherwise interacts with non-contiguous anino acid residues that are brought together by folding or other aspects of the secondary and/or tertiary structure of the polypeptide chain. Preferably, the one or more portions of mature TFPI (for example, Figure 3A) are selected from the group consisting of: amino acids 148-170, amino acids 150-170, amino acids 155-175, arnino acids 160-180, arnino acids 165-185, arnino acids 170-190, amino acids 175-195, amino acids 180-200, amino acids 185-205, amino acids 190-210, amino acids 195 215, amino acids 200-220, amino acids 205-225, amino acids 210-230, amino acids 215-235. amino acids 220-240, amino acids 225-245, amino acids 230-250, amino acids 235-255, amino acids 240-260, amino acids 245-265, amino acids 250-270, amino acids 255-275, amino acids 260-276, amino acids 148-175, amino acids 150-175, amino acids 150-180, amino acids 150 185. amino acids 150-190, amino acids 150-195, amino acids 150-200, amino acids 150-205. amino acids 150-210, amino acids 150-215, amino acids 150-220, amino acids 150-225, amino acids 150-230. amino acids 150-235, amino acids 150-240. amino acids 150-245. amino acids 150-250, amino acids 150-255, amino acids 150-260, amino acids 150-265, amino acids 150 270, amino acids 150-275, amino acids 150-276., amino acids 190-240. amino acids 190-276. amino acids 240-276, amino acids 242-276, amino acids 161-181, amino acids 162-181, amino acids 182-240, amino acids 182-241. and amino acids 182-276. The aptamer preferably comprises a dissociation constant for human TFPI or a variant or one or more portions thereof, of 81 WO 2012/109675 PCT/US2012/024916 less than 100 M, less than 1 pM, less than 500 nM, less than 100 nM, preferably 50 nM or less, preferably 25 nM or less, preferably 10 nM or less, preferably 5 nM or less, more preferably 3 nM or less, even more preferably 1 nM or less, and most preferably 500 pM or less. [003511 The invention also provides methods for identifying aptamers that bind at least in part to or otherwise interact with one or more portions of TFPI, which comprise the steps of (a) contacting a mixture of nucleic acids with full-length TFPI or one or more portions of TFPI under conditions in which binding occurs; (b) partitioning unbound nucleic acids from those nucleic acids that have bound to full-length TFPI or one or more portions of TFPI; (c) specifically eluting the bound nucleic acids with full-length TFPI or a portion of TFPI, or a ligand that binds to full-length TFPI or a portion of TFPI; (d) amplifying the bound nucleic acids to yield a ligand-enriched mixture of nucleic acids; and, optionally, (e) reiterating the steps of contacting, partitioning, eluting and amplifying through as many cycles as desired to obtain aptamer(s) that bind to one or more portions of TFPI. For example, the TFPI aptarners may bind to or otherwise interact with a linear portion or a confonnational portion of TFPI. A TFPI aptamer binds to or otherwise interacts with a linear portion of TFPI when the aptamer binds to or otherwise interacts with a contiguous stretch of amino acid residues that are linked by peptide bonds. A TFPI aptamer binds to or otherwise interacts with a conformational portion of TFPI when the aptamer binds to or otherwise interacts with non-contiguous arnino acid residues that are brought together by folding or other aspects of the secondary and/or tertiary structure of the polypeptide chain. Preferably, the one or more portions of mature TFPI (for example, Figure 3A) are selected from the group consisting of: amino acids 148-170, amino acids 150-170, amino acids 155-175. amino acids 160-180, amino acids 165-185, amino acids 170-190, amino acids 175-195, amino acids 180-200, amino acids 185-205, amino acids 190-210, amino acids 195 215, amino acids 200-220, amino acids 205-225. amino acids 210-230, amino acids 215-235, amino acids 220-240, amino acids 225-245, amino acids 230-250, amino acids 235-255, amino acids 240-260, amino acids 245-265. amino acids 250-270. amino acids 255-275. amino acids 260-276, amino acids 148-175, amino acids 150-175, amino acids 150-180, amino acids 150 185, amino acids 150-190, amino acids 150-195., amino acids 150-200, amino acids 150-205, amino acids 150-210, amino acids 150-215, amino acids 150-220, amino acids 150-225, amino acids 150-230, amino acids 150-235, amino acids 150-240, amino acids 150-245, amino acids 150-250, amino acids 150-255, amino acids 150-260, amino acids 150-265, amino acids 150 82 WO 2012/109675 PCT/US2012/024916 270, amino acids 150-275, amino acids 150-276, amino acids 190-240, amino acids 190-276. amino acids 240-276, amino acids 242-276, amino acids 161-181, amino acids 162-181, amino acids 182-240, amino acids 182-241. and amino acids 182-276. The aptamer preferably comprises a dissociation constant for human TFPI or a variant or one or more portions thereof of less than 100 pM, less than 1 pM, less than 500 nM, less than 100 nM, preferably 50 nM or less, preferably 25 nM or less, preferably 10 nM or less, preferably 5 nM or less, more preferably 3 nM or less, even more preferably 1 nM or less, and most preferably 500 pM or less. [00352] The invention further provides methods for identifying aptamers that bind at least in part to or otherwise interact with one or more portions of TFPI, which comprise the steps of (a) contacting a mixture of nucleic acids with full-length TFPI or one or more portions of TFPI under conditions in which binding occurs in the presence of a TFPI ligand (a ligand that binds to TFPI) that blocks one or more epitopes on TFPI from aptarner binding; (b) partitioning unbound nucleic acids from those nucleic acids that have bound to full-length TFPI or one or more portions of TFPI; (c) amplifying the bound nucleic acids to yield a ligand-enriched mixture of nucleic acids; and, optionally, (d) reiterating the steps of contacting, partitioning and amplifying through as rnany cycles as desired to obtain aptaier(s) that bind to one or more portions of TFPJ. In other embodiments of this method, inclusion of a TFPI ligand that blocks one or more portions on TFPI from aptamer binding can occur during the contacting step, the partitioning step, or both. For example, the TF1I aptamers may bind to or otherwise interact with a linear portion or a conformational portion of TFPI. A TFPI aptamer binds to or otherwise interacts with a linear portion of TFPI when the aptamer binds to or otherwise interacts with a contiguous stretch of amino acid residues that are linked by peptide bonds. A TFPI aptamer binds to or otherwise interacts with a conformational portion of TFPI when the aptamer binds to or otherwise interacts with non-contiguous amino acid residues that are brought together by folding or other aspects of the secondary and/or tertiary structure of the polypeptide chain. Preferably, the one or more portions of mature TFPI (for example, Figure 3A) are selected from the group consisting of: amino acids 148-170, amino acids 150-170, amino acids 155-175, amino acids 160-180, amino acids 165-185, amino acids 170-190, amino acids 175-195, amino acids 180 200, amino acids 185-205, amino acids 190-210, amino acids 195-215, amino acids 200-220, amino acids 205-225, amino acids 210-230, amino acids 215-235, amino acids 220-240, amino acids 225-245, amino acids 230-250, amino acids 235-255, amino acids 240-260, amino acids 83 WO 2012/109675 PCT/US2012/024916 245-265. amino acids 250-270. amino acids 255-275, amino acids 260-276. amino acids 148 175, amino acids 150-175, amino acids 150-180, amino acids 150-185, amino acids 150-190, amino acids 150-195, amino acids 150-200, amino acids 150-205, amino acids 150-210, amino acids 150-215, amino acids 150-220, amino acids 150-225, amino acids 150-230, amino acids 1 50-235, amino acids 150-240, amino acids 1 50-245, amino acids 1 50-250, amino acids 150 255, amino acids 150-260, amino acids 150-265, amino acids 150-270, amino acids 150-275, amino acids 150-276, amino acids 190-240, amino acids 190-276, amino acids '240-276, amino acids 242-276, amino acids 161-181, amino acids 162-181, amino acids 182-240, amino acids 182-241, and amino acids 182-276. The aptamer preferably comprises a dissociation constant for human TFPI or a variant or one or more portions thereof of less than 100 iM, less than 1 PM, less than 500 nM, less than 100 nM, preferably 50 nM or less, preferably 25 nM or less, preferably 10 nM or less, preferably 5 nM or less, more preferably 3 nM or less, even more preferably I nM or less, and most preferably 500 pM or less. [00353] The invention further provides methods for identifying aptamers that bind at least in part to or otherwise interact with one or more portions of TFPI, which comprise the steps of (a) contacting a mixture of nucleic acids with full-length TFPI or one or more portions of TFPI under conditions in which binding occurs; (b) partitioning unbound nucleic acids from those nucleic acids that have bound to full-length TFPI or one or more portions of TFPI; (c) partitioning bound nucleic acids that have a desired functional property from bound nucleic acids that do not have a desired functional property; (d) amplifying the bound nucleic acids that have a desired functional property to yield a ligand-enriched mixture of nucleic acids; and, optionally, (e) reiterating the steps of contacting, partitioning, partitioning and amplifying through as many cycles as desired to obtain aptamer(s) that bind to one or more portions of TFPI. Steps (b) and (c) can occur sequentially or simultaneously. For example, the TFPI aptamers may bind to or otherwise interact with a linear portion or a conformational portion of TFPI. A TFPI aptamer binds to or otherwise interacts with a linear position of TFPI when the aptamer binds to or otherwise interacts with a contiguous stretch of amino acid residues that are linked by peptide bonds. A TFPI aptamer binds to or otherwise interacts with a conformational portion of TFPI when the aptamer binds to or otherwise interacts with non-contiguous amino acid residues that are brought together by folding or other aspects of the secondary and/or tertiary structure of the polypeptide chain. Preferably, the one or more portions of mature TFPI (for example, Figure 84 WO 2012/109675 PCT/US2012/024916 3A) are selected from the group consisting of: amino acids 148-170. amino acids 150-170. amino acids 155-175, amino acids 160-180, amino acids 165-185, amino acids 170-190, amino acids 175-195, amino acids 180-200, amino acids 185-205, amino acids 190-210, amino acids 195 215, amino acids 200-220, amino acids 205-225, amino acids 210-230, amino acids 215-235, amio acids '20-'240, amino acids 225-245, amino acids 230-250, amino acids 235-255, amino acids 240-260, amino acids 245-265, amino acids 250-270, amino acids 255-275, amino acids 260-276, amino acids 148-175, amino acids 150-175, amino acids 150-180, amino acids 150 185, amino acids 150-190, amino acids 150-195, amino acids 150-200, amino acids 150-205, amino acids 150-2 10, amino acids 150-215, amino acids 150-220, amino acids 150-225, amino acids 150-230, amino acids 150-235, amino acids 150-240, amino acids 150-245, amino acids 150-250, amino acids 150-255, amino acids 1 50-260, amino acids 150-265, amino acids 150 270, arnino acids 150-275, arnino acids 150-276, arnino acids 190-240, arnino acids 190-276, aimno acids 240-276, amino acids 2421276, amino acids 161-181, amino acids 162-181, amino acids 182-240, amino acids 182-241, and amino acids 182-276. The aptamer preferably comprises a dissociation constant for hurnan TFPI or a variant or one or more portions thereof of less than 100 paM, less than 1 FM, less than 500 nM, less than 100 nM, preferably 50 nM or less, preferably 25 nM or less, preferably 10 nM or less, preferably 5 nM or less, more preferably 3 nM or less, even more preferably 1 nM or less, and most preferably 500 pM or less. 1003541 The invention also provides an aptamer that binds to a human tissue factor pathway inhibitor (TFPI) polypeptide having the amino acid sequence of SEQ ID NO: 11, wherein the aptamer modulates TFPI-mediated inhibition of blood coagulation, and wherein the aptamer competes for binding to TFPI with a reference aptamer comprising a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 4 (ARC 19499). SEQ ID NO: 1 (ARC26835), SEQ ID NO: 2 (ARC17480), SEQ ID NO: 3 (ARC19498), SEQ ID NO: 5 (ARC9500), SEQ ID NO:6 (ARC19501), SEQ ID NO: 7 (ARC31301), SEQ ID NO: 8 (ARC18546), SEQ ID NO: 9 (ARC19881) and SEQ ID NO: 10 (ARC1988Z2). Preferably, the reference aptamer comprises the nucleic acid sequence of SEQ ID NO: 4 (ARC19499). [003551 The invention further provides an aptamer that binds to a human tissue factor pathway inhibitor (TFPI) polypeptide having the amino acid sequence of SEQ ID NO: 11, wherein the aptamer binds to a linear portion or a conformational portion of TFPI in which at least a portion of the region recognized by the aptamer is different than the TFPI region bound 85 WO 2012/109675 PCT/US2012/024916 by Factor VIla, Factor Xa, or both Factor VIla and Factor Xa. Preferably, the aptamer binds to one or more regions comprising at least a portion of the amino acid sequence of SEQ ID NO: 11 selected from the group consisting of: amino acid residues 148-170, amino acid residues 150 170, amino acid residues 155-175, amino acid residues 160-180, amino acid residues 165-185, amino acid residues 170-190, amino acid residues 175-195, amino acid residues 180-200, amino acid residues 185-205, amino acid residues 190-210, amino acid residues 195-215, amino acid residues 200-220, amino acid residues 205-225, amino acid residues 210-230, amino acid residues 215-235, amino acid residues 220-240, amino acid residues 225-245, amino acid residues 230-250, amino acid residues 235-255, amino acid residues 240-260, amino acid residues 245-265, amino acid residues 250-270, amino acid residues 255-275, amino acid residues 260-276, amino acid residues 148-175, amino acid residues 150-175, amino acid residues 150-180, amino acid residues 150-185, amino acid residues 150-190, amino acid residues 150-195, amino acid residues 150-200, amino acid residues 150-205, amino acid residues 150-210, amino acid residues 150-215, amino acid residues 150-220, amino acid residues 150-225, amino acid residues 150-230, amino acid residues 150-235, amino acid residues 150-240, amino acid residues 150-245., amino acid residues 150-250, amino acid residues 150-255, amino acid residues 150-26(, amino acid residues 150-265, amino acid residues 150-270, amino acid residues 150-275, amino acid residues 150-276, amino acid residues 190-240, amino acid residues 190-276, amino acid residues 240-276, amino acid residues 242-276, amino acid residues 161-181, amino acid residues 162-181, amino acid residues 182-240, amino acid residues 182-241, and amino acid residues 182-276. More preferably, the aptamer competes with a reference aptamer comprising the nucleic acid sequence of SEQ ID NO: 4 (ARC19499) for binding to TFPL [003561 The invention also provides an aptamer that binds to the same region on a human tissue factor pathway inhibitor (TFPI) polypeptide having the amino acid sequence of SEQ ID NO: 11 as the region bound by a TFPI aptamer comprising the nucleic acid sequence of SEQ ID NO: 4 (ARC19499). [003571 The invention further provides an aptamer that binds to a region on a human tissue factor pathway inhibitor (TFPI) polypeptide comprising one or more portions of SEQ ID NO: 11, wherein the one or more portions is selected from the group consisting of: amino acid residues 148-170, amino acid residues 150-170, amino acid residues 155-175, amino acid residues 160 86 WO 2012/109675 PCT/US2012/024916 180, amino acid residues 165-185. amino acid residues 170-190, amino acid residues 175-195, amino acid residues 180-200, amino acid residues 185-205, amino acid residues 190-210, amino acid residues 195-215, amino acid residues 200-220, amino acid residues 205-225, amino acid residues 210-230, amino acid residues 215-235. amino acid residues 220-240, amino acid residues 225-245, amino acid residues 230-250, amino acid residues 235-255, amino acid residues 240-260, amino acid residues 245-265, amino acid residues 250-270, amino acid residues 255-275, amino acid residues 260-276, amino acid residues 148-175, amino acid residues 150-175, amino acid residues 150-180, amino acid residues 150-185, amino acid residues 150-190, amino acid residues 150-195, amino acid residues 150-200, amino acid residues 150-205, amino acid residues 150-210, amino acid residues 150-215, amino acid residues 150-220, amino acid residues 150-225, amino acid residues 150-230, amino acid residues 150-235, amino acid residues 150-240, amino acid residues 150-245, amino acid residues 150-250, arnino acid residues 150-255, amino acid residues 150-260, amino acid residues 150-265, amino acid residues 150-270, amino acid residues 150-275, amino acid residues 150-276, arnino acid residues 190-240, amino acid residues 190-276, amino acid residues 240-276, amino acid residues 242-276, amino acid residues 161-181, amino acid residues 162-181, arnino acid residues 182-240, arnino acid residues 182-241, and amino acid residues 182-276. 1003581 The invention additionally provides an aptamer that binds to human tissue factor pathway inhibitor (TFPI) and exhibits one or more of the following properties: a) competes for binding to TFPI with any one of SEQ ID NOs: 1-10; b) inhibits TFPI inhibition of Factor Xa; c) increases thrombin generation in hemophilia plasma; d) inhibits TFPI inhibition of the intrinsic tenase complex; e) restores normal hemostasis, as measured by thromboelastography (TEG) in whole blood and plasma; f) restores normal clotting, as indicated by shorter clot time, more rapid clot formation or more stable clot development, as measured by thromboelastography (TEG) or rotational thromboelastometry (ROTEM) in whole blood and plasma; or g) decreases the clot time, as measured by dilute prothrombin time (dPT), tissue factor activated clotting time (TF-ACT) or any other TFPI-sensitive clot-time measurement. [003591 The invention also provides an aptamer that binds to human tissue factor pathway inhibitor wherein the aptamer competes for binding to TFPI with a reference aptamner selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 87 WO 2012/109675 PCT/US2012/024916 SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. [003601 The invention further provides an aptamer that binds to tissue factor pathway inhibitor (TFPI) wherein the aptamer competes, either directly or indirectly, for binding to TFPI with a reference antibody selected from the group consisting of: AD4903. [003611 The invention also provides an aptamer that binds to human tissue factor pathway inhibitor (TFPI) and comprises a stein and loop motif having the nucleotide sequence of SEQ ID NO: 4, wherein: a) any one or more of nucleotides 1,2, 3, 4, 6, 8, 1-1, 12, 13, 17, 20, 21, 22, 24, 28, 30 and 32 may be modified from a 2'-OMe substitution to a 2'-deoxy substitution; b) any one or more of nucleotides 5, 7, 15, 19, 23, 27, 29 and 31 may be modified from a 2'-OMe uracil to either a 2'-deoxy uracil or a 2'-deoxy thymine; c) nucleotide 18 may be modified from a 2'-OMe uracil to a 2'-deoxy uracil; and/or d) any one or more of nucleotides 14, 16 and 25 may be modified from a 2'-deoxy cytosine to either a 2'-OMe cytosine or a 2-fluoro cytosine. [00362] The invention additionally provides an aptamer that binds to human tissue factor pathway inhibitor (TFPI) and comprises nucleotides 7-28 of SEQ ID NO: 2. [00363] The invention further provides a method for treating a bleeding disorder comprising administering any one of the above aptamers. [00364] The invention futher provides an aptamer that binds to tissue factor pathway inhibitor (TFPI), wherein the aptamer comprises a primary nucleic acid sequence selected from the group consisting of SEQ ID NOs.: 4, 1, 2, 3, 5, 6, 7, 8, 9 and 10. A primary nucleic acid sequence of an aptamer refers solely to the nucleotides (adenine, guanine, cytosine, uracil, thymine), without any modifications (such as a 2'-O Methyl, 2%fluoro modification, 3T or PEG). APTAMER MEDICINAL CHEMISTRY [003651 Once aptamers that bind to TFPI are identified, several techniques may be optionally performed in order to further increase binding, stability, potency and/or functional characteristics of the identified aptamer sequences. [003661 Aptamers that bind to TFPI may be truncated in order to obtain the minimal aptamer sequence having the desired binding and/or functional characteristics (also referred to herein as a "minimized construct" or a "minimized aptamer"). One method of accomplishing this is by using folding programs and sequence analysis, e.g., aligning clone sequences resulting from a selection to look for conserved motifs and/or covariation to inform the design of minimized 88 WO 2012/109675 PCT/US2012/024916 constructs. Suitable folding programs include, for example, the RNA structure program (Mathews, D.H.; Disney, M.D.; Childs, J.L.; Schroeder, SJ.; Zuker, M.; and Turner, D.H,, "Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure," 2004. Proceedings of the National Academv of Sciences. US, 101, 7287-7292). Biochemical probing experiments can also be performed in order to determine the 5' and 3' boundaries of an aptamer sequence to inform the design of minimized constructs. Minimized constructs can then be chemically synthesized and tested for binding and functional characteristics, as compared to the non-minimized sequence from which they were derived. Variants of an aptamer sequence containing a series of 5', 3' and/or internal deletions may also be directly chemically synthesized and tested for binding and/or functional characteristics, as compared to the non-minimized aptamer sequence from which they were derived. [00367] Additionally, doped reselections may be used to explore the sequence requirements within a single active aptamer sequence or a single minimized aptamer sequence. Doped reselections are performed using a synthetic, degenerate pool that has been designed based on the single sequence of interest. The level of degeneracy usually varies 70% to 85% from the wild type sequence, i.e., the single sequence of interest. In general, sequences with neutral mutations are identified through the doped reselection process, but in some cases sequence changes can result in improvements in affinity. The composite sequence information from clones identified using doped reselections can then be used to identify the minimal binding motif and aid in optimization efforts. 1003681 Aptamer sequences and/or minimized aptamer sequences may also be optimized post SELEX using aptamer medicinal chemistry to perform random or directed mutagenesis of the sequence to increase binding affinity and/or functional characteristics, or alternatively to determine which positions in the sequence are essential for binding activity and/or functional characteristics. [003691 Aptamer medicinal chemistry is an aptamer improvement technique in which sets of variant aptamers are chemically synthesized. These sets of variants typically differ from the parent aptamer by the introduction of a single substituent, and differ from each other by the location of this substituent. These variants are then compared to each other and to the parent. 89 WO 2012/109675 PCT/US2012/024916 Improvements in characteristics may be profound enough that the inclusion of a single substituent may be all that is necessary to achieve a particular therapeutic criterion. [003701 Alternatively the information gleaned from the set of single variants may be used to design further sets of variants in which more than one substituent is introduced simultaneously. In one design strategy, all of the single substituent variants are ranked, the top 4 are chosen and all possible double (6), triple (4) and quadruple (1) combinations of these 4 single substituent variants are synthesized and assayed. In a second design strategy, the best single substituent variant is considered to be the new parent and all possible double substituent variants that include this highest-ranked single substituent variant are synthesized and assayed. Other strategies may be used, and these strategies may be applied repeatedly such that the number of substituents is gradually increased while continuing to identify further-improved variants. 1003711 Aptamer medicinal chemistry may be used particularly as a method to explore the local, rather than the global, introduction of substituents. Because aptamers are discovered within libraries that are generated by transcription, any substituents that are introduced during the SELEX 'process must be introduced globally. For example, if it is desired to introduce phosphorothioate linkages between nucleotides then they can only be introduced at every A (or every G'3, C, T, U, etc.) if globally substituted. Aptamers that require phosphorothioates at some As (or some G, C, T, U, etc.) (locally substituted) but cannot tolerate it at other As (or some G, C, T, U, etc.) cannot be readily discovered by this process. 1003721 The kinds of substituents that can be utilized by the aptamer medicinal chemistry process are only limited by the ability to introduce them into an oligomer synthesis scheme. The process is certainly not limited to nucleotides alone. Aptamer medicinal chemistry schemes may include substituents that introduce steric bulk, hydrophobicitv, hydrophilicity, lipophilicity, lipophobicity, positive charge, negative charge, neutral charge, zwitterions, polarizability, nuclease-resistance, conformational rigidity, conformational flexibility, protein-binding characteristics, mass, etc. Aptamer medicinal chemistry schemes may include base modifications, sugar-modifications or phosphodiester linkage-modifications. [003731 When considering the kinds of substituents that are likely to be beneficial within the context of a therapeutic aptamer, it may be desirable to introduce substitutions that fall into one or more of the following categories: 90 WO 2012/109675 PCT/US2012/024916 (1) Substituents already present in the body, e.g., 2'-deoxy, 2'-ribo, 2'-O-methyl nucleotides, inosine or 5-methyl cytosine; (2) Substituents already part of an approved therapeutic, eg., 2 t fluoro nucleotides; or (3) Substituents that hydrolyze, degrade or metabolize to one of the above two categories, e.g., methylphosphonate-linked oligonucleotides or phosphorothioate-linked oligonucleotides. [003741 The aptarners of the invention include aptarners developed through aptamer medicinal chemistry, as described herein. [003751 Target binding affinity of the aptamers of the invention can be assessed through a series of binding reactions between the aptamer and the target (e .,a protein) in which trace 3P labeled aptamer is incubated with a dilution series of the target in a buffered medium and then analyzed by nitrocellulose filtration using a vacuum filtration manifold. Referred to herein as the dot blot binding assay, this method uses a three layer filtration medium consisting (from top to bottom) of nitrocellulose, nylon filter and gel blot paper. Aptarner that is bound to the target is captured on the nitrocellulose filter, whereas the non-target bound aptamer is captured on the nylon filter. The gel blot paper is included as a supporting rnedium for the other filters. Following filtration, the filter layers are separated, dried and exposed on a phosphor screen and quantified using a phosphorimaging system. The quantified results can be used to generate aptamer binding curves from which dissociation constants (K,)) can be calculated. In a preferred embodiment, the buffered medium used to perform the binding reactions is 1x Dulbecco's PBS (with Ca" 4 and Mg ) plus 0.1 mg/mL BSA. 1003761 Generally, the ability of an aptamer to modulate the functional activity of a target can be assessed using in vitro and in vivo models, which will vary depending on the biological function of the target. In some embodiments, the aptamers of the invention may inhibit a known biological function of the target. In other embodiments, the aptamers of the invention may stimulate a known biological function of the target. The functional activity of aptamers of the invention can be assessed using in vitro and in vivo models designed to measure a known function of TFPI. [003771 Aptamer sequences and/or minimized aptamer sequences may also be optimized using metabolic profile directed aptamer medicinal chemistry for site-specific identification of 91 WO 2012/109675 PCT/US2012/024916 cleavage sites and modifications to optimize stability of the aptamer sequences and/or minimized aptamer sequences. [003781 Metabolic profile directed aptamer medicinal chemistry involves incubating a parent aptamer with a test fluid to result in a mixture. Then, the mixture is analyzed to determine the rate of disappearance of the parent aptamer or the amount or percentage of aptamer remaining after incubation, the specific aptamer metabolic profile and the specific aptamer metabolite sequences. Knowledge of the sequences of the specific metabolites formed allows one to identify the sites of nuclease cleavage based on the mass of the metabolite(s). After systematically conducting metabolic profiling and identifying specific aptamer cleavage sites, the method involves introducing chemical substitutions or modifications at or near the cleavage sites that are designed to optimize the stability of the aptamer sequences and/or minimized aptamer sequences. [00379] In one embodiment, an aptamer is identified and modified by a) incubating a parent aptamer with a test fluid to result in a mixture; b) analyzing the mixture to identify metabolites of the parent aptamer, thereby detecting at least one aptamer cleavage site in the parent aptarner; and c) introducing a chemical substitution at a position proximal to the at least one aptamer cleavage site to result in a modified aptamer. This enhances the stability of the aptamer, and, in particular, the stability of the aptamer to endonucleases and exonucleases. 1003801 In some embodiments, the test fluid is a biological matrix, particularly a biological matrix selected from the group consisting of one or more of: serum; plasma; cerebral spinal fluid; tissue extracts, including cytosolic fraction, S9 fraction and microsomal fraction; aqueous humour; vitreous humour and tissue homogenates. In some embodiments, the biological matrix is derived from a species selected from the group consisting of one or more of: mouse, rat, monkey, pig, human, dog, guinea pig and rabbit. In some embodiments, the test fluid comprises at least one purified enzyme, particularly at least one purified enzyme selected from the group consisting of: snake venom phosphodiesterase and DNAse L, [003811 In some embodiments, the analyzing step includes analyzing the resulting aptamer using liquid chromatography and mass spectrometry, particularly electron spray ionization liquid chromatography mass spectrometry, polyacrylamide gel electrophoresis or capillary electrophoresis to determine a position of at least one aptamer cleavage site. In some embodiments, the analyzing step includes analyzing the resulting aptamer using a bioanalytical 92 WO 2012/109675 PCT/US2012/024916 method selected from the group consisting of one or more of: denaturing polyacrylamide gel electrophoresis (PAGE); capillary electrophoresis; high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC/MS), particularly LC/MS/MS or LC/MS/MS/MS, and more particularly electrospray ionization LC/MS (ESI-LC/MS), ESI LC/MS/MS and ESI-LC/MS/MIS/MS. [00382] In some embodiments, the proximal position includes a position selected from the group consisting of: a position immediately 5' to the aptamer cleavage site, a 5' position at or within three nucleotides of the aptamer cleavage site, a position immediately 3' to the aptamer cleavage site, a 3' position at or within three nucleotides of the aptamer cleavage site, and at the cleaved internucleotide linkage. [003831 In some embodiments, the chemical substitution is selected from the group consisting of: a chernical substitution at a sugar position; a chemical substitution at a base position and a chernical substitution at an internucleotide linkage. More particularly, a substitution is selected from the group consisting of: a nucleotide substituted for a different nucleotide; a purine substitution for a pyrimidine; a 2 -amine substitution for any nucleotide; a 2 '-deoxy dihydrouridine substitution for a uridine; a 2'-deoxy-5-methyl cytidine for a cytidine; a 2-amino purine substitution for a purine; a phosphorothioate substituted for a phosphodiester; a phosphorodithioate substituted for a phosphodiester; a 2'-deoxy nucleotide substituted for a 2' OH nucleotide, a 2'-OMe nucleotide or a 2'-fluoro nucleotide; a 2'-OMe nucleotide substituted for a 2'-OH- nucleotide, a 2'-deoxy nucleotide, or a 2'-fluoro nucleotide; a 2'-fluoro nucleotide substituted for a 2'-OH nucleotide, a 2'-deoxy nucleotide or a 2'-OMe nucleotide; or a 2'-0 methoxyethyl nucleotide substituted for a 2'-OH, 2'-fluoro or 2'-deoxy nucleotide; a 2'-0 methoxyethyl nucleotide or deoxy nucleotide for a 2'-fluoro nucleotide; and the addition of one or more PEG or other polymers or other PIC or distribution-influencing entity. [003841 In additional embodiments, the introducing step of these methods further includes introducing more than one chemical substitution at one or more cleavage sites or at a single cleavage site or both. [003851 In another embodiment, wherein more than one aptamer cleavage site is detected, the introducing step of these methods further includes introducing at least one chemical substitution at the associated proximal position of the aptamer cleavage site determined to occur first in time 93 WO 2012/109675 PCT/US2012/024916 during the incubating step or at any other cleavage site(s) that provides the desired properties upon introduction of a chemical substitution. [003861 In other embodiments, these methods further include the step of testing the stability of the modified aptamer in the test fluid. In some embodiments, aptamer stability is assessed by determining the percent of modified aptamer that remains intact in the test fluid as compared to the percent of the parent aptamer that remains intact in the test fluid. In some embodiments, the percent of intact aptamer is assessed by a bioanalytical method selected from the group consisting of one or more of: denaturing polyacrylamide gel electrophoresis (PAGE); capillary electrophoresis; IPLC and LC/MS, particularly LC/MIS/MS or L/MS/MS/MS, and more particularly ESI-LC/MS, ESI-LC/MS/MS and ESI-LC/MS/MS/MS. In other embodiments, the modified aptamer is more stable in the test fluid than the parent aptamer, preferably at least 2 fold, more preferably at least 5 fold and most preferably at least 10 fold more stable. [003871 In additional embodiments, these methods further include determining a dissociation constant or ICo of the modified aptamer for its target. In some embodiments, chemical substitutions are introduced singly at each position or in various combinations in the aptarner, and the dissociation constant or ICSI for each resulting aptamer is determined. Chemical substitutions are introduced at a position proximal to the aptamer cleavage site such that a single chemical modification results in a dissociation constant for the modified aptamer that is the same or less than that of the parent aptamer. In another embodiment of the invention, the method includes selecting a modified aptamer having a dissociation constant or ICso for its target that is the same or less than that for the parent aptamer. 1003881 In other embodiments, the modified aptamer binds to a target having a biological activity, and the method further includes testing the biological activity of the target in the presence and absence of modified aptamer. In another embodiment, the method further includes selecting a modified aptamer that binds to a target having a biological activity that is the same or better than that of the parent aptamer. The biological activity may be measured in any relevant assay, such as an ELISA assay or a cell-based assay. [003891 In some embodiments, the incubating, analyzing, introducing and testing steps are repeated iteratively until the desired stability is achieved. [003901 The aptamers of the invention may be routinely adapted for diagnostic purposes according to any member of techniques employed by those skilled in the art. Diagnostic 94 WO 2012/109675 PCT/US2012/024916 utilization may include both in vivo or in vitro diagnostic applications. Diagnostic agents need only be able to allow the user to identify the presence of a given target at a particular locale or concentration. Simply the ability to form binding pairs with the target may be sufficient to trigger a positive signal for diagnostic purposes. Those skilled in the art would also be able to adapt any aptamer by procedures known in the art to incorporate a labeling tag to track the presence of such ligand. Such a tag could be used in a number of diagnostic procedures. APTAMERS HANG IMMUNOSTIMULATORY MOTIFS [00391] The invention provides aptamers that bind to TFPI and modulate its biological function. [00392] Recognition of bacterial DNA by the vertebrate immune system is based upon the recognition of unmethylated CG dinucleotides in particular sequence contexts ("CpG motifs"). One receptor that recognizes such a motif is Toll-like receptor 9 ("TLR 9"), a member of a family of Toll-like receptors (- 0 rnembers) that participate in the innate immune response by recognizing distinct microbial components. TLR 9 is activated by unmethylated oligodeoxynucleotide ("ODN") CpG sequences in a sequence-specific manner. The recognition of CpG motifs triggers defense mechanisms leading to innate and ultimately acquired immune responses. For example, activation of TLR 9 in mice induces activation of antigen presenting cells, up-regulation of MiC class I and II molecules, and expression of important co-stimulatory molecules and cytokines including IL-12 and IL-23. This activation both directly and indirectly enhances B and T cell responses, including a robust up-regulation of the THI cytokine IFN gamma. Collectively, the response to CpG sequences leads to: protection against infectious diseases, improved immune response to vaccines, an effective response against asthma, and improved antibody-dependent cell-mediated cytotoxicity. Thus, CpG ODNs can provide protection against infectious diseases, function as immuno-adjuvants or cancer therapeutics (monotherapy or in combination with a mAb or other therapies), and can decrease asthma and allergic response. [003931 A variety of different classes of CpG motifs have been identified, each resulting upon recognition in a different cascade of events, release of cytokines and other molecules, and activation of certain cell types. See, eg., CpG Motifs in Bacterial DNA and Their Immune Effects, Annu. Rev. Immunol 2002, 20:709-760, which is incorporated herein by reference. Additional immunostimulatory motifs are disclosed in the following U.S. Patents, each of which 95 WO 2012/109675 PCT/US2012/024916 is incorporated herein by reference: U.S. Patent No. 6,207,646; U.S. Patent No. 6,239,116; U.S. Patent No. 6,429,199; U.S. Patent No. 6,214,806; U.S. Patent No. 6,653,292; U.S. Patent No. 6,426,334; U.S. Patent No. 6,514,948 and U.S. Patent No. 6,498,148. Any of these CpG or other immunostimulatory motifs can be incorporated into an aptamer. The choice of aptamers is dependent upon the disease or disorder to be treated. Preferred immunostimulatory motifs are as follows (shown 5' to 3', left to right) wherein "r" designates a purine, "y" designates a pyrimidine, and "X" designates any nucleotide: AACGTTCGAG (SEQ ID NO: 12); AACGTT; ACGT; rCGy; rrCGyy; XCGX; XXCGXX; and XiX 2

CGYY

2 ; wherein X 1 is G or A. X2 is not C, Y 1 is not G and Y 2 is preferably T. [003941 In those instances where a CpG motif is incorporated into an aptamer that binds to a specific target other than a target known to bind to CpG motifs, and upon binding stimulates an inunune response (a "non-CpG target"), the CpG is preferably located in a non-essential region of the aptamer. Non-essential regions of aptamers can be identified by site-directed niutagenesis, deletion analyses and/or substitution analyses. However, any location that does not significantly interfere with the ability of the aptamer to bind to the non-CpG target may be used. In addition to being embedded within the aptarner sequence, the CpG motif may be appended to either or both of the 5' and 3' ends or otherwise attached to the aptaner. Any location or rneans of attachment may be used as long as the ability of the aptamer to bind to the non-CpG target is not significantly interfered with. 1003951 As used herein, "stimulation of an immune response" can mean either (1) the induction of a specific response (e.g., induction of a Th response) or the production of certain molecules, or (2) the inhibition or suppression of a specific response (e.g., inhibition or suppression of the Th2 response) or of certain molecules. [003961 Aptamers of the invention, including aptamers having one or more CpG or other immunostimulatory sequences, can be identified or generated by a variety of strategies using, e.g., the SELEX" process described herein, The incorporated immunostimulatory sequences can be DNA, RNA, substituted DNA or RNA, and/or a combination of substituted or unsubstituted DNA/RNA. In general, the strategies can be divided into two groups. For both groups of strategies, the CpG motifs are included to: a) stimulate the immune response to counteract situations where a repressed immune response is relevant to disease development (i.e., immune deficiency diseases such as AIDS), and b) to focus a stimulated immune response against a 96 WO 2012/109675 PCT/US2012/024916 particular target or cell type (i.e., cancer cells), or to bias an immune response towards a TH1I state and away from TH2 or TH17 state (i.e., including CpG motifs in an aptamer against an anti allergy target such as IgE to counteract an allergic condition). [003971 In group one, the strategies are directed to identifying or generating aptamers including both a CpG motif or other immunostimulatory sequence as well as a binding site for a target, where the target (hereinafter "non-CpG target") is a target other than one known to recognize CpG motifs or other immunostimulatory sequences. In some embodiments of the invention, the non-CpG target is a TFPI target. The first strategy of this group includes performing SELEX' to obtain an aptamer to a specific non-CpG target, preferably a target using an oligonucleotide pool wherein a CpG motif has been incorporated into each member of the pool as, or as part of, a fixed region, e.g., in some embodiments the randomized region of the pool members includes a fixed region having a CpG motif incorporated therein, and identifying an aptarner including a CpG motif. The second strategy of this group includes performing SELEX to obtain an aptamer to a specific non-CpG target, and following selection, appending a CpG motif to the 5' and/or 3' end or engineering a CpG motif into a region, preferably a non essential region, of the aptamer. The third strategy of this group includes performing SELEX" M to obtain an aptarner to a specific non-CpG target, wherein during synthesis of the pool the molar ratio of the various nucleotides is biased in one or more nucleotide addition steps so that the randomized region of each member of the pool is enriched in CpG motifs, and identifying an aptamer including a CpG motif. The fourth strategy of this group includes performing SELEX to obtain an aptamer to a specific non-CpG target, and identifying an aptamer including a CpG motif. The fifth strategy of this group includes performing SELEX M to obtain an aptamer to a specific non-CpG target, and identifying an aptamer which, upon binding, stimulates an immune response but that does not include a CpG motif. [003981 In group two. the strategies are directed to identifying or generating aptamers including a CpG motif and/or other sequences that are bound by the receptors for the CpG motifs (e.g.. TLR9 or the other toll-like receptors) and upon binding stimulate an immune response. The first strategy of this group includes performing SELEX" to obtain an aptamer to a target known to bind to CpG motifs or other immunostimulatory sequences and upon binding stimulate an immune response using an oligonucleotide pool wherein a CpG motif has been incorporated into each member of the pool as, or as part of, a fixed region, e.g., in some embodiments the 97 WO 2012/109675 PCT/US2012/024916 randomized region of the pool members include a fixed region having a CpG motif incorporated therein, and identifying an aptamer including a CpG motif. The second strategy of this group includes performing SELEXI to obtain an aptamer to a target known to bind to CpG motifs or other immunostimulatory sequences and upon binding stimulate an immune response and then appending a CpG motif to the 5' and/or 3' end or engineering a CpG motif into a region, preferably a non-essential region, of the aptamer. The third strategy of this group includes performing SELEX M to obtain an aptamer to a target known to bind to CpG motifs or other immunostimulatory sequences and upon binding stimulate an immune response, wherein during synthesis of the pool the molar ratio of the various nucleotides is biased in one or more nucleotide addition steps so that the randomized region of each member of the pool is enriched in CpG motifs, and identifying an aptamer including a CpG motif. The fourth strategy of this group includes performing SELEX * to obtain an aptamer to a target known to bind to CpG motifs or other immunostimulatory sequences and upon binding stimulate an immune response, and identifying an aptaier including a CpG motif. The fifth strategy of this group includes performing SELEX TM to obtain an aptamer to a target known to bind to CpG motifs or other inununostimulatory sequences, and identifying an aptamer which, upon binding, stimulates an immune response but that does not include a CpG motif. MODULA TION OF PHARMACOK INETICS AND BIODISTRIBUTION OF APTAMER THERAPE 1/TICS 1003991 It is important to match the pharmacokinetic properties for all oligonucleotide-based therapeutics, including aptamers, to the desired pharmaceutical application. Aptamers must be able to be distributed to target organs and tissues, and remain in the body (unmodified) for a period of time consistent with the desired dosing regimen. [004001 The invention provides materials and methods to affect the pharmacokinetics of aptamer compositions and, in particular, the ability to tune aptamer pharmacokinetics. The tunability of (i.e., the ability to modulate) aptamer pharmacokinetics is achieved through conjugation of modifying moieties (e.g., PEG polymers) to the aptamer and/or the incorporation of modified nucleotides (e.g., 2'-fluoro or 2'-0-methyl) or modified internucleotide linkages to alter the chemical composition of the aptamer. The ability to tune aptamer pharmacokinetics is used in the improvement of existing therapeutic applications, or alternatively, in the development of new therapeutic applications. For example, in some therapeutic applications, e.g., in anti 98 WO 2012/109675 PCT/US2012/024916 neoplastic or acute care settings where rapid drug clearance or turn-off may be desired, it is desirable to decrease the residence times of aptamers in the circulation, Alternatively, in other therapeutic applications, e.g., maintenance therapies where systemic circulation of a therapeutic is desired, it is desirable to increase the residence times of aptamers in the circulation. [004011 In addition, the tunability of aptamer pharmacokinetics is used to modify the disposition, for example the absorption, distribution, metabolism and elimination (ADME) of an aptamer to fit its therapeutic objective in a subject. Tunability of the pharmacokinetics of an aptainer can affect the manner and extent of absorption of the aptamer, the distribution of an aptamer throughout the fluids and tissues of the body, the successive metabolic transformations of the aptamer and its inetabolite(s) and finally, the elimination of the aptamer and its metabolite(s). For example, in some therapeutic applications, it may be desirable to alter the biodistribution of an aptamer therapeutic in an effort to target a particular type of tissue or a specific organ (or set of organs), or to increase the propensity to enter specific cell types. In these applications, the aptamer therapeutic preferentially distributes into specific tissues and/or organs and accumulates therein to cause a therapeutic effect. In other therapeutic applications, it may be desirable to target tissues displaying a cellular marker or a symptom associated with a given disease, cellular injury or other abnormal pathology, such that the aptamer therapeutic preferentially accumulates in the affected tissue. For example, PEGylation of an aptarner therapeutic (e.g., PEGylation with a 20 kDa PEG polymer or other polymer or conjugation entity) is used to target inflamed tissues, such that the PEGylated aptamer therapeutic preferentially accumulates in the inflamed tissue. 1004021 To determine the pharmacokinetic profiles of aptamer therapeutics (e.g., aptamer conjugates or aptamers having altered chemistries, such as modified nucleotides), a variety of parameters are studied in normal subjects, e.g., test animals or humans, or in diseased subjects, e.g.. TFPI-specific animal models, such as animal models of hypercoagulation or hypocoagulation, or humans with a coagulation deficiency. Such parameters include, for example, the distribution or elimination half-life (tm 12 ), the plasma clearance (CL), the volume of distribution (Vss), the area under the concentration-time curve (AUC), the maximum observed serum or plasma concentration (Cmk), and the mean residence time (MRT1) of an aptamer composition. As used herein, the term "AlC" refers to the area under the plasma concentration curve of an aptamer therapeutic versus the time after aptamer administration. The AUC value is 99 WO 2012/109675 PCT/US2012/024916 used to estimate the exposure of the aptamer and also used to determine the bioavailability of an aptamer after an extravascular route of administration, such as, e.g., subcutaneous administration. Bioavailability is determined by taking the ratio of the AUC obtained after subcutaneous administration to the AUC obtained after intravenous administration and normalizing them to the doses used after each administration (i.e., the percent ratio of aptamer administered after subcutaneous administration as compared to the same aptamer administered by intravenous administration at the same dose or normalized dose). The CL value is the measurement of the removal of the parent aptamer therapeutic from the systemic circulation. The volume of distribution (Vd) is a term that relates the amount of aptamer in the body at one time to its plasma concentration. The Vd is used to determine how well a drug is removed from the plasma and distributed to tissues and/or organs. A larger Vd implies wide distribution, extensive tissue binding, or both a wide distribution and extensive tissue binding. There are three basic volurnes of distribution: (i) the apparent or initial volume of distribution at time zero obtained from back extrapolation of the concentration-time curve; (ii) the volume calculated once distribution is complete, approximating to Vdss, where the area volume is dependent upon the elimination kinetics; and (iii) the volume of distribution calculated once distribution is complete. The parameter that should ideally be measured is the Vdss because this parameter is independent of the elimination kinetics. If the Vss for the aptamier is larger than the blood volume, it suggests that the aptamer is distributed outside of the systemic circulation and is likely to be found in the tissues or organs. Pharmacodynamic parameters may also be used to assess drug characteristics. 1004031 To determine the distribution of aptamer therapeutics (e.g., aptamer conjugates or aptamers having altered chemistries, such as modified nucleotides), a tissue distribution study or quantitative whole body autoradiography using a radiolabeled aptamer that is administered to a normal animal or a diseased target specific animal model is used. The accumulation of the radiolabeled-aptamer at a specific site can be quantified. [004041 The pharmacokinetics and biodistribution of an aptamer described herein, such as a stabilized aptamer, can be modulated in a controlled manner by conjugating an aptamer to a modulating moiety, such as, but not limited to, a small molecule, peptide, or polymer, or by incorporating modified nucleotides into an aptamer. The conjugation of a modifying moiety 100 WO 2012/109675 PCT/US2012/024916 and/or altering nucleotide chemical composition alters fundamental aspects of aptamer residence time in circulation and distribution within and to tissues and cells. [004051 In addition to metabolism by nucleases, oligonucleotide therapeutics are subject to elimination via renal filtration. As such, a nuclease-resistant oligonucleotide administered intravenously typically exhibits an in vivo half-life of <30 minutes, unless filtration can be blocked. This can be accomplished by either facilitating rapid distribution out of the blood stream into tissues or by increasing the apparent molecular weight of the oligonucleotide above the effective size cut-off for the glomerulus. Conjugation of small molecular weight therapeutics to a PEG polymer (PEGylation), as described below, can dramatically lengthen residence times of aptamers in the circulation, thereby decreasing dosing frequency and enhancing effectiveness against vascular targets. [00406] Modified nucleotides can also be used to modulate the plasma clearance of aptamers. For example, an unconjugated aptaner that incorporates for example, 2'-fluoro, 2'-OMe, and/or phosphorothioate stabilizing chemistries, which is typical of current generation aptarners as it exhibits a high degree of nuclease resistance in vitro and in vivo, displays rapid distribution into tissues, primarily into the liver and kidney, when compared to unmodified aptarner. PAG-DERIVATIZED NUCLEIC ACIDS [00407] As described above and as shown in Figure 11, derivatization of nucleic acids with high molecular weight non-immunogenic polymers has the potential to alter the pharmacokinetic and pharmacodynamic properties of nucleic acids making them more effective and/or safer therapeutic agents. Favorable changes in activity can include increased resistance to degradation by nucleases, decreased filtration by the kidneys, decreased exposure to the immune system, and altered distribution of the therapeutic through the body. [004081 The aptamer compositions of the invention may be derivatized with one or more polyalkylene glycol ("'PAG") moieties, Typical polymers used in the invention include polyethylene glycol ("PEG"), also known as polyethylene oxide ("PEO") and polypropylene glycol (including poly isopropylene glycol). Additionally, random or block copolymers of different alkylene oxides can be used in many applications. In a common form, a polyalkylene glycol, such as PEG, is a linear polymer terminated at each end with hydroxyl groups: HO

CH

2 CH20-(CH2C 2 0) i.-CHI 2 CH2-OH. This polymer, alpha-, omega-dihydroxvlpolyethyiene glycol, can also be represented as HO-PEG-Oil, where it is understood that the -PEG- symbol 101 WO 2012/109675 PCT/US2012/024916 represents the following structural unit: -CH 2 CH1 2

O-(CH

2

CH

2 O) -CH 2 CH-, where n typically ranges from 4 to 10,000. [004091 PAG polymers suitable for therapeutic indications typically have the properties of solubility in water and in many organic solvents, lack of toxicity, and lack of immunogenicity. One use of PAGs is to covalently attach the polymer to insoluble molecules to make the resulting PAG-molecule "conjugate" soluble. For example, it has been shown that the water-insoluble drug paclitaxel, when coupled to PEG, becomes water-soluble. Greenwald, et at J Org. Chem., 60:33 1-336 (1995). PAG conjugates are often used not only to enhance solubility and stability, but also to prolong the blood circulation half-life of molecules and later distribution within the body. [004101 The PAG derivatized compounds conjugated to the aptamers of the invention are typically between 5 and 80 kDa in size, however any size can be used, the choice dependent on the aptamer and application. Other PAG derivatized compounds of the invention are between 10 and 80 kDa in size. Still other PAG derivatized compounds of the invention are between 10 and 60 kDa in size. In some embodiments, the PAG moieties derivatized to compositions of the invention are PEG moieties having a molecular weight ranging from 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 kDa in size. In some embodiments, the PEG is linear PEG, while in other embodiments, the PEG is branched PEG. In still other embodiments, the PEG is a 40kDa branched PEG as depicted in Figure 6. In some embodiments, the 40 kDa branched PEG is attached to the 5' end of the aptamer as depicted in Figure 7. 1004111 Production of high molecular weight PEGs (>10 kDa) can be difficult, inefficient and expensive. To synthesize high molecular weight PEG-nucleic acid conjugates, higher molecular weight activated PEGs are generated. Methods for generating such molecules involve the formation of a linear activated PEG, or a branched activated PEG in which two or more PEGs are attached to a central core carrying the activated group. The terminal portions of these higher molecular weight PEG molecules, i.e., the relatively non-reactive hydroxyl (--OH) moieties, can be activated or converted to functional moieties for attachment of one or more of the PEGs to other compounds at reactive sites on the compound. Branched activated PEGs will have more than two termini, and in cases where two or more termini have been activated, such activated higher molecular weight PEG molecules are herein referred to as, multi-activated PEGs. In some cases, not all termini in a branched PEG molecule are activated. In cases where any two termini 102 WO 2012/109675 PCT/US2012/024916 of a branched PEG molecule are activated, such PEG molecule is referred to as a bi-activated PEG. In some cases where only one terminus in a branched PEG molecule is activated, such PEG molecule is referred to as mono-activated. In other cases, the linear PEG molecule is di functional and is sometimes referred to as "PEG diol". The terminal portions of the PEG molecule are relatively non-reactive hydroxyl moieties, the -OHl groups, that can be activated or converted to functional moieties for attachment of the PEG to other compounds at reactive sites on the compounds. Such activated PEG diols are referred to herein as homo bi-activated PEGs. The molecules are generated using any of a variety of art-recognized techniques. In addition to activating PEG using one of the previously described methods, one or both of the terminal alcohol functionalities of the PEG molecule can be modified to allow for different types of conjugation to a nucleic acid. For example, converting one of the terminal alcohol fiunctionalities to an amine or a thiol allows access to urea and thiourethane conjugates. Other functionalities include, e.g., maleimides and aldehydes. [00412] In rnany applications, it is desirable to cap the PEG molecule on one end with an essentially non-reactive moiety so that the PEG molecule is mono-functional (or mono activated). In the case of protein therapeutics, which generally display multiple reaction sites for activated PEGs, horno bi-functional activated PEGs lead to extensive cross-linking, yielding poorly functional aggregates. To generate mono-activated PEGs, one hydroxyl moiety on the terminus of the PEG diol molecule typically is substituted with a non-reactive methoxy end moiety, -OCI 13. In this embodiment, the polymer can be represented by MeO-CH2C-1 2 0 (CI-l 2

CH

2

O),CH

2

CH

2 -OI and is commonly referred to as "mPEG", where n typically ranges from 4 to 10,000. [004131 The other, un-capped terminus of the PEG molecule typically is converted to a reactive end moiety that can be activated for attachment at a reactive site on a surface or a molecule, such as a protein, peptide or oligonucleotide. [004141 In some cases, it is desirable to produce a hetero bi-functional PEG reagent, where one end of the PEG molecule has a reactive group, such as an N-hydroxysuccinimide or nitrophenyl carbonate, while the opposite end contains a maleimide or other activating group. In these embodiments, two different functionalities, for example, amine and thiol, may be conjugated to the activated PEG reagent at different times. 103 WO 2012/109675 PCT/US2012/024916 PHARMACEUTICAL COMPOSITIONS [004151 The invention also includes pharmaceutical compositions comprising an aptamer that binds to TFPI. In some embodiments, the compositions include a therapeutically effective amount of a pharmacologically active TFPI aptamer or a pharmaceutically acceptable salt thereof, alone or in combination, with one or more pharmaceutically acceptable carriers or diluents. [004161 The compositions may comprise one or more TFPI aptamers. For example, the compositions may comprise ARC 19499. Alternatively, the compositions may comprise ARC19882. Alternatively, the compositions may comprise AR-C19499 and another TFPI aptamer. In embodiments where the composition includes at least two aptamers that can be the same aptamer or two different aptamers, the aptamers may, optionally, be tethered or otherwise coupled together. Preferably, the compositions comprise ARC19499, either alone or in combination with another TFPI aptamer. Alternatively, the compositions comprise a TFPI aptamner in combination with another agent. Preferably, the compositions comprise ARC1 9499 in combination with another agent. 1004171 As used herein, the terms pharmaceuticallyy acceptable salt" refers to salt forms of the active compound that are prepared with counter ions that are non-toxic under the conditions of use and are compatible with a stable formulation. Examples of pharmaceutically acceptable salts of TFPI aptamers include hydrochlorides, sulfates, phosphates, acetates, ftmarates, maleates and tartrates. 1004181 The terms pharmaceuticallyy acceptable carrier", "pharmaceutically acceptable medium" or "pharmaceutically acceptable excipient", as used herein, means being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Pharmaceutically acceptable carriers are well known in the art. Examples of pharmaceutically acceptable carriers can be found, for example, in Goodman and Gillmans, The Pharmacological Basis of Therapeutics, latest edition. [004191 The pharmaceutical compositions will generally include a therapeutically effective amount of the active component(s) of the therapy, e.g., a TFPI aptamer of the invention that is dissolved or dispersed in a pharmaceutically acceptable carrier or medium. Examples of preferred pharmaceutically acceptable carriers include, but are not limited to, physiological saline solution, phosphate buffered saline solution, and glucose solution. However it is 104 WO 2012/109675 PCT/US2012/024916 contemplated that other pharmaceutically acceptable carriers may also be used. Examples of other pharmaceutically acceptable media or carriers include any and all solvents, dispersion media, coatings. antibacterial agents, antifungal agents, isotonic and absorption delaying agents and the like. Pharmaceutically acceptable carriers that may be used in the compositions include, but are not limited to, ion exchangers, aluniina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethyiene-polyoxypropylene- block polymers, polyethylene glycol and wool fat. The use of such rnedia and agents for pharmaceutically active substances is well known in the art. [00420] The pharmaceutical compositions may also contain pharmaceutically acceptable excipients, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts, or buffers for modifying or maintaining pi, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution or absorption of the formulation. For solid compositions, excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate and the like. 1004211 The pharmaceutical compositions are prepared according to conventional mixing, granulating or coating methods, and typically contain 0.1% to 99.9%, for example, 0.1% to 75%, 0.1% to 50 %, 0.1% to 25%, 0.1% to 10%, 0.1 to 5%, preferably 1% to 50%, of the active component. [00422] The formulation of pharmaceutical compositions is known to one of skill in the art. Typically, such compositions may be formulated as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection; as tablets or other solids for oral administration; as time release capsules for slow release formulations; or in any other form currently used, including eye drops, creams, lotions, salves, inhalants and the like. The compositions may also be formulated as suppositories, using for example, polyalkylene glycols as the carrier. In some embodiments, suppositories are prepared from fatty emulsions or suspensions. The use of sterile formulations, such as saline-based 105 WO 2012/109675 PCT/US2012/024916 washes, by surgeons, physicians or health care workers to treat a particular area in the operating field may also be particularly useful. [004231 The compositions may be formulated as oral dosage forms, such as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions. For instance, for oral administration in the form of a tablet or capsule (e.g., a gelatin capsule), the active drug component can be coined with an oral, non-toxic, pharmaceutically acceptable carrier, such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include starch, magnesium aluimfinum silicate, starch paste, gelatin, methylcellulose, sodiurn carboxymethyicellulose and/or polyvinylpyrrolidone, natural sugars, such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethylene glycol and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum starches, agar, alginic acid or its sodium salt, or effervescent mixtures and the like. Diluents, include, e. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine. 1004241 Pharmaceutical compositions can also be formulated in liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids containing cholesterol, stearylamine or phosphatidyleholines. In some embodiments, a film of lipid components is hydrated with an aqueous solution of drug to a form lipid layer encapsulating the drug, as described in U.S. Pat. No. 5,262,564. For example, the aptamers described herein can be provided as a complex with a lipophilic compound or non-immunogenic, high molecular weight compound constructed using methods known in the art. Additionally, liposomes may bear aptamers on their surface for targeting and carrying cytotoxic agents internally to mediate cell killing. An example of nucleic acid associated complexes is provided in U.S. Patent No. 6,011,020. [004251 The compositions of the invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxyethylaspan ami dephenol, or 106 WO 2012/109675 PCT/US2012/024916 polyethyleneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compositions of the invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross linked or amphipathic block copolymers of hydrogels. [004261 The compositions of the invention may also be used in conjunction with medical devices. [004271 The quantity of active ingredient and volume of composition to be administered depends on the host animal to be treated. Precise amounts of active compound required for administration depend on the judgment of the practitioner and are peculiar to each individual. [004281 A minimal volume of a composition required to disperse the active compounds is typically utilized. Suitable regimes for administration are also variable, but would be typified by initially administering the compound and monitoring the results and then giving further controlled doses at further intervals. ADMINISTRATION 1004291 The compositions may be administered to a vertebrate, preferably a rnammal, and more preferably a human. The terms "patient" and "subject" are used interchangeably throughout the application, and these terns include both human and veterinary subjects. 1004301 In embodiments where the TFP1 aptamers are antagonist aptamers, the TFPI aptamer compositions provided herein are administered to subjects in an amount effective to inhibit, reduce, block or otherwise modulate TFPI-mediated inhibition of blood coagulation. The TFPI aptamer compositions may completely or partially inhibit, reduce, block or otherwise modulate TFPI-mediated inhibition of blood coagulation. The TFPI aptaners are considered to inhibit or otherwise modulate TFPI activity when the aptamers cause an increase in thrombin generation (such as, for example, peak thrombin, endogenous thrombin potential or lag time) over hemophilic plasma that is equivalent to at least 1-2% of factor replacement. [004311 The compositions may be administered by numerous routes of administration. Such routes of administration include, but are not limited to, oral routes; topical routes, such as intranasally, vaginally or rectally; and parenteral routes, such as intravenous, subcutaneous, intradermal, intramuscular, intraarticular and intrathecal administration. Suitable routes of administration may also be used in combination, such as intravenous administration followed by 107 WO 2012/109675 PCT/US2012/024916 subcutaneous administration. The route of administration, however, is determined by the attending physician. Preferably, the formulations are administered intravenously. Most preferably, the formulations are administered subcutaneously. [004321 Oral dosage forms may be administered as tablets, capsules, pills, powders, granules. elixirs, tinctures, suspensions, syrups or emulsions. [004331 Topical dosage forms include creams, ointments, lotions, aerosol sprays and gels for intranasal vehicles, inhalants or transdermal patches. [004341 Parenteral dosage forms include pre-filled syringes, and solutions and iyophilized powders that are reconstituted prior to administration. [004351 The dosage regimen utilizing the aptamers of the invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular aptarner or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine arid prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. [00436] The pharmaceutical compositions may be administered using various treatment regirnens. For example, the compositions may be administered as a maintenance therapy at a defined dose for a defined period of tirne, such as when a patient is riot suffering frorn a bleeding episode. Alternatively, the compositions may be administered on demand, i.e., as needed, such as when a patient is suffering from a bleeding episode. In a further alternative embodiment, the compositions may be administered as a combination of maintenance therapy and on demand therapy. In such an embodiment, the compositions may be administered as a maintenance therapy at a defined dose for a defined period of time until a bleed occurs, in which case the dosage of the compositions would be increased on an as needed basis until the bleeding stopped, at which point the dosage of the compositions would be decreased back to the prior maintenance level. In another such embodiment, the compositions may be administered as a maintenance therapy at a defined dose for a defined period of time until a bleed occurs, in which case another bleeding disorder therapy would be administered to the patient (such as Factor VIII) until the bleeding stopped, at which point the other bleeding disorder therapy would be discontinued. During this entire time, the compositions would continue to be administered as a maintenance therapy. In yet another such embodiment, the compositions may be administered as a 108 WO 2012/109675 PCT/US2012/024916 maintenance therapy at a defined dose for a defined period of time until a bleed occurs, in which case the dosage of the compositions would be decreased and another bleeding disorder therapy would be administered to the patient (such as Factor ViII) until the bleeding stopped, at which point the dosage of the compositions would be increased back to the prior maintenance level and the other bleeding disorder therapy would be discontinued. In another such embodiment, another bleeding disorder therapy (such as Factor VIII) may be administered as a maintenance therapy at a defined dose for a defined period of time until a bleed occurs, in which case the compositions would be administered to the patient until the bleeding stopped, at which point therapy with the compositions would be discontinued. During this entire time, the other bleeding disorder therapy would continue to be administered as a maintenance therapy. In yet another such embodiment, another bleeding disorder therapy (such as FVI) may be administered as a maintenance therapy at a defined dose for a defined period of time until a bleed occurs, in which case the dosage of the other bleeding disorder therapy would be decreased and the compositions would be administered to the patient until the bleeding stopped, at which point the dosage of the other bleeding disorder therapy would be increased back to the prior maintenance level and therapy with the compositions would be discontinued. INDICATIONS [00437] The compositions are used to treat, prevent, delay the progression of or ameliorate tissue factor pathway inhibitor (TFPI)-mediated pathologies, including the treatment of bleeding disorder pathologies involving TFPn-mediated inhibition of blood coagulation. The pathologies to be treated, prevented, delayed or ameliorated are selected from the group consisting of coagulation factor deficiencies, congenital or acquired, mild or moderate or severe, including hemophilia A (Factor ViII deficiency), hemophilia B (Factor IX deficiency) and hemophilia C (Factor XI deficiency); hemophilia A or B with inhibitors; other factor deficiencies (V, VII, X, XIII, prothrombin, fibrinogen); deficiency of a2-plasmin inhibitor; deficiency of plasminogen activator inhibitor 1; multiple factor deficiency; functional factor abnormalities (e.g., dysprothrombinemia); joint hemorrhage (hemarthrosis), including, but not limited to, ankle, elbow and knee; spontaneous bleeding in other locations (muscle, gastrointestinal, mouth, etc.); hemorrhagic stroke; intracranial hemorrhage; lacerations and other hemorrhage associate with trauma; acute traumatic coagulopathy; coagulopathy associated with cancer (e.g., acute promyelocytic leukemia); von Willebrand's Disease; disseminated intravascular coagulation; 109 WO 2012/109675 PCT/US2012/024916 liver disease; menorrhagia; thrombocytopenia and hemorrhage associated with the use of anticoagulants (e.g., vitamin K antagonists, FXa antagonists, etc.). [004381 The compositions may also be administered prior to, during and/or after a medical procedure. For example, the pharmaceutical compositions may be administered in conjunction (before, during and/or after) with medical procedures, such as: prophylaxis and/or treatment associated with bleeding caused by dental procedures, orthopedic surgery including but not limited to arthroplasty (e.g., hip replacement), surgical or radionuclide synovectomy (RSV), major surgery, venipuncture, transfusion and amputation. THERAPEUTIC RATIONALE [004391 Without wishing to be bound by theory regarding mechanism of action, the following therapeutic rationale is offered by way of example only. [00440] Inhibitors of tissue factor pathway inhibitor (TFPI) would be expected to enhance the generation of thrombin via the tissue factor/Factor VIa pathway (also known as the extrinsic pathway). In a normal individual, activation of the extrinsic pathway stimulates initiation of the thrombin generation response, resulting in a small amount of activated thrombin. Following initiation, this pathway is rapidly deactivated by the inhibitory action of TFPI. Subsequent propagation of the thrombin generation response depends upon thrombin-mediated feedback activation of the intrinsic pathway, which includes Factor VIII (FVIII) and Factor IX (FIX). Propagation is necessary to generate a sufficiently large quantity of thrombin to catalyze the formation of a stable clot. Individuals with a deficiency of either Factor VIII (hemophilia A) or Factor IX (hemophilia B) have an impaired propagation response. Individuals with a severe deficiency (<1V%) cannot produce thrombin via the intrinsic pathway that is dependent on these proteins. This condition results in the inability to produce sufficient thrombin to have adequate platelet activation, fibrin generation and stable clot formation. However, these individuals have an intact extrinsic pathway. Inhibition of TFPI could permit continuation of the initiation response and enable propagation to occur via the extrinsic pathway, permitting sufficient thrombin generation to partially or completely replace the deficient intrinsic pathway and thus reduce bleeding risk. Individuals with mild or moderate deficiencies in these factors, who are also at risk for increased bleeding, may also benefit from the enhanced blood coagulation caused by TFPI inhibition. In addition, in patients with normal intrinsic and extrinsic pathways who are bleeding for other reasons, such as trauma, TFPI inhibition may provide a hemostatic stimulus 110 WO 2012/109675 PCT/US2012/024916 that could control bleeding. Patients with other deficiencies of clotting factors, platelet deficiencies, and vascular defects associated with bleeding, might also benefit from a treatment that would inhibit TFPL COMBINATION THERAPY [004411 One embodiment of the invention comprises a TFPI aptamer or a salt thereof or a pharmaceutical composition used in combination with one or more other treatments for bleeding diseases or disorders, such as other procoagulant factors or other inhibitors of coagulation cascade regulatory molecules. The pharmaceutical compositions may also be administered in combination with another drug, such as: activated prothrombin complex concentrates (APCC), Factor Eight Inhibitor Bypass Agent (FEIBA") recombinant Factor Vila (e.g., Novoseven", recombinant Factor VIII (Advate*, Kogenate" Recombinate", iHelixate*, ReFacto"), plasma derived Factor VII (I Humate P* I lemofiI M*), recombinant Factor IX (BeneFIX*), plasma derived Factor IX (Bebulin VIH", Konyne*, Mononine*), cryoprecipitate, desnopressin acetate (DDAVP), epsilon-aminocaproic acid or tranex amic acid. Alternatively, the pharmaceutical compositions may be administered in combination with another therapy, such as: blood or blood product transfusion, plasmapheresis, immune tolerance induction therapy with high doses of replacement factor, inunune tolerance therapy with immunosuppressive agents (e.g., prednisone, rituximab) or pain therapy. In general, the currently available dosage forms of the known therapeutic agents and the uses of non-drug therapies for use in such combinations will be suitable. 1004421 "Combination therapy" (or "co-therapy") includes the administration of a TFPI aptamer and at least a second agent or treatment as part of a specific treatment regimen intended to provide the beneficial effect from the co-action of these therapeutic agents or treatments. The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agent or treatments. Administration of these therapeutic agents or treatments in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected). [004431 Combination therapy may, but generally is not, intended to encompass the administration of two or more of these therapeutic agents or treatments as part of separate monotherapy regimens that incidentally and arbitrarily result in the combinations of the

III

WO 2012/109675 PCT/US2012/024916 invention. Combination therapy is intended to embrace administration of the therapeutic agents or treatments in a sequential manner. That is, wherein each therapeutic agent or treatment is administered at a different time, as well as administration of these therapeutic agents or treatments, or at least two of the therapeutic agents or treatments, in a substantially simultaneous manner, Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single injection having a fixed ratio of each therapeutic agent or in multiple, single injections for each of the therapeutic agents, [004441 Sequential or substantially simultaneous administration of each therapeutic agent or treatment can be effected by any appropriate route including, but not limited to, topical routes, oral routes, intravenous routes, subcutaneous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The therapeutic agents or treatments can be administered by the same route or by different routes. For example, a first therapeutic agent or treatment of the combination selected may be administered by injection while the other therapeutic agents or treatments of the combination may be administered subcutaneously. Alternatively, for example, all therapeutic agents or treatments may be administered subcutaneously or all therapeutic agents or treatments may be administered by injection. The sequence in which the therapeutic agents or treatments are administered is not critical unless noted otherwise. 100445] Combination therapy also can embrace the administration of the therapeutic agent or treatments as described above in further combination with other biologically active ingredients. Where the combination therapy comprises a non-drug treatment, the non-drug treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agent and non-drug treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non-drug treatment is temporally removed from the administration of the therapeutic agent, perhaps by days or even weeks. REVERSAL AGENTS [004461 The invention further relates to agents that reverse the effects of the TFPI aptamers, referred to herein as "TFPI reversal agents", The agent can be any type of molecule, such as a protein, antibody, small molecule organic compound or an oligonucleotide. [004471 Preferably, a TFPI reversal agent is a nucleic acid that is 10-15 nucleotides in length. However, there are no limits to the length of the reversal agent. 112 WO 2012/109675 PCT/US2012/024916 [004481 Preferably, a TFPI reversal agent binds to a TFPI aptamer. A TFPI reversal agent may bind to a full length TFPI aptamer or a fragment thereof. Such binding may be through ionic interactions, covalent bonding, complementary base pairing, hydrogen bonding, or any other type of chemical bond. Preferably, such binding is via complementary base pairing. Without wishing to be bound by theory, a TFPI reversal agent acts by hybridizing to a TFPI aptamer, thereby disrupting the TFPI aptamer's secondary and tertiary structure and preventing the binding of the TFPI aptamer to TFPI. By preventing the binding of the TFPI aptamer to TFPI, the effect of the binding interaction, e.g., therapeutic effect, and/or stimulation or inhibition of the molecular pathway, can be modulated, providing a means of controlling the extent of the binding interaction and the associated effect. [004491 A TFPI reversal agent may be a ribonucleic acid, deoxyribonucleic acid or mixed ribonucleic and deoxyribonucleic acid. Preferably, a TFPI reversal agent is single stranded. Preferably, a TFPI reversal agent comprises all 2'-) Methyl residues and a 3'-inverted deoxythymidine. However, a TFPI reversal agent may contain any nucleotides, modified or unmodified, along with any other 3' or 5' modifications that may be found on aptaners. [00450] Examples of TFPI reversal agents include, but are not limited to: SEQ ID NO: 15, which is ARC23085; SEQ ID NO: 16, which is ARC23087; SEQ ID NO: 17, which is ARC23088; and SEQ ID NO: 18, which is ARC23089. 1004511 Preferably, the TFPI reversal agent is a nucleic acid comprising the structure set forth below: mA-mGii-mC-mC-mA-mA-mi-mU-mA-mnU-mA-mU-mU-'mC-mC (SEQ ID NO: 15), wherein "mN" is a 2'-O Methyl containing residue (which is also known in the art as a 2'-OMe , 2' methoxy or 2'-OCH3 containing residue). [004521 Alternatively, the TFPI reversal agent is a nucleic acid comprising the structure set forth below: mU-mA-mU-mA-mU -mA-mC-mG-mC-mA-mC-mC-mU-mA-mA (SEQ ID NO: 16). wherein "mN" is a 2'-O Methyl containing residue. [004531 Alternatively, the TFPI reversal agent is a nucleic acid comprising the structure set forth below: mC-mU-mA-mA-mC-mG-mA-mG-mC-mC (SEQ ID NO: 17), wherein "mN" is a 2'-O Methyl containing residue, 113 WO 2012/109675 PCT/US2012/024916 [004541 Alternatively, the TFPI reversal agent is a nucleic acid comprising the structure set forth below: mC-mA-mC-mC-mU-mA-mA-mC-mG-mA-mG-mC-mC-mA-mA (SEQ ID NO: 18), wherein "mN" is a 2'-O Methyl containing residue. [004551 The chemical name of ARC23085 is 2'-OMe-adenyiyl-(3'->5')- 2'-OMe-guanylyl (3'->5')- 2'-OMe-eytidylyl-(3'-45')- 2'-OMe-cytidylyl-(3'-+5')- 2'-OMe-adenylyl-(3'-+5') 2'-OMe-adenylyi-(3'-45')- 2'-OMe-guanvyll-(3'->5')- 2'-OMe-uracylyi-(3'-45')- 2'-OMe adenyiyl-(3'->5')-2'-OMe-uracylyi-(3'->5')- 2'-OMe-adenylyl-(3'-+5')- 2'-OMe-uracylyl (3'->5')- 2'-OMe-uracylyl-(3'->5')- 2'-OMe-cytidyiyl-(3'->5')- 2'-OMe-cytidylyl. [004561 The chemical name of ARC23087 is 2'-OMe-uriacylyl-(3'-+5 ')- '2'-OMe-adenylyl (3'->5')- 2'-OMe-uracyiyi-(3'->5')- 2'-OMe-adenyIyi-(3'-45')- 2'-OMe-uracylyl-(3'-+5')- 2' OMe-adenylyl-(3 '-+5')- 2 '-OMe-cytidylyl-(3 '-->5')- 2'-OMe-guanylyl-(3' ->5')- 2'-OMe cytidylvl-(3 '-+5')- 2'-OMe-adenylyl-(3'--+5')- 2'-OMe-cytidylyl-(3'--+5')- 2'-OMe-cytidylyl (3' -5 ')- 2'-OMe-uracylyl-(3 '-+-5')- 2'-OMe-adenylyl-(3 '-->5')- 2'-OMe-adeniyl. [004571 The chemical narne of ARC23088 is 2'-OMe-cvtidylyl-(3 '-+-5')- 2'-OMe-uracylyl (3' ---5 ')- 2'-OMe-adenylyl-(3 '-+-5')- 2'-OMe-adenylyl-(3 '-->5')- 2'-OMe-cytidylyl-(3 '-+-5')- 2' OMe-guanylyl-(3 '-+-5')- 2'-OMe-adenylyl-(3 '-+-5')- 2'-OMe-guanylyi-(3'-+5')- 2'-OMe cytidylyl-(3 '-->5')- 2'-OMe-cytidylyl. 1004581 The chemical name of ARC23 089 is 2'-OMe-cytidylyl-(3 '-5')- 2'-OMe-adenylyl (3' --+5')- 2'-OMe-cytidylyl-(3 '-+5')- 2'-OMe-cytidylyl-(3' --+5')- 2'-OMe-uracylyl-(3'-+5')- 2' OMe-adenylyl-(3 '-+-5')- 2'-OMe-adenylyl-(3'-+-5')- 2'-)Me-cytidylyl-(3'-+-5')- 2'-OMe guanylyl-(3'-+-5')- 2'-)Me-adenylyl-(3 '-+-5')- 2'-OMe-guanylyl-(3'-+5')- 2'-OMe-cytidylvl (3'--5')- 2'-OMe-cytidylyl-(3'--5')- 2 '-OMe-adenylyl-(3'-+5')- 2'-OMe-adenylyl. [004591 The invention also includes TFPI reversal agents that have 70% identity or more to any one of SEQ ID NOs: 15, 16, 17 or 18. For example, the TFPI reversal agents may have 70. 75. 80, 85, 90, 95 or 100% identity to one of SEQ ID NOs: 15, 16, 17 or 18. [004601 The invention also includes pharmaceutical compositions containing TFPI reversal agents that bind to TFPI aptamers. In some embodiments, the compositions include an effective amount of a pharmacologically active TFPI reversal agent or a pharmaceutically acceptable salt thereof, alone or in combination, with one or more pharmaceutically acceptable carriers. The compositions may contain one or more different TFPI reversal agents. The TFPI reversal agents 114 WO 2012/109675 PCT/US2012/024916 are administered to subjects in an amount effective to reverse the therapeutic effect of the TFPI aptamer. The compositions may be administered by numerous routes of administration, such as, for example, topically, intranasally or parenterally. The dosage regimen for a TFPI reversal agent will depend on a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration, the renal and hepatic function of the patient; the amount of TFPI aptainer used to treat a patient; and the particular TFPI reversal agent or salt thereof employed. An ordinary skilled physician or veterinarian can readily determine and prescribe the effective amount of the TFPI reversal agent required to reverse the therapeutic effect of a TFPI aptamer. [004611 The invention further includes agents that neutralize the hemostatic activity of the TFPI aptamer. Such an agent may bind to TFPI and prevent its inhibition by the TFPI aptamer, or the agent may inhibit downstream coagulation factors (e.g., FXa or thrombin) in a manner that counteracts the hemostatic activity of the TFPI aptarner. Such an agent may include, but is not limited to, anticoagulants, such as unfractionated heparin or low molecular weight heparin. A study by Wesselschmidt (Wesselschmidt el al., "Structural requirements for tissue factor pathway inhibitor interactions with factor Xa and heparin", Blood Cioaguil Fibrinolysis, vol. 4, pp. 661-669 (1993)) shows that heparin binds to TFPI through interactions with the K1 and C terminal domains, which could interfere either directly or indirectly with the ability of TFPI aptamers to bind to TFPJ. Moreover, in the same study, heparin-binding was observed to facilitate the FXa inhibitory activity of TFPI, which would tend to further counteract the hemostatic activity of TFPI aptamers. Finally, heparin is well known to inhibit thrombin, FXa and other coagulation factors through an antithrombin-dependent mechanism. These activities could neutralize the ability of TFPI aptamers to stimulate thrombin generation and clot formation. In the event that the hemostatic effects of TFPI aptamers were to induce thrombosis, one of these agents could be administered to arrest its progression. An ordinary skilled physician or veterinarian can readily determine and prescribe the effective amount of the anticoagulant or other neutralizing agent required to reverse the hemostatic effect of a TFPI aptamer. KITS [004621 'The pharmaceutical compositions may also be packaged in a kit. 'The kit will comprise the composition, along with instructions regarding administration of the TFPI aptamer. The kit may also comprise one or more of the following: a syringe or pre-filled syringe, an 115 WO 2012/109675 PCT/US2012/024916 intravenous bag or bottle, a vial, the same TFPI aptamer in a different dosage form or another TFPI aptamer. For example, the kit may comprise both an intravenous formulation and a subcutaneous formulation of a TFPI aptamer of the invention. Alternatively, the kit may comprise lyophilized TFPI aptamer and an intravenous bag of physiological saline solution or phosphate buffered saline solution. The kit form is particularly advantageous when the separate components must be administered in different dosage forms (i.e., parenteral and oral) or are administered at different dosage inteials. The kit may further comprise a TFPI reversal agent, along with instructions regarding administration of the reversal agent. The kit may contain both an intravenous formulation and a subcutaneous formulation of the TFPI reversal agent. Alternatively, the kit may contain lyophilized TFPI reversal agent and an intravenous bag of solution. [00463] Preferably, the kits are stored at 5+3 0 C. The kits can also be stored at room temperature or frozen at -20 0 C. REGULATING TFPI [00464] The invention also provides a method for regulating TFPI in which a molecule binds or otherwise interacts with one or more portions of TFPI, wherein at least one portion is outside of the K] and K2 domains of TFPI, such as the K3/C terminal region. The molecule can be any type of molecule, such as, for example, a small molecule organic compound, an antibody, a protein or peptide, a nucleic acid, a siRNA, an aptamer, or any combination thereof. Preferably, the molecule is a small molecule organic compound. More preferably, the molecule is an antibody. Most preferably, the molecule is an aptamer. For example, the molecule may bind to or otherwise interact with a linear portion or a conformational portion of TFPL A molecule binds to or otherwise interacts with a linear portion of TFPI when the molecule binds to or otherwise interacts with a contiguous stretch of amino acid residues that are linked by peptide bonds. A molecule binds to or otherwise interacts with a conformational portion of TFPI when the molecule binds to or otherwise interacts with non-contiguous amino acid residues that are brought together by folding or other aspects of the secondary and/or tertiary structure of the polypeptide chain. Preferably, the molecule binds at least in part to one or more portions of mature TFPI (for example, Figure 3A) that are selected from the group consisting of: amino acids 148-170. amino acids 150-170. amino acids 155-175. amino acids 160-180, amino acids 165-185, amino acids 170-190, amino acids 175-195, amino acids 180-200, amino acids 185 116 WO 2012/109675 PCT/US2012/024916 205, amino acids 190-210. amino acids 195-215. amino acids 200-220. amino acids 205-225. amino acids 210-230, amino acids 215-235, amino acids 220-240, amino acids 225-245, amino acids 230-250, amino acids 235-255. amino acids 240-260, amino acids 245-265. amino acids 250-270, amino acids 255-275, amino acids 260-276, amino acids 148-175, amino acids 150 175, amino acids 150-180, amino acids 150-185, amino acids 150-190, amino acids 150-195, amino acids 150-200, amino acids 150-205, amino acids 150-210, amino acids 150-215, amino acids 150-220, amino acids 150-225, amino acids 150-230, amino acids 150-235, amino acids 150-240, amino acids 150-245, amino acids 150-250, amino acids 150-255, amino acids 150 260, amino acids 150-265, amino acids 150-270, amino acids 150-275, amino acids 1 50-276, amino acids 190-240, amino acids 190-276, amino acids 240-276, amino acids 242-276, amino acids 161-181, amino acids 162-181, amino acids 182-240, amino acids 182-241, and amino acids 182-276. The molecule preferably comprises a dissociation constant for human TFPI, or a variant thereof, of less than 100 pM, less than 1 IM, less than 500 nM, less than 100 nM, preferably 50 nM or less, preferably 25 nM or less, preferably 10 nM or less, preferably 5 nM or less, more preferably 3 nM or less, even more preferably I nM or less, and most preferably 500 pM or less. [00465] Many modifications and variations of the invention can be made without departing front its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. 1004661 All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The invention having now been described by way of written description, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples below are for purposes of illustration and not limitation of the claims that follow. 117 WO 2012/109675 PCT/US2012/024916 EXAMPLES 1004671 In the examples, one or more of the following TFP1 aptamers were used to conduct the various experiments. ARC26835 is the aptamer described in SEQ ID NO: 1. ARC 17480 is the aptamer described in SEQ ID NO: 2. ARC 19498 is the aptamer described in SEQ ID NO: 3. ARC19499 is the aptamer described in SEQ ID NO: 4. ARC19500 is the aptamer described in SEQ ID NO: 5. ARC19501 is the aptamer described in SEQ ID NO: 6. ARC26835 is the core aptamer sequence for each of ARCI17480, ARC19498, ARC19499, ARC19500 and ARC19501. ARC31301 is the aptamer described in SEQ ID NO: 7. ARC18546 is the aptamer described in SEQ ID NO: 8. ARC19881 is the aptamer described in SEQ ID NO: 9. ARC19882 is the aptamer described in SEQ ID NO: 10. ARC31301 is the core aptamer sequence for each of ARC18546, ARC19881 and ARC19882. EXAMPLE 1 [004681 This example demonstrates how ARCI 9499 was generated. [004691 In vitro selection experiments were performed using a pool of modified oligonucleotide molecules, each of which contained dC, mA, mG and iUl residues, and recombinant human tissue factor pathway inhibitor (TFPI), which was obtained from American Diagnostica (catalog #4500PC, Stamford, CT). Iterative rounds of selection for binding to TFPI, followed by amplification, were performed to generate ARC14943, an 84 nucleotide long precursor to ARC19499. ARC14943 was minimized from 84 nucleotides to 32 nucleotides (ARC26835) using computational folding prediction programs and systematic deletion. Appendage of a 3'inverted deoxythymidine residue to ARC26835 resulted in ARC 17480, a 33 nucleotide long molecule. Addition of a 5'-hexylamine group to ARCi 7480 resulted in ARC19498. This molecule was then PEGylated via the 5'-hexylamine group with a 40 kDa PEG moiety to result in ARC19499. EXAMPLE 2 1004701 This example demonstrates that ARC17480 binds tightly to TFPI in vitro in a dot-blot binding experiment, in both the absence and presence of competitor tRNA. [004711 Radiolabeled ARC 17480 was incubated with different concentrations of TFPI. ARC 17480 bound to TFPI was then captured on a nitrocellulose filter membrane. The ratio of radiolabeled ARC 17480 bound to the nitrocellulose filter over total radiolabeled ARC 17480 added was determined and plotted as the percentage ofARC17480 bound as a function of protein 118 WO 2012/109675 PCT/US2012/024916 concentration. An example of an ARC 17480/TFPI binding plot is shown in Figure 12A. The data were fit to models for monophasic and biphasic aptamer-protein binding. This experiment was repeated eleven times and Kos using both monophasic and biphasic binding models were determined for each data set. The mean Ku determined using a monophasic fit was 4.0 - 1.5 nM and using a biphasic fit was 1 .7 ± 0.7 nM. Both monophasic and biphasic fits to the data assume different models for the interaction of ARC 7480 to TFPI, although the fits in and of themselves do not explicitly support either binding model. Regardless of the model used to fit the data, the

K

0 determined for binding of ARC] 7480 to TFPI was essentially the same. When the Kns determined from both the monophasic and biphasic fits were taken into consideration, the mean

K

0 of ARCi17480 binding to TFPI was 2.9 + 1.6 nM. This mean K 0 does not assume a mode of binding interaction between ARC] 7480 and TFPI and, as such, is the most robust determination of the binding interaction between the aptamer and the protein. ARC17480 maintained binding to human TFPI in the presence of tRNA, indicating that the binding was specific. A shift in binding affinity of ARC 1480 to TFPI was observed in the presence of 0.1 mg/mL tRINA with a mean KD of 42 i: 12 nM. An example plot of ARC 17480 binding to TFPI in both the presence and absence of tRNA is shown in Figure 12B. EXAMPLE 3 1004721 This example demonstrates that unlabeled ARC?] 480, ARC19498, ARC19499, ARC26835, AR C19500, ARC19501, ARC31301, ARC18546, ARC19881 and ARC19882 compete with radiolabeled ARC17480 for binding to TFPI. This example also demonstrates that all of these aptamers have affinities for TFPI that are similar to that observed with ARC 17480. 1004731 Each aptamer was evaluated for binding to TFPJI in a binding-competition assay. For these experiments, human TFPI (American Diagnostica, Stamford, CT, catalog #4500PC) was incubated with trace amounts of radiolabeled ARC 17480 and different concentrations of unlabeled competitor aptamer (5000 nM-0.25 nM for all aptamers except ARC19499; 1000 nM 0.05 nM for ARC19499). For experiments with ARC17480 and ARC19499 in Figure 13A, 60 nM TFPI was used. For all other experiments (Figure 13B-E), 10 nM TFPI was used. ARC 17480 was included as a competitor in every experiment as a control. For each aptamer, the percentage of radiolabeled ARC 17480 bound at each competitor aptamer concentration was used for analysis. The percentage of radiolabeled ARC7480 bound was plotted as a function of aptamer concentration and fit to the equation y=(nax/(i + x/IC 5 o)) + int, where y=the percentage 119 WO 2012/109675 PCT/US2012/024916 of radiolabeled ARC 17480 bound, x=the concentration of aptamer, max=the maximum radiolabeled ARC 17480 bound, and int=the y-intercept, to generate an IC 50 value for binding competition. Figure 13A-E shows graphs of competition experiments with ARC 17480, ARC19498. ARC19499, ARC26835, ARC19500, ARC19501, ARC31301, ARC18546, ARC19881 and ARC] 9882. These molecules all competed similarly with radiolabeled ARC17480 for binding to TFPI. These experiments demonstrate that ARC 17480, ARC19498, ARC19499, ARC26835, ARCi 9500, ARC9501, ARC3 1301, ARC1 8546, ARC1 9881 and ARC1 9882 all bind similarly to full-length TFPI. EXAMPLE 4 [004741 This example demonstrates that the TFPI aptamers bind specifically to TFPI. [004751 In this experiment, ARC] 7480 was tested for binding to a variety of proteins that are key molecules in the coagulation cascade, molecules whose inhibition would show a similar profile to TFPI inhibition, or molecules that are similar in structure or function to TFPI. Proteins investigated were TFPI, Factor Va (FVa), Factor XII (FXII), antithrombin (ATIII), heparin cofactor II (11CI), alpha-thrombin, prothrombin, Factor VIa (FVIa), Factor IXa (FIXa), Factor Xa (FXa), Factor XIa (FXIa), kallikrein, plasmin, alpha-i antitrypsin (serpin-Al), TFPI-2, and GST-TFPI-2. In the presence of up to 0.5-1 gM of each protein, ARCI 7480 did not have any significant affinity for the sequence- and mechanistically-related proteins tested (Figure 14A-D). ARC17480 showed some binding to FXII at higher concentrations of protein. This binding was eliminated in the presence of 0.1 mg/mL tRtNA (Figure 14D), indicating that the binding was likely non-specific. 1004761 These experiments indicate that any effects on coagulation that are mediated by the TFPI aptamers are likely due to direct binding and inhibition of TFPI. EXAMPLE 5 [004771 This example demonstrates that ARC19499 binds tightly to TFPI in a plate-based binding assay. This example also demonstrates that ARC 19498 binds tightly to TFPI due to its competition with ARC 19499 for binding to TFPI in a plate-based binding assay. [004781 In order to assess the binding affinity of ARC 19499 to TFPI, recombinant human TFPI protein (0.5 mg/nL) was diluted in Dulbecco's Phosphate-buffered Saline (DPBS) to a final concentration of 15 pg/mL, and 100 pL was added to a 96-well Maxisorb plate and incubated overnight at 4 'C. The TFPI solution was then removed and the plate was 120 WO 2012/109675 PCT/US2012/024916 subsequently washed 3 times with 200 PL wash buffer (DPBS + 0.05% Tween 20) at room temperature. The plate was then blocked with 200 pL of 10 mg/mL bovine serum albumin (BSA) in DPBS for 30 minutes at room temperature. The BSA blocking solution was then removed and the plate was washed again 3 times with 200 piL wash buffer. Serially diluted ARC 19499 in DPBS with 0.1% BSA was then added to the plate and incubated for 3 hours at room temperature. After washing 3 times with 200 paL wash buffer, 100 pL of 0.5 pg/miL rabbit monoclonal anti-PEG antibody (Epitomics) was added to the plate and incubated for 60 minutes at room temperature. The anti-PEG antibody solution was then removed and the plate was washed as described above. Then, 100 pL anti-rabbit IgG-HRP secondary antibody (Cell Signaling Technology), diluted 1000-fold in assay buffer, was added to each well and incubated for 30 minutes. After washing 3 times with 200 pL wash buffer, 100 pL of TMB solution (Pierce) was added to each well and incubated for 2 minutes before adding 100 pL stop solution (2N H 2S04) to each well to stop the reaction. The assay plate was then read at 450 nm using a Victor 3 V 1420 multilabel counter (Perkin Elmer). Five different binding experiments suggested that the binding affinity between ARC 19499 and recombinant TFPI is 30 nM in the plate-based binding assay. The data from one of these experiments is shown in Figure 15. [004791 In order to assess the affinity of ARC 19498 towards TFPI protein, an ARC19499:TFPI binding competition assay was set up. Recombinant human TFPI protein (0.5 mg/mL) was diluted in DPBS to a final concentration of 15 gg/nL, and 100 p.L was added to a 96-well Maxisorb plate and incubated overnight at 4 'C. The TFPI solution was then removed and the plate was subsequently washed 3 times with 200 L wash buffer (DPBS + 0.05% Tween 20) at room temperature. The plate was then blocked with 200 pL of 10 mg/mL BSA in DPBS for 30 minutes at room temperature. The BSA blocking solution was then removed and the plate was washed again 3 times with 200 iiL wash buffer. ARC19498 was serially diluted and mixed at different concentrations with 20 nM ARC19499 in DPBS in 0.1% BSA. The ARC19498:ARC19499 mixtures were added to the plate and incubated for 3 hours at room temperature. After washing 3 times with 200 pL wash buffer, 100 L of 0.5 pg/mL rabbit monoclonal anti-PEG antibody (Epitomics) was added to the plate and incubated for 60 minutes at room temperature. The anti-PEG antibody solution was then removed and the plate was washed as described above. Then, 100 pL anti-rabbit IgG-HRP secondary antibody (Cell 121 WO 2012/109675 PCT/US2012/024916 Signaling Technology), diluted 1000-fold in assay buffer, was added to each well and incubated for 30 minutes. After washing 3 times with 200 piL wash buffer, 100 pL of TMB solution (Pierce) was added to each well and incubated for 2 minutes before adding 100 PL stop solution (2N 11 2 SO4) to each well to stop the reaction. The assay plate was then read at 450 nm using a Victor 3 V 1420 multi label counter (Perkin Elmer). The percent inhibition of ARC 9499 binding was calculated using 0 nM ARC] 9498 in 20 nM ARC 9499 as 0% inhibition, and 0 nM ARC] 9498 and 0 rnM ARC] 9499 as 100% inhibition. The ICO was calculated based on 4 parameter logistics using Graphpad Prism 4 software. Figure 16 shows two replicates of this experiment, both of which gave an IC 0 o of 20 nM for ARC19498 competition with ARC19499 in this assay. These results suggest that ARC 19498 has a binding affinity for TFPI in the plate based binding assay that is similar to that observed for ARC 19499. EXAMPLE 6 1004801 This example examines the regions on TFPI where ARC 17480 binds. Dot blot binding experiments were carried out with radiolabeled ARC 17480 and various truncated TFPI proteins, and binding-competition experiments were carried out with radiolabeled ARC 17480, TFPI, and heparin or low molecular weight heparin (LMWH). The proteins used for binding experiments are described in Table I below. [004811 Trace amounts of radiolabeled ARC 17480 were incubated with different concentrations (500 nM-0.7 nM) of full-length TFPI and TFPI-His (Table 1). Figure 17A shows that ARC 17480 had reduced binding to TFPI-His when compared to its binding to full-length TFPI. This experiment suggested that the C-terminal 20 amino acids of TFPI, which are missing in TFPI-His but present in full-length TFPI, contribute to binding of ARC 17480 to TFPI. [004821 Trace amounts of radiolabeled ARC17480 were incubated with different concentrations of truncated TFPI-K I K2 (500 nM-0,008 nM) and the K3-C-terninal domain protein (500 nM-0.7 nM) (Table 1). Figure 17B shows that ARC17480 had no detectable binding to truncated TFPI-KI K2 and very weak binding to the K3-C-terminal domain protein that was only detectable at higher concentrations of the protein. Trace amounts of radiolabeled ARC 1480 were incubated with different concentrations of the C-terminal peptide (10 pM-0.17 nM) (Table 1). Neutravidin (-100 nM monomer) was then added to the binding solution to assist in the capture of aptamer:peptide complexes on a nitrocellulose filter. The amount of radiolabeled aptamer captured on a nitrocellulose filter was quantitated and compared to the total 122 WO 2012/109675 PCT/US2012/024916 amount of radiolabeled aptamer to generate a binding curve, which is shown in Figure 17B, ARC 17480 showed weak binding to the C-terminal peptide at high concentrations of peptide. [004831 Trace amounts of radiolabeled ARC 17480 were incubated with different concentrations of full-length TFPi (500 nM-0.008 nM) in the absence or presence of 0.1 mg/nIL unfractionated heparin (Figure 18A). The inclusion of heparin in the binding experiment completely abolished ARC17480 binding to TFPl. In a separate experiment, trace amounts of radiolabeled ARC17480 were incubated with 12.5 nM full-length TFPI and different concentrations (5 pM-0.25 nM) of unfractionated heparin or low molecular weight heparin (LMWH). Figure 18B shows that both unfractionated heparin and LMWH competed with ARC 17480 for binding to TFPI in a concentration-dependent manner. Heparin was a more effective competitor than LMWH. The K3-C-terminal regions of TFPI have been implicated in glyocalyx binding, and this is the region of the protein where heparin and LMWH should bind. These experiments suggest that the K3-C-terminal region of TFPI is important for ARC17480 binding to TFPI. [004841 Taken together, these experiments demonstrate that the C-terminal domain likely participates in ARC17480 binding to TFPI. These experiments also demonstrate that the ARC]17480 binding region on TFPI is not entirely contained within the K-1-K2 region of the protein, or within the K3-C-terminal region of the protein. The regions required for ARC17480 binding likely spans more than one domain of the protein. Table 1: Proteins used for binding experiments Protein Description Amino acids of mature TFPI Full-length TFPJ American Diagnostica, E. coli expressed 1-276 R&D systems. murine myeloma cell line T PI-Hi_ _ ______ _expresed 1256 _ _C-_2 terminalI 0-is tag_ Truncated TFPI- American Diagnostica, E. coli expressed KIK2 1-161 K3-C-terminal E. cob expressed N terminal 6- His tag + 182 domain 276 G-Ienmln Ipetd ytei N-terminal hiotin +242-2761 EXAMPLE 7 [004851 This example examines the regions on TFPI where ARC1 7480 and ARC19499 bind. For these experiments, antibodies that bind to different regions on TFPJ were used to compete for binding to TFPI with ARC1 9499 in a plate-based binding assay, or to compete for binding to 123 WO 2012/109675 PCT/US2012/024916 TFPI with ARC 17480 in a dot-blot binding assay. The antibodies used for competition are shown in T'able 2 below. [004861 This example demonstrates that antibody AD4903 (American Diagnostica, catalog #4903) competed for binding of ARC19499 to TFPI in a plate-based binding assay and competed for binding of ARC17480 to TFPI in a dot blot-binding assay (Figure 19A and Figure 20C). Antibody AD4903 was raised against a fragment of TFPI containing K1 domain amino acid residues 22-87, and binds to TFPI somewhere in this region (Table 2). This example also demonstrates that antibody ACJK-4, which was raised against a peptide that contained amino acid residues 148-162 that are part of the intervening region between the K2 and K3 domains of TFPI, competed weakly with ARC17480 for binding to TFPI in a dot-blot binding assay (Figure 20B). This example also demonstrates that antibodies ACJK-1 and ACJK-2, which were raised against peptides that contained arnino acid residues 261-276 and 245-262, respectively, that are part of the C-terminal domain of TFPI, partially competed for ARCI 9499 binding to TFPI in a plate-based binding assay (Figure 1913). This example further demonstrates that several other antibodies that bind to different regions of TFPI did not compete with ARC 19499 binding to TFPI in a plate-based binding assay, and did not compete with ARC 1480 for binding to TFPI in a dot blot-based binding assay. The antibodies used for the competition experiments are shown in Table 2 below. 1004871 For plate-based binding experiments, 400 ng/well of TFPI (American Diagnostics, cat# 4900PC) in 100 uL Dulbecco's Phosphate-buffered Saline (DPBS) was used to coat a 96 well Maxisorb plate at 4 C. The TFPI solution was then removed and the plate was subsequently washed 3 times with 200 oL wash buffer (DPBS + 0.05% Tween 20) at room temperatnre. The plate was then blocked with 200 IlL of 10 mg/mL bovine serum albumin (BSA) in DPBS for 30 minutes at room temperature. The BSA blocking solution was then removed and the plate was washed 3 times with 200 iL wash buffer. The competing antibodies were serially diluted and mixed with ARCI 9499 at the final concentration of 25 nM ARC19499 and 0.1% B3SA in DPBS, and the mixture was then added to the assay plate and incubated for 3 hours at room temperature. ARCI 9498 was similarly mixed with ARC19499 and used as a positive control in the antibody competition assay. Wells were then washed, as described above. For experiments using antibodies AD4903, AD4904 and 7035-AO1 for competition, 100 pL of 0.5 ug/mL rabbit mnonoclonal anti-PEG antibody (Epitomics, cat # 2061-1) in assay buffer was 124 WO 2012/109675 PCT/US2012/024916 added to the plate and incubated for 3 hours at room temperature. Anti-PEG antibody solution was then removed and the plate was washed as described above, followed by addition of 100 piL of 1:1 000-diluted anti-rabbit IgG--LRP secondary antibody in assay buffer to each well (Cell Signaling Technology, cat # 7074) and incubated for 30 minutes. The secondary antibody solution was removed and the plate was washed, as described above. For antibodies ACJKl ACJK5, 0.5 pg/niL of 100 piL biotinylated rabbit monoclonal anti-PEG antibody (Epitomics, cat # 2173) in assay buffer was added to the assay plate and incubated for 3 hours at room temperature, followed by washing, as described above. The antibody was then removed and the plate was washed as described above, followed by addition of 100 pL of streptavidin-IRP (4800-30-06) from R&D Systems (Minneapolis, MN) diluted 200-fold in DPBS and incubated for an additional 1 hour at room temperature. The streptavidin-HRP was then removed and the plate was washed as described above. Then 100 uL of TMIB solution (Pierce, #34028) solution was added to each well and incubated for 2 minutes, followed by addition of 100 pL stop solution (2N 11 2

SO

4 ) to each well to stop the reaction. The assay plate was then read at 450 nm using a Victor3V 1420 multilabel counter (Perkin Elmer). Percent inhibition of binding was calculated using 0 nM antibody in 25 nM ARC19499 as 0% inhibition, and 0 nM antibody and 0 nM ARC19499 as 100% inhibition. The IC 50 was calculated based on 4-parameter logistics using Prism 4 Graphpad software. 1004881 As shown in Figure 19A, antibody AD4903, which binds within the Ki domain of TFPI, competed with ARC 19499 for binding to recombinant TFPI in the plate-based binding assay. Antibodies ACJK-1 and ACJK-2. which bind to regions within the C-terminal region of TFPI, partially competed for ARC19499 binding to TFPI in the plate-based binding assay (Figure 19B). -Antibodies AD4904. ACJK-3, ACJK-4, ACJK-5 and 7035-AOl showed no competition for ARC19499 binding to TFPI in the plate-based binding assay (Figure 19A and B). These experiments suggest that the Ki region and the C-terminal region of TFPI are involved in ARC 19499 binding to TFPI. [004891 The antibodies in Table 2 were also tested in a dot blot-based competition binding assay. In these experiments, trace amounts of radiolabeled ARC 17480 were incubated with 10 nM recombinant TFPI, with or without the addition of antibody. Antibodies were tested at 1000 nM, 333 nM, Ill nM, 37.0 nM, 12.4 nM, 4.12 nM, 1,37 nM, 0.46 nM, 0.15 nM and 0.051 nM. ARC 17480 was included as a competitor in every experiment as a control. For each molecule, 125 WO 2012/109675 PCT/US2012/024916 the percentage of radiolabeled ARC 17480 bound at each competitor aptamer concentration was used for analysis. The percentage of radiolabeled ARC 17480 bound was plotted as a function of aptamer concentration and fit to the equation y=(max/(i + x/1C 0 )) + int, where y=the percentage of radiolabeled ARC 17480 bound, x=the concentration of aptamer, max=the maximum radiolabeled ARC17480 bound, and int=the y-intercept, to generate an ICo value for binding competition. Figure 20 shows the binding-competition experiments carried out with ACJK-1, ACJK-2, ACJK-3, ACJK-4, ACJK-5, AD4903 and AD4904. These experiments demonstrate that antibody AD4903 competed for ARC 17480 binding to TFPI in the dot blot competition assay (Figure 20C), ACJK-4 partially competed for binding in this assay (Figure 20B), while ACJK-l, ACJK-2, ACJK-3, ACJK-5 and AD4904 showed no significant competition for binding with ARC1 7480 (Figure 20A-C). These experiments suggest that the K1 region and the K2-K3 intervening regions of TFPI are involved in ARC17480 binding to TFPL. [00490] Taken together, these experiments demonstrate that the K1 region of TFPI is likely involved in ARCI17480/ARC 19499 binding to TFPI. These experiments also suggest that the C terminal and the K2-K3 intervening regions of TFPI may be involved in aptamer binding. Lack of binding competition of antibodies to other regions of TFPI does not preclude their involvement in aptamer binding. Table 2: Antibodies used in ARC 19499 competition assays 1Region of mature TFPi Type used as an antigen for Antibody target region Antibody antibody generation in TFPI AD4903 nouse monoclonal 22-87 K1 region AD4904 mouse monociora 88-160 K2 region ACJK-1 rabbit polyclonal 261-276 C-terminal peptide AC)K-2 _ r__bit, polycloal 245-62__C-erminaleprie_ ACJK-3 rabbit polyclonal 192-204 K3 peptide K2-K3 intervening ACJK-4 rabbit polyclonal 148-162 peptide ACJK-5 Irabbit polyclonal 54-69 K1 peptide _ 2AO1 ___En Ouse polyelonal__j___g1_____g~~eg tein 7035Al IfOSpOylii 152-252 K.3--r-erinal regiont EXAMPLE [00491] This example demonstrates that ARC19499 has in vitro activity inhibiting TFPI in the extrinsic tenase (Xase) inhibition assay, 126 WO 2012/109675 PCT/US2012/024916 [004921 In this assay, tissue factor ('IF) was mixed with Factor VIla (FVIIa) and phospholipid vesicles. Factor X (FX) was added and aliquots were removed and quenched at various time points. At this point, a chromogenic substrate for Factor Xa (F'Xa) was added and absorbance at 405 nm was measured over a course of an hour in order to determine rates of FXa generation. When I nM TFPI was included, the rate of FXa generation was significantly decreased. This was seen in Figure 21A when comparing the filled diamonds (no TFPI) with the empty circles (1 nM TFPi). When increasing concentrations of ARC19499 were also included along with the 1 nM TFPI, there was a dose-dependent improvement on the rate of FXa generation. 1000 nM ARC19499 (empty diamonds) resulted in rates of FXa generation that were close to the rate of FXa generation in the absence of TFPI (filled diamonds) (Figure 21A). These rates were normalized by dividing the rate at one specific time point (in this case, at 4 minutes) by the rate achieved with no TFPI at that same tirne point (Figure 21B). In this manner, TFPI reduced the rate of FXa generation at 4 minutes by nearly 70%. Increasing concentrations of ARC19499 improved this rate, reaching levels close to that of no TFPI with 10-1000 nM aptamer (Figure 2113). [00493] This experiment indicates that ARC 19499 inhibits TFPI in an in vitro extrinsic tenase inhibition assay. EXAMPLE 9 1004941 This example demonstrates that ARC26835, ARC17480, ARC1 9498, ARC 19499. ARC19500, ARC19501, ARC31301, ARC18546, ARC19881 and ARC19882 have TFPI inhibitory activity in the Factor Xa (FXa) activity assay. 1004951 Each aptamer was evaluated for inhibition of TFPI in a Factor Xa (FXa) activity assay. The ability of FXa to cleave a chromogenic substrate was measured in the presence and absence of TFPI, with or without the addition of aptamer. For these experiments, 2 nM human FXa was incubated with 8 nM human TFPI. Then, 500 piM chromogenic substrate and aptamers were added, and FXa cleavage of the substrate was measured by absorbance at 405 nm (A 40 5 ) as a function of tirne. Aptamers were tested at 500 nM, 125 nM, 31.25 nM, 7.81 nM, 1.95 nM and 0.49 nM concentrations. ARC 17480 was included as a control in each experiment. For each aptamer concentration, the A 405 was plotted as a function of time and the linear region of each curve was fit to the equation y=mx + b, where y=A 40 5 , x:the aptamer concentration, m=-the rate of substrate cleavage, and b=the y-intercept, to generate a rate of FXa substrate cleavage. The 127 WO 2012/109675 PCT/US2012/024916 rate of FXa substrate cleavage in the presence of TFPI and the absence of aptamer was subtracted from the corresponding value in the presence of both TFPI and aptamer for each aptamer at each concentration. Then, the adjusted rates were plotted as a function of aptamer concentration and fit to the equation y=(Vmax/( 1 + ICso/x)), where y=the rate of substrate cleavage, x=concentration of aptamer, and Vmax=the maximum rate of substrate cleavage, to generate a IC 0 s and maximum (Vmax) value. Figure 22A-C show graphs of FXa activity assays with ARC26835, ARC 17480, ARC19498, ARC 19499, ARC19500, ARC19501, ARC3 1301, ARC] 8546, ARC19881 and ARC19882. These aptam ers all inhibited TFPI in these assays, as evidenced by an increase in FXa activity as a function of aptamer concentration. These aptamers all had similar activity in the FXa assay. EXAMPLE 10 [00496] This example demonstrates that ARC19499 protects Factor Xa (FXa) from inhibition by TFPI in a chromogenic assay with purified components. [00497] FXa (I nM), TFPI (2.5 nM), ARC19499 (0-500 nM) and Spectrozyme Xa (American Diagnostica) chrornogenic substrate (200 pM) were incubated in HEPES-buffered saline (20 mM IEPES, 150 mM NaCl, pH1 7.4) containing 2 mM CaCk and 0.1% PEG-6,000 (HBSP2 buffer) at 37 C until equilibrium was achieved (5 minutes). The rate of Spectrozyme FXa hydrolysis was determined using a ThermoMax instrument (Molecular Devices) and plotted as the %FXa activity compared to no TFPI (100%). Increasing concentrations of ARC19499 caused an increase in FXa activity (Figure 23), demonstrating that ARCi 9499 protected FXa from the inhibition by TFPI. Based on this data, the apparent dissociation constant (Ku) of ARCI 9499 for TFPI was 1.8 nM. ARC19499 is specific for TFPI and did not inhibit FXa activity in the absence of TFPI (data not shown). EXAMPLE 11 [004981 This example demonstrates that ARC 19499 protects the extrinsic FXase complex, which is composed of tissue factor, Factor Vila (FVIIa) and Factor Xa (FXa), from inhibition by TFPI in a chromogenic activity assay with purified components. [004991 Relipidated tissue factor (TF; 20 pM), FVIIa (1 nM), PCPS vesicles (75% phosphatidyl choline/25% phosphatidyl shrine; 20 pM) and ARC19499 (0-1000 nM) were incubated in HBSP2 buffer at 37 'C for 10 minutes, followed by the simultaneous addition of FX (1 p M) and TFPI (2.5 nM). Aliquots were removed every 30 seconds for 5 minutes and 128 WO 2012/109675 PCT/US2012/024916 quenched into HBS buffer containing 20 mM EDTA and 0.1% PEG. Spectrozyme FXa substrate (200 pM) was added, the rate of substrate hydrolysis was measured, and the concentration of active FXa was estimated from a calibration curve. Increasing concentrations of ARC19499 caused an increase in FXa activity (Figure 24) tip to 70% of the rate measured in the absence of TFPI, demonstrating that ARC19499 substantially protected the extrinsic FXase complex from the inhibition by TFPI. EXAMPLE 12 [00500] This example demonstrates that ARC19499 protects the tissue factor:FVIIa complex from TFPI inhibition in a fluorogenic assay of tissue factor:FVIIa activity carried out with purified components. [005011 Tissue factor (TF; 1 nM), FVIla (2 nM) and ARCI 9499 (0-7.5 nM) were incubated in H-lBSP2 at 37 0 C for 10 minutes, followed by the simultaneous addition of a fluorogenic substrate SN-17c (50 pM) and TFPI (8 nM). The rate of substrate hydrolysis was measured in a fluorescence plate reader (BioTek). TFPI inhibited approximately 50% of TF:FVIIa activity under these conditions (Figure 25). The addition of a stoichiornetric concentration of ARC19499 (8 nM) completely restored full TF:FVIIa activity compared to a no TFPI control (Figure 25), demonstrating that ARC19499 efficiently protected the TF:FVTa complex from TFPI inhibition. A titration of increasing ARC1 9499 concentrations in the presence of 8 nM TFPI increased the activity of the TF:FVlIa complex in an ARC 19499 concentration-dependent manner, reaching the nid-point of activity at ~1 nM ARC 19499. Data analysis indicated that the apparent KJ) of ARC19499 for TFPI in this assay was 1.2 nM. ARC19499 is specific for TFPI and did not inhibit TF:FVIIa activity in the absence of TFPI (data not shown). EXAMPLE 13 [005021 This example demonstrates that ARC19499 inhibits TFPI in a synthetic coagulation proteome that models hemophilia A and hemophilia B. These data show that ARC19499 restored normal thrombin generation in the presence of complete (0%) Factor VIII (FVIII) or Factor IX (FIX) deficiency. ARC19499 also restored normal thrombin generation in the presence of incomplete (2%, 5% or 40%) FVIII deficiency. [005031 Thrombin generation was initiated with 5 pM relipidated tissue factor (TF) added to a mixture of procoagu plants and coagulation inhibitors (Factors V, VII, Vlla, VIII, IX, X, XI, prothrombin, antithronbin and TFPI; all at mean physiologic concentrations) and 50 AM PCPS 129 WO 2012/109675 PCT/US2012/024916 (75% phosphatidyl choline/25% phosphatidyl shrine). Thrombin generation over time was measured in a chromogenic assay using the Spectrozyme TH substrate (American Diagnostica). ARC 19499 was tested at increasing concentrations of I nM, 2.5 nM, 5 nM and 10 nM in a fully reconstituted system (healthy control) or in reconstituted systems in which either FVIII (severe hemophilia A) or FIX (severe hemophilia B) was omitted. [00504] In the presence of all proteins at their mean physiologic concentrations ("healthy control"; Figure 26), the initiation (lag) phase of thrombin generation initiated with 5 pM relipidated TF was approximately 6 minutes, ad the maximum concentration of active thrombin observed was 270 nM (filled diamonds). The omission of TFPI significantly shortened the initiation phase (to 2 minutes) and increased maximum thrombin concentration to 374 nM (filled circles). Additions of increasing ARC19499 concentrations in the presence of 2.5 nM TFPI decreased the duration of the initiation phase and increased maximum thrombin concentration in an ARC 1 9499-dependent rnainer, and at 10 rnM (empty squares) the ARC 19499 thrombin generation profile was almost identical to that observed in the absence of both TFPI and ARC1 9499 (Figure 26). [00505] In the absence of FVI II, TF initiated thrombin generation was significantly suppressed (Figure 27). The initiation phase was extended from 6 minutes in the "healthy control" (filled diamonds) to 10 minutes in hemophilia A (empty diamonds), and maximum thrombin activity decreased from 270 nM to 34 nM. The omission of TFPI in the absence of FVII I restored normal thrombin generation (maximum concentration of active thrombin increases to 264 nM) and the duration of the initiation phase decreased to 2 minutes (filled circles). In the absence of FVIII and in the presence of 2.5 nM TFI, the addition of 5 nM ARC 19499 (asterisks) restored thrombin generation to the level observed in the "healthy control" with the initiation phase of 3 minutes. In the presence of 10 nM ARC 19499 (empty squares) and 2. nMTFPI and in the absence of FVIII. the thrombin generation profile became similar to that observed in the absence of TFPL FVIII and ARC 19499. [005061 The effect of TFPI inhibition by ARC 19499 in a hemophilia B (no FIX) synthetic coagulation proteome was similar to that observed for the hemophilia A model, i., ARC19499 at 5 nM concentration and in the absence of FIX restored thrombin generation to the level similar to that observed in "healthy control" (Figure 28). 130 WO 2012/109675 PCT/US2012/024916 [005071 The effect of ARC 19499 on TF-initiated thrombin generation was evaluated in the synthetic coagulation proteome model at 0%, 2% (0.014 nM), 5% (0.035 nM), 40% (0.28 nM) and 100% (0.7 nM) FViiiL The selected FVIII concentrations covered the range observed in severe (<1%), moderate (1-5%) and mild (5-40%) hemophilia A patients. In the absence of ARC19499 (Figure 29), thrombin generation was suppressed at all FVIii concentrations tested, up to and including 40%. The peak thrombin level observed in the 40% FVIII proteome (asterisks) was approximately 50% of the"healthy control" (filled diamonds). The addition of ARC19499 at a concentration of I nM was sufficient to significantly boost thrombin generation by shortening the initiation phase and increasing peak thrombin levels (Figure 30). The addition of 2.5 nM ARC1 9499 essentially normalized thrombin generation in the presence of 0-5% FVIii (Figure 31), while at 40% and 100% FVIII 2 5 nM ARC19499 induced further shortening of the initiation phase and increased peak thrombin, nearly to the extent of the "No TFPI" control. 1005081 Figure 32 shows additional synthetic coagulation proteome data for 0% FVIII in the presence of a series of ARC19499 concentrations (0, 1, 2.5, 5 and 10 nM) compared to a "healthy control" and a "No TFPI" control. Figure 33 shows the data for 100% FVIII for the same range of ARC 19499 concentrations. Figures 34, 35 and 36 show the data for 2%, 5% and 40% FVIII in the presence of 0, 1 and 2.5 nM AR C19499, respectively. Under all conditions, ARC 19499 showed a significant procoagulant response, causing the initiation phase (lag time) to decrease and the peak thrombin to increase. In all cases of FVIII deficiency (0-40% FVIII), ARC 19499 was able to restore a normal thrombin generation profile. EXAMPLE 14 1005091 This example demonstrates that in vitro activity of ARC19499 is specific for the presence of TFPL [005101 In this experiment, the ability of ARC 19499 to affect thrombin generation in the calibrated automated thrombogram (CAT) assay, which measures the generation of thrombin over time following initiation of the tissue factor coagulation pathway, was tested in three different plasma conditions. In the first condition, increasing concentrations of ARC 19499 were added to pooled normal plasma (PNP) and mixed with a solution containing tissue factor (TF) and phospholipids so that the TF concentration was either 0.1 or 1.0 pM in the final reaction volume (Figure 37). Thrombin generation was initiated by the addition of a mixture containing calcium chloride and a fluorogenic substrate for thrombin. The reaction took place at 37 'C, and 131 WO 2012/109675 PCT/US2012/024916 fluorescence intensity was measured periodically over 1 hour. ARC 19499 was tested at the following concentrations in the plasma: 0.1, 1, 10, 100 and 1000 nM. [005111 With either TF concentration, increasing ARC 19499 increased the generation of thrombin in PNP plasma (Figure 37A-B), Both the endogenous thrombin potential (ETP area under the curve) and peak thrombin (highest level of thrombin produced at any one point in the assay) values increased in a dose-dependent manner with ARC19499 (Figure 37)C-D). The lag time (time it takes for thrombin generation to begin) decreased in a dose-dependent manner with ARC1 9499 (Figure 37E). These results were observed at both concentrations of TF. [005121 The CAT assay measuring ARC19499 activity was repeated in TFPI-depleted plasma. Plasma that was immunodepleted for TFPI and lyophilized was obtained from American Diagnostica (Stamford, CT) and resuspended prior to use. Thrombin generation was measured as described above with 0.01, 0.1 or 1.0 pM TF. The results in Figure 38A show that the thrombin generation cunes measured for each TF concentration were distinct frorn each other, but within a specific TF concentration there was essentially no difference in thrombin generation as the ARC19499 concentration was increased. This was also seen in the parameters measured in the CAT assay. There was little or no change in ETP, peak thrombin or lag time as the ARC19499 concentration increased (Figure 38B-D), independent of TF concentration. [00513] ARC19499 activity was tested in a third set of plasma conditions. In this case, PNP was incubated with a polyclonal antibody against TFPI, in order to neutralize all TFPI activity. ARC19499 was then added to this antibody-treated plasma (Figure 39). Again, thrombin generation was initiated with either 0.01, 0.1 or 1.0 pM 'FE. Addition of the polyclonal antibody enhanced thrombin generation at all three TF concentrations because the TFPI was neutralized; however, increasing concentrations of ARC 19499 appeared to cause no further increases in thrombin generation (Figure 39A-C). There was little to no effect on the ETP, peak thrombin or lag time when ARC 19499 was added (Figure 39D-F), [005141 These experiments indicate that ARC19499 only has procoagulant activity when functioning TFPI is present in the plasma, and therefore, ARC 19499 is specific for TFPI. EXAMPLE 15 [005151 'This example demonstrates that ARC17480. ARC19498 and ARC19499 inhibit TFPI activity in vitro. 132 WO 2012/109675 PCT/US2012/024916 [005161 In this experiment, the inhibitory activity of TFPI aptamers (ARC 17480, ARC 19498 and ARC 19499) were measured in vitro in pooled hemophilia A (Factor VIII-deficient) plasma in a calibrated automated thrombogram (CAT) assay. Aptamers were titrated at different concentrations in pooled hemophilia A plasma, and the amount of thrombin generated was compared to a pooled normal plasma control (Figure 40A-C), using 1.0 pM TF in the final reaction. Both the endogenous thrombin potential (ETP), which is the area under the thrombin generation versus time curve, and the peak throibin, which is the highest concentration of thrombin generated over the course of the experiment, provided indirect measures of aptamer inhibition of TFPI. All three aptamers had similar activity in this assay, with ARC19499 having slightly higher activity than the other two. ARC 19499 corrected the ETP to near normal levels by 30 nM (Figure 40D). Peak thrombin levels also increased with increasing concentrations of aptamer (Figure 40E). [005171 These results show thatARC1480, ARC19498 and ARC19499 inhibit TFPI activity in vitro. EXAMPLE 16 [00518] This example demonstrates that ARC] 9499 increases thrombin generation in normal human plasma treated with an anti-Factor VIII antibody to generate a hemophilia A-like state. [00519] Platelet-poor plasma from a normal, healthy volunteer was treated with an anti-FVIII antibody to generate a hemophilia A-like state. Thrombin generation in this antibody-treated plasma was similar to that observed with hemophilia A plasma (Figure 41). Addition of ARC 19499 to the antibody-treated plasma resulted in a dose-dependent increase in thrombin generation. These results demonstrate that ARC19499 can correct thrombin generation in plasma with low FViII levels that results from treatment with an anti-F'VIII antibody. EXAMPLE 17 [005201 'This example demonstrates that ARC 17480 and ARC 19499 inhibit TFPI activity in vitro and have biological activity. [005211 The effects of the non-PEGylated core TFPI inhibitory aptamer ARC 17480 and the PEGylated aptamer ARC 19499 were evaluated for in vitro thrombin generation activity in hemophilia B (FIX-deficient) plasma using the calibrated automated thrombogram (CAT) assay. [005221 These studies were performed in plasma pooled from two hemophilia B patients with <1% Factor IX levels (commercially available from George King Bio-Medical, Inc, Overland 133 WO 2012/109675 PCT/US2012/024916 Park, KS). In this assay, plasma and aptarner were mixed together and added to a reagent containing phospholipids and tissue factor. Thrombin generation was initiated by the addition of a mixture containing calcium chloride and a fluorogenic substrate for thrombin. The reaction took place at 37 'C, and fluorescence intensity was measured periodically over 1 hour. The final concentrations of tissue factor and phospholipids were 1 pM and 4 pM, respectively. Aptamers were tested at the following concentrations in the plasma: 0.3, 1, 3, 10, 30, 100, 300 and 1000 nM. Individual thrombin generation curves are shown in Figure 42, illustrating the effects of increasing concentrations of ARC19499 (Figure 42A) or AR C17480 (Figure 42B) on the extent of thrombin generation in hemophilia B plasma compared to pooled normal plasma. The plot of ETP, peak thrombin and lag time are shown in Figure 43. Results with ARC19499 are plotted on the left side, and results with ARC17480 are plotted on the right side. [00523] The ETP and peak thrombin levels decreased ~-85% and ~95%, respectively, in plasma from the hemophilia B pool compared to the pooled normal plasma, consistent with a deficiency in thrombin generation due to the loss of Factor IX. Both ARC17480 (triangles) and ARC19499 (diamonds) largely corrected the defect in thrombin generation, as measured by both of these parameters (Figure 43). By 100 rM, both aptarners demonstrated an ETP nearly equivalent to that achieved with pooled normal plasma, and a nearly equivalent peak thrombin by 300 nM ARC17480 (Figure 43). Peak thrombin plateaued by 300 nM ARC19499. ARC 17480 and ARC 19499 decreased the lag time in hemophilia B plasma, below what was achieved with pooled normal plasma and hemophilia B plasma without any drug (Figure 43). 1005241 These results show that AR 17480 and ARC 19499 inhibit TFPI with similar potency in hemophilia B plasma in vitro. EXAMPLE 18 [005251 This example demonstrates that ARC19499 inhibits TFPI activity in vitro and has biological activity compared to a negative control aptamer. [005261 The ability of ARC 19499 to enhance thrombin generation was tested in three platelet poor hemophilia plasmas: plasma pooled from 7-8 patients with severe hemophilia A (<1% FVIII levels; referred to as "hemophilia A plasma"), plasma from three different hemophilia A patients with high titers of anti-FVIII antibodies (>160 Bethesda units (BU)/mL; referred to as "inhibitor plasma"), and plasma pooled from two patients with severe hemophilia B (<1% FIX levels; referred to as "hemophilia B plasma"). All plasmas were from George King Bio-Medical 134 WO 2012/109675 PCT/US2012/024916 (Overland Park, KS). Thrombin generation was measured using the calibrated automated thrombogram (CAT) assay. In this assay, plasma and aptamer were mixed together and added to a reagent containing phospholipids and tissue factor. Thrombin generation was initiated by the addition of a mixture containing calcium chloride and a fluorogenic substrate for thrombin. The reaction took place at 37 C, and fluorescence intensity was measured periodically over 1 hour. The final concentrations of tissue factor and phospholipids were 1 pM and 4 M, respectively. Thrombin generation in the presence of ARCi9499 (0.3, 1, 3, 10, 30, 100, 300 and 1000 nM ) was compared to negative control aptamer (0,1, 1, 10, 100 and 1000 nIM). Plots of ETP, peak thrombin and lag time (mean ± s.e.m.) are shown in Figure 44. [005271 Hemophilia A plasma had a slightly shorter lag time and a markedly decreased ETP and peak thrombin (~50% and -75%, respectively) compared to normal plasma. Increasing concentrations of ARC19499 largely corrected the defect in thrombin generation. ETP was corrected to near-normal levels with 3 nM ARC1 9499, and peak thrombin was corrected with 100 nM aptamer (Figure 44A). Inhibitor plasma also had decreased ETP and peak thrombin (-50% and -70%, respectively) compared to normal plasma. As with the severe hemophilia A plasma, ARC19499 increased thrombin generation in this plasma. With 30 nM ARC19499, both the ETP and peak thrombin were at normal levels (Figure 44B). Hemophilia B had an even greater defect in thrombin generation, with significantly decreased ETP and peak thrombin (0% and -90%, respectively) and an increased lag time. As with the hemophilia A plasmas, increasing concentrations of ARC 19499 improved thrombin generation in this plasma, achieving normal ETP levels with 30 nM ARC 19499, and normal peak thrombin levels with 100-300 nM aptamer (Figure 44C). Taken together, these results demonstrate that 30-100 nM ARC 9499 is effective in restoring coagulation in three different types of hemophilia plasma. A negative control aptamer was also tested in the three different plasmas and demonstrated no correction of thrombin generation (Figure 44). [005281 The sequence of the negative control aptamer, ARC32603, used in this example was: mG-mG-mA-mA-mU -mA-mU-mA-dC-mU-nmU-mG-mG-dC-mU-mG-dC-mU-niU-mA-mG mG-mU-mG-dC-mG-mU-mA-mU -mA-mU -mA- 3 T (SEQ ID NO: 15z). 135 WO 2012/109675 PCT/US2012/024916 EXAMPLE 19 [005291 This example demonstrates that ARC17480. ARC26835, ARC19500, ARC19501. ARC31301, ARC18546, ARC19881 and ARC19882 have biological activity in the calibrated automated thrombogram (CAT) assay. [00530] The TFPI-inhibitory activity of each aptamer was evaluated in the CAT assay in pooled hemophilia A plasma at 500 nM, 167 nM, 55.6 nM, 8.5 nM, 6.17 nM and 2.08 nM aptamer concentration. ARC1 7480 was included in every experiment as a control. For each aptamer, the endogenous thrombin potential (ETP) and peak thrombin values at each aptamer concentration were used for analysis. The ETP or peak thrombin value for hemophilia A plasma alone was subtracted from the corresponding value in the presence of aptamer for each molecule at each concentration. Then, the adjusted ETP and peak values were plotted as a function of aptamer concentration and fit to the equation y=(maxi( + IC 5 o/x)) - int, where y:::ETP or peak thrombin, concentration of aptamer, max:::the maximum ETP or peak thrombin, and int:::the y intercept, to generate an ICO value for both the ETP arid the peak thrombin. Figure 45A-D and Figure 46A-B show graphs of CAT experiments with ARC1 7480, ARC26835, ARC19500, ARC19501, ARC3 1301, A RC18546, ARC19881 and ARCi9882. Both the adjusted endogenous thrombin potential (ETP) and peak thrombin are shown. These experiments demonstrate that ARCI 7480, ARC26835, ARC19500, ARCI9501, ARC3 1301, A RCi8546, ARC19881 and ARC19882 all ftmnctionally inhibited TFPI in the CAT assay, as evidenced by a concentration-dependent increase in both ETP and peak thrombin in hemophilia A plasma. These molecules all have similar activity in the CAT assay. EXAM-PLE 20 [00531] This example demonstrates that the TFPI aptamers have biological activity. [005321 In this experiment, the ability of ARC19499 to affect thrombin generation compared to that of NovoSeven" was tested using the calibrated automated thrombogram (CAT) assay. The CAT assay generates a number of parameters to compare thrombin generation. The lag time is a measure of the length of time that it takes for thrombin generation to begin. Peak thrombin is a measure of the highest amount of thrombin to be generated at any one point. The endogenous thrombin potential (ETP) is the area under the thrombin generation curve. [00533I These studies were performed in the presence of three different plasmas: platelet-poor plasma from healthy volunteers, a pool of plasma from hemophilia A patients with <1% Factor 136 WO 2012/109675 PCT/US2012/024916 VIII levels (commercially available from George King Bio-Medical, Inc, Overland Park, KS), and plasma from hemophilia A patients with a high titer of inhibitor antibody to Factor VIII (commercially available from George King Bio-Medical, Inc, Overland Park, KS). In the CAT assay, plasma and drug (either ARC 19499 or NovoSeven") were mixed together and added to a reagent containing phospholipids and tissue factor. Thrombin generation was initiated by the addition of a mixture containing calcium chloride and a fluorogenic substrate for thrombin. The reaction took place at 37 C, and fluorescence intensity was measured periodically over 1 hour. The final concentrations of tissue factor and phospholipids were 1 pM and 4 gM, respectively. The drugs were tested at the following concentrations in the plasma: 0.3, 1, 3, 10, 30, 100 and 300 nM. [005341 In the plasma from healthy volunteers, there was no change in the ETP over the range of concentrations tested with both ARCI 9499 and NovoSeven" (Figure 47A). The peak thrombin levels increased slightly at the higher doses, with ARC 19499 and NovoSeven" behaving in a nearly identical manner (Figure 4713). ARC19499 had no effect on the lag time of thrombin generation, while NovoSeven* demonstrated a dose-dependent decrease in lag time, reaching a minimum lag time by 30 nM (Figure 47C). [00535] The ETP and peak thrombin levels decreased -40% and ~75%, respectively, in plasma frorn the hemophilia A pool, consistent with a deficiency in thrombin generation due to the loss of Factor VIII. ARC 19499 and N ovoSeven* largely corrected the defect in thrombin generation, as measured by both of these parameters. These agents demonstrated a nearly equivalent effect on ETP, reaching a maximum ETP by 30 nM (Figure 48A). NovoSeven* had a slightly higher effect on peak thrombin reaching a maximal level by 30 nM. ARC19499 reached the same level of peak thrombin by 300 nM (Figure 48B). As seen in the plasma from healthy volunteers. ARC 19499 had no effect on the lag time, while NovoSeven" showed a dose dependent decrease in lag time, reaching a maximum effect by 30 nM (Figure 48C). [005361 Similar results were seen in the plasma from patients with a high tier antibody. Both drugs increased ETP and peak thrombin in the same manner (Figures 49A-B). Again, ARC 19499 had no effect on the lag time, while NovoSeven" showed a dose-dependent decrease of lag time (Figure 49C). Standard error associated with the inhibitor plasma was higher than that seen in the healthy plasma or hemophilia A pool. This was most likely due to the difference in titers between the three inhibitor patients (160 BU/mL, 533 BU/mL and 584 BU/mL). 137 WO 2012/109675 PCT/US2012/024916 [005371 Overall, with the exception of the lag time. ARC 19499 and NovoSeven* had very comparable effects on thrombin generation in all plasmas tested. EXAMPLE 21 [005381 This example demonstrates that the TFPI aptamers have biological activity. [005391 In this experiment, the ability of ARC19499 to affect clot formation compared to that of NovoSeven® was tested using the thromboelastography (TEG") assay. The TEG* assay measures the mechanical properties of a developing clot. In the TEG assay, a cup containing the blood product and any activators oscillates freely around a pin that is attached to a torsion wire. As a clot develops, newly formed fibrin strands connect the oscillating cup to the stationary pin and begin to pull on the pin, thus generating force on the torsion wire. This force is converted to a signal by the computer to monitor clot formation, and is displayed as a tracing of signal height versus time. From this tracing, one can extract a number of parameters to measure various aspects of clot formation. The R-value measures the time that it takes for an initial clot to develop. The angle is a measure of the rate at which the clot forms. The maximum amplitude (MA) is a measure of clot strength and stability. [00540] These studies were performed in citrated whole blood from healthy volunteers. In the first assay, the drugs were tested in untreated whole blood. In the second assay, the blood was first treated with a sheep polyclonal antibody against human Factor VIII for three hours at 37 C prior to drug addition. In both assays, NovoSeven* or ARC 19499 were added to the blood (antibody-treated or not) at final blood concentrations of 0.01, 0.1, 1, 10 or 100 nM. Activation of clotting occurred upon addition of tissue factor (Innovin) at a final dilution of 1:200000 (~ fM) and calcium chloride at a final concentration of 11 mM. [00541] In untreated blood, both ARC19499 and NovoSeven' demonstrated a dose dependent, moderate decrease in R-value that appeared to reach a minimum value by 10 nM. (Figure 50A). The angle and MA values remained unchanged over the concentrations tested (Figure 50B-C). [005421 In the blood treated with Factor VIII antibody, both drugs had similar effects on the R-value. The R-value was prolonged in blood treated with antibody compared to untreated blood. As the ARC19499 or NovoSeven" concentration increased, the R-value was restored to the same level it was in untreated blood (Figure 51A). Antibody treatment decreased the rate of clot formation in the blood, which is reported as the angle. NovoSeven* had a strong effect on 138 WO 2012/109675 PCT/US2012/024916 the angle, increasing it linearly from 0.1 to 100 nM of NovoSeven". This increase surpassed the angle achieved with untreated blood. ARC 19499 also increased the angle, but the value appeared to plateau by 10 nM of aptamer, at a similar level as that achieved with untreated blood (Figure 51B). The effect on MA was minimal with both drugs, primarily because there does not appear to be a large difference in the MA of whole blood, with or without FVIii antibody treatment. Both drugs resulted in MA values that fell between those achieved with untreated blood and those achieved with antibody-treated blood (Figure 5 1 C). [00543] As seen in the CAT assay, ARCI19499 and NovoSeven" had very comparable effects on clot formation in whole blood, whether the blood was lacking Factor VIII or not. The main difference between the two drugs was seen in the effect on the rate of clot formation (angle), with NovoSeven* showing a more linear increase in rate as the concentration increased, but ARC 19499 did increase the rate as well. EXAMPLE 22 [00544] This example demonstiates that the TFPI aptamers have biological activity. [00545] In this experiment, the synergy between ARCi 9499 and Factor VIII in thrombin generation was tested using the calibrated automated thrombogram (CAT) assay. These studies were performed in the presence of a pool of plasma from hemophilia A patients with <1 %/X Factor VIII (FV III) levels (commercially available from George King Bio-Medical, Inc, Overland Park, KS). Increasing concentrations of ARC19499 (from I to 300 nM) were analyzed in the presence of 0, 1.4, 2.5, 5, 14 and 140% Factor VIII (World Health Organization International Standard). The results were compared to the baseline responses for hemophilia A and pooled normal plasmas in the absence of ARC 19499. [00546] Assuming the normal and hemophilia A plasmas are otherwise equivalent, the absence of Factor VIII in the hemophilia A plasma caused a marginal decrease in the baseline lag time for thrombin generation compared to normal plasma (Figure 52A), a 3-4 fold decrease in peak thrombin concentration (Figure 52B) and a 1.5-1.6 fold decrease in endogenous thrombin potential (ETP) (Figure 52C) in this experiment. In the absence of Factor VIII, ARC19499 had little or no effect on the lag time for thrombin generation, but caused a dose-dependent increase in peak thrombin concentration and ETP. The addition of exogenous Factor VIII caused incremental changes in all parameters, with the largest effects observed on the peak thrombin concentration (Figure 52B). Reconstitution with 140% Factor VIII restored this parameter to a 139 WO 2012/109675 PCT/US2012/024916 level similar to that observed in normal plasma, with smaller improvements observed at 14% Factor VIII and below. Moreover, the incremental increase in peak thrombin caused by each Factor VIII concentration was nearly identical at all concentrations of ARC19499, suggesting that the effects of the two agents on thrombin generation are additive rather than synergistic. With ETP, Factor VIII flattened the ARC 19499 dose-response curve with an additive effect observed only at the lower concentrations of ARC19499 (Figure 52C). Once 10 nM of ARC19499 was reached, additional Factor VIII did not appear to have a benefit. Figure 53A shows the ETP of hemophilia A plasma with different concentrations of FVIII added (dashed lines). Addition of ARC19499 resulted in a dose-dependent increase in thrombin generation in hemophilia A plasma and in hemophilia A plasma with 5% FVIII added. ARC19499 mediated procoagulant effect in hemophilia A plasma that was similar to 14% FVIII at 1-10 nM aptamer when ETP was evaluated (Fi gure 53 A) or 1 0---30 nM when peak thrombin was evaluated. When a saturating amount of ARC19499 (300 nM) was added to plasma with different concentrations of FVIII, thrombin generation levels were near to that observed with normal plasma, indicating that ARC 19499 does not have a severe prothrombotic effect (Figure 5313). Even with 140% Factor VIII, ETP levels of normal plasma were never reached. By this measure, therefore, the addition of exogenous Factor VIII appeared to obviate the need for a bypassing agent like ARC 19499, rather than facilitate its action. Interestingly, ARC19499 appeared to decrease lag time at the higher concentrations of Factor VIII (Figure 52A). 1005471 The inhibition of TFPI in this case may enable more rapid, Factor VIII-dependent propagation of thrombin generation. EXAM-PLE 23 [00548] This example demonstrates that ARC 19499 can improve coagulation in a spatial model of clot formation, in hemophilia plasma activated with immobilized tissue factor. [005491 The key property of the spatial experimental model is that blood plasma clotting is activated by a surface covered with immobilized tissue factor (TF). The fibrin gel then propagates into the bulk of plasma. Clotting takes place in a specially designed chamber (Figure 54A). Plasma samples are loaded into the well of the chamber that is subsequently placed in the thermostat. All experiments are performed at 37 'C. Clotting is initiated by immersion of an insert with TF immobilized on its end face into the chamber. Clot formation is registered by light scattering from fibrin gel using a CCD camera (Figure 54B). The chamber is uniformly 140 WO 2012/109675 PCT/US2012/024916 illuminated with monochromatic light and images are captured every 15 seconds. The acquired series of images is then processed by computer and parameters of spatial dynamics of blood clotting are calculated. [005501 For the purposes of this set of experiments, surfaces were derivatized with TF densities in the range of 1-100 pmole/m'. The density of TF on the surface was characterized by the ability to activate Factor X (Enzyme Research Laboratories) in the presence of excess Factor Vta (Novoseven"; Novo Nordisk) using a chromogenic Factor Xa substrate S-2765 (Chromogenix). The rate of S-2765 cleavage was measured by light absorption (405 nm) and compared to a calibration curve prepared using a set of TF standard solutions (American Diagnostica) to calculate TF concentration. [005511 Each light scattering image was processed by calculating the mean light scattering intensity (based on pixel intensity) along a perpendicular drawn to activating surface. The data from each image was depicted as a single contour line on a plot of light scattering intensity versus distance from the activating surface (Figure 55). Clot propagation was depicted qualitatively by successive contour lines of increasing light scattering intensity, determined from images taken at consecutive tirepoints up to 90 minutes (Figure 55), or quantitatively, by plotting clot size versus tire (Figure 56). The clot size for each image was determined as the coordinate (in micrometers or millimeters) along the contour line where the scattering intensity is half-maximal. Based on clot size versus time plots, the following parameters were calculated: lag time (delay between contact of plasma with activator and beginning of clot formation), initial velocity of clot growth (a, or Vinit.ai; mean slope of the clot size versus time curve over the first 10 minutes after the lag time), spatial or stationary velocity of clot growth (j3 or Vstationary; mean slope over the next 30 minutes) and clot size after 60 minutes of the experiment (an integral parameter of clot formation efficiency). For each experiment, four perpendiculars were drawn from different points along the activator surface. Profiles of clot size versus time were analyzed and four values of each clotting parameter were obtained and then averaged to obtain means. [005521 This study was conducted primarily using freshly prepared plasma (rather than commercial or frozen plasma) from normal donors and hemophilia A patients. Blood was collected from healthy volunteers and hemophilia A patients at a 9:1 v/v ratio into a solution containing 3.8% sodium citrate plus 0.2 mg/ml CTI (Institute of Protein Research, Russian Academy of Sciences), then it was processed by centuifigation at 1,500 g for 15 minutes to 141 WO 2012/109675 PCT/US2012/024916 obtain platelet-poor plasma. It was additionally centrifuged at 10,000 g for 5 minutes to obtain platelet-free plasma. Fresh pools of normal plasma were prepared from 3 healthy donors each. At 15 minutes before an experiment, 300 gL of plasma was supplemented with 18 gL of ARC 19499 or recombinant Factor VIla (alternatively designated rVIIa or Novoseven"). In control experiments lacking ARC]19499 or Factor rVIIa, plasma was supplemented with the same volume of phosphate buffered saline, Plasma was recalcified by the addition of 6 pL I M CaCl 2 , mixed, and 300 uL of recalcified plasma was placed in the experimental chamber. The insert with the TF-derivatized surface was then placed in the chamber to initiate clotting (Figure 54A). [005531 The result of a typical spatial clot formation experiment, activated by 1 pmole/m t of TF density in normal pooled plasma, without and with 300 nM of ARC19499 is shown in Figure 55A and B, respectively. The plots show contours of light scattering as a function of distance from the activator. The time between two contours is 2.5 minutes and the total time of each experiment is 90 minutes. The enhancement in light scattering at each timepoint in Figure 55B compared to Figure 55A indicates that addition of ARC19499 improved spatial clot formation. However, the effects of ARC 19499 on clot formation are more clearly seen in a plot of clot size versus tirne (Figure 56) derived from the processed scattering data, where improvements in lag time. V

M

ial (u) and clot size at 60 minutes are observable. [005541 Clot formation parameters were plotted as a function of TF surface density in Figure 57. Vertical error bars indicate standard deviations (SD) for clot parameters while "n" is the number of experiments performed at a specific TF density. Horizontal bars are SD for determinations of TF density (n='2 for each activator series). Figure 57A shows averaged lag time dependence on activator TF density. The magnitude of TFPI inhibition by ARC 19499 depended on TF density and became more significant as the density decreased (up to 2.5-fold shortening of the lag time at TF densities of 1-3 pmole/m 2 ). Figure 57B shows the averaged initial clot growth velocity dependence on activator TF density. Again, the effect of ARC19499 was significant only at low TF densities (1-3 pmole/m 2 ) where a -1.8-fold increase of the initial velocity was observed. Figure 57C illustrates the stationary clot growth velocity dependence on activator IF density; ARC 19499 had little effect on clot propagation velocity throughout the entire range of activators. Finally, Figure 57D shows the averaged clot size after 60 minutes. Inhibition of TF'PI affected clot size at densities of 1-4 pmole/m 2 TF; ARC 19499 effects became 142 WO 2012/109675 PCT/US2012/024916 insignificant as the TF density increased. Based on these data, two TF densities were chosen for further studies of ARC19499: low, 1-2 pmoie/m2, and medium, 10-20 pmoie/ m2. Several lots of activators were prepared for each of these TF densities; the mean values for the low and medium density activators were 2.0 0.68 (n=22 lots) and 20.6 ± 8.90 pmole/m (n=5 lots), respectively. [00555 The influence of different ARC] 9499 concentrations (from 0 to 1000 nM) on spatial clotting in normal pooled plasma was evaluated to examine the dose-dependence of ARC19499 effects. Figure 58 shows means and standard errors of the mean (SEM) for experiments with different normal plasma pools (n=4) and low surface TF densities. Lag time (Figure 58A) decreased with increasing ARC 19499 concentration up to 30 nM, and then stabilized. Initial velocity (Figure 5813) increased by ~30% with increasing ARC19499 concentration, while stationary velocity (Figure 58C) was not significantly affected throughout the entire range of concentrations. There was a detectable increase in clot size at 60 minutes (Figure 58D) with increasing ARC] 9499 concentration. For all affected parameters, maximal effects of ARC 19499 were clearly achieved by 300 nM, and the concentration of half-maximal effect was <10 nM. Figure 59 shows means (±SEM) of clotting parameters for 0 and 300 nM of ARC 1 9499 at low TF density combining the raw data from Figures 57 and 58 (n::::6). To calculate the statistical significance of the ARC] 9499 effect, the difference between each parameter value, with and without ARC19499, was calculated for each experiment, and the distribution of these differences was compared with zero using the t-test. Asterisks indicate statistical significance (P<0.05), that the difference between values ARC19499 was different from zero. The effects on all four parameters were statistically significant, although the effects on lag time and clot size were the largest. [00556] Figure 60 shows mean parameters (ISEM) for experiments with different normal plasma pools (n=3) and medium surface TF densities, plotted as a function of ARC19499 concentration. The effects of ARC 19499 on clotting were less substantial in this experiment. Figure 61 shows the statistical analysis comparing 0 and 300 nM ARC 19499 for all four clotting parameters. Although some of the differences appear statistically significant (indicated by asterisk), the effects of ARC 19499 on clotting in normal plasma activated by medium TF density were very small. [005571 Typical spatial clot formation activated by low density TF in hemophilia A plasma is shown in Figure 62. The plots show profiles of light scattering as a function of distance from the 143 WO 2012/109675 PCT/US2012/024916 activator for hemophilia A plasma alone (Figure 62A) and hemophilia A plasma containing 100 nM ARC19499 (Figure 62B) or 100 nM rVIIa (Figure 62C). Example light scattering images from which this data was derived are shown in Figure 63, and a plot of clot size versus time derived from the processed data is shown in Figure 64, with a normal plasma profile included for comparison. Based on these data, ARC19499 improved spatial clot formation by shortening lag time and increasing clot size. As shown in Figure 64, 100 nM ARC 19499 partially normalized clot formation, facilitating clot propagation from the activating surface. In contrast, 100 nM rVIIla stimulated potent TF-independent clotting. Rather than stimulating normalization of spatial clot propagation from the activating surface, rVIia at this concentration induced clotting throughout the reaction chamber. [005581 Further experiments characterized the concentration-dependent effects of ARC 19499 in various hemophilia patient plasimas. The demographics of the patient pool from which samples were drawn are shown in the table in Figure 65. All of the patients had severe (<1%) or moderate (1-5%) FVIII deficiencies. Figures 66, 67 and 68 show the effects of ARCl 9499 and rVla on spatial clot formation activated by low density TF in plasmas of patients #1, #2 and #3, respectively. The error bars henceforth indicate SEM for n:::4 regions along the propagating fibrin clot front within a single experiment. Panels A and B of these figures show the lag time dependence on ARCI 9499 and rVIla concentrations, respectively. The lag time decreased 2-fold with increasing concentrations of ARCI19499 from 0 up to 30 nM, with no significant further change in lag time at higher ARC19499 concentrations. Panels C and D of the same figures show the initial velocity dependence on ARC19499 and rVIla concentrations, respectively. The initial velocity increased 2-fold with increasing concentrations of ARC] 9499 from 0 up to 30 nM, with no significant further change at higher ARC19499 concentrations. Figure 69 shows stationary clot growth velocity dependence on ARC 19499 and rVIIa concentrations for all 3 patients in one graph. ARC19499 had no effect on stationary velocity through the whole investigated range of concentrations, while addition of rVIla led to a strong increase of this parameter. Finally, Figure 70 shows the dependence of clot size at 60 minutes on ARC 19499 (Figure 70A) and rVIIa (Figure 70B). The clot size at 60 minutes increased 1.5-2 fold with increasing concentrations of ARC19499 from 0 up to 30 nM, with no significant further change at higher ARC19499 concentrations. A statistical analysis of the low TF density data, comparing each parameter for 0 and 300 nM ARC19499, is shown in Figure 71. ARC19499 had significant 144 WO 2012/109675 PCT/US2012/024916 effects on lag time (Figure 71A), initial velocity (Figure 71B) and clot size at 60 minutes (Figure 71D), but no effect on stationary velocity (Figure 71C). [005591 Figures 72. 73 and 74 show the effects of ARC 19499 and rVIIa on spatial clot formation activated by medium density TF in plasmas of patients #4, #5 and #6, respectively. ARC19499 had little effect on clotting parameters for the medium density activator through the entire range of concentrations tested. A statistical analysis of the medium TF density data comparing each parameter for 0 and 300 nM ARC19499 is shown in Figure 75. ARC19499 had no significant effect on any of the four clotting parameters under these conditions. [005601 To estimate the extent of clotting normalization by ARC19499 under conditions of low TF density, Figure 76 shows the mean parameters of clotting for hemophilia A and hemophilia A with 300 nM of ARC 19499 in comparison to normal plasma. ARC1 9499 shortened the lag time below the normal level and normalized the initial velocity, but had no effect on stationary velocity. ARC 19499 increased the clot size at 60 minutes approximately 2 fold from 30% up to 60% of the normal value. In order to check whether ARCi 9499 effects differ in normal and hemophilia A plasma, the ratios with and without 300 nM of ARC19499 were plotted for all four clotting parameters [ratio:::(Parameter)ARC 19499 / (Parameter)_jxc 19499] (Figure 77). In both hemophilia A and normal plasmas, the lag time ratio was ~).5, indicating that the addition of 300 nM ARC19499 decreased the lag time by about half in each. The ratios for Vati were also similar between plasmas. However, larger ratios were observed for Vinitia and clot time at 60 minutes in hemophilia A plasma compared to normal plasma, suggesting that the maximal effect of ARC 19499 in hemophilia A plasma slightly exceeded that in normal plasma. [00561] To determine an IC.

5 for ARC 19499 in hemophilia A plasma, we plotted mean lag time and clot size as a function of ARC 19499 concentration (up to 10 nM) for low TF density (Figure 78). The half-maximal effect, calculated by curve-fitting, was -0.7 nM for both parameters. [005621 Figure 79 compares clotting parameters for hemophilia A plasma alone with 300 nM ARC 19499 or with 30 nM rVIIa. Figure 79A-D show lag time, initial clot growth velocity, stationary velocity and clot size after 60 minutes, respectively. In contrast to rVIIa, ARC 19499 increased clot size primarily by shortening the lag time and increasing initial velocity-; it had no effect on spatial propagation stage (Vstationr. 145 WO 2012/109675 PCT/US2012/024916 [005631 Experiments at low TF density in TFPI-depleted plasma were also performed to gain insight into the mechanism of action of ARC 19499 and into the regulation of spatial clotting by TFPI. Lyophilized TFPI-depleted plasma was purchased from American Diagnostica, resuspended in demonized water, and CTI added to 0.2 mg/mL. Recombinant TFPI (rTFPI; R&D Systems) was added into plasma at the concentration of 0 or 10 nM, with or without ARC 19499 (0 or 300 nM), for measurement of spatial clot formation in the presence of low TF surface density. The addition of rTFPI significantly increased the lag time (Figure 80) in the absence of ARC19499, but had no effect in the presence of 300 nM ARCI19499, suggesting that it was completely inhibited. ARCI 9499 had no effect on clotting in TFPI-depleted plasma in the absence of supplementary rTFPI, indicating that its effects are TFPI-specific. Neither rTFPi or ARC19499 had any effect on initial velocity in this experiment. [00564] In conclusion, ARC19499 significantly improved clotting in normal and hemophilia A plasma in the spatially heterogeneous system at low TF density (1-3 pmole/m2). The lag time was shortened, and initial velocity of spatial propagation and clot size at 60 minutes were increased by ARC 19499 up to 2-fold, with little effect on spatial propagation velocity far from the activator. In hemophilia A plasma, this resulted in complete normalization of the lag time and initial velocity parameters, while clot size at 60 minutes was partially normalized (increases from 30% to 60% of normal upon addition of ARC] 9499). With increases in TF density, the effects of the aptamer became smaller and there was almost no effect at 'F> 20 pmole/m2. The action of ARC] 9499 on clotting in this experiment at low TF density was TFPI-specific, since ARC19499 had no effect on clotting in TFPI-deficient plasma. EXAMPLE 24 [00565] This example demonstrates that ARC 19499 can improve clotting in whole blood and cell-free (plasma) clot-time assays, in samples collected from hemophilia A and hemophilia B patients. [005661 Blood samples (20 mL) were collected from 12 subjects, including seven severe hemophilia A (subjects #1, 3, 5, 8, 10, 11 and 12) subjects, two severe hemophilia B (#4 and 9) subjects and three healthy controls (#2, 6 and 7). Blood was collected into 0.5 mM EDTA and 0.1 mg/mL corn trypsin inhibitor (CTI; Haematologic Technologies Inc.). Approximately half of each sample was used for whole blood assays, while the other half was centrifuged to prepare platelet poor plasma (PPP). 146 WO 2012/109675 PCT/US2012/024916 [005671 The TF-activated clotting time (TF-ACT) is a whole blood assay performed using the Hemochron* Response Whole Blood Coagulation System (International Technidyne Corp.), a commonly used system for measuring patient responses to unfractionated heparin and protamine. Standard ACTs measured by this instrument use tubes containing an activator of the "contact" or "intrinsic" pathway of coagulation (g celite or kaolin). However, for TF-ACTs, the tubes designed for measuring standard ACTs were rinsed of contact-activating reagent. In its place was added 12 uL of I M CaCl, a desired amount of ARC]9499, and 2 pL of 5 nM relipidated, recombinant TF (Haematologic Technologies). Upon the addition of 2 mL whole blood to this mixture, the clot time was measured on the Hiemochron* Response instrument as for a standard ACT. The results are shown in tabular format in Figure 81. An average baseline TF-ACT of 335 + 22 seconds was observed in normal subjects. Moderate decreases in TF-ACT (up to 75 seconds), indicative of a procoagulant effect, were observed in these individuals for ARC1 9499 concentrations ranging from 44 to 700 nM. The two hemophilia B subjects showed baseline TF ACTs of 528 and 580 seconds. The TF-ACT decreased by 160-205 seconds at 88 nM ARC19499 in these two individuals, then increased moderately at higher ARCi 9499 concentrations (up to 350 nM). A relatively broad range of baseline TF-ACT values was observed in the hemophilia A group, with an average value of 578 + 140 seconds. Substantial ARC1 9499-dependent shortening of the TF-ACT was observed in 6 of 7 of these individuals, and in two of these (subjects #1 and 11) values in the normal range or below were observed. Only a moderate decrease of up to 47 seconds was observed in subject #12, but this subject also displayed the shortest baseline TF-ACT (328 seconds) of the group. These data suggest that TFPI suppression by ARC 19499 was able to improve clotting activity in a simple, whole blood clotting assay. [005681 Dilute prothrombin time (dPT) assays were performed on PPP prepared from the same blood samples as described for the TF-ACT assays. 'The standard prothrombin time (PT) is performed by adding thromboplastin. consisting of tissue factor (~1 nM), calcium chloride and phospholipids, to plasma to evaluate the integrity of the "tissue factor" or "extrinsic" pathway of coagulation. The clot time in a normal plasma sample measured using the standard PT protocol is typically - 1 seconds. The PT is commonly used for measuring patient responses to warfarin. and is largely insensitive to deficiencies in contact pathway factors like FVIl and FIX. In contrast to the standard PT, the dPT uses a very low TF concentration and clot times measured 147 WO 2012/109675 PCT/US2012/024916 by this assay are sensitive to factors in both the IF and contact pathways. In this particular experiment, thromboplastin reagent (Innovin; Dade-Behring) was diluted in tris-buffered saline (20 mM tris, pH 7.5, 150 mM NaCl) to reach a concentration of 0.3 pMTF. The dPT was performed by mixing 120 pL of PPP with 60 tL of the dilute TF solution and incubating at 37 'C for 3 minutes before adding 60 aL of 25 mM CaCl2 to the plasma/TF mixture. Clotting time was recorded on an ACL-8000 coagulometer (from Instrumentation Laboratory, Bedford, MA) and the data for all subjects is shown in tabular format in Figure 82. Baseline clot times in PPP samples from all normal and hemophilia B subjects, and 6 of 7 hemophilia A subjects were >360 seconds, which was the pre-set, maximum measurable clot time on the coagulometer. One hemophilia A subject (#10) displayed a baseline dPT of 169 seconds, Increasing concentrations of ARC19499 added to the PPP typically resulted in decreased dPT clot times. In PPP from normal subjects, 2 nM ARCI 9499 was sufficient to significantly lower the clot tirne (avg = 278 A 15 seconds) relative to baseline, but had no apparent effect in plasma from hemophilia A or B subjects. However, 8 nM ARCI 9499 lowered the clot time in PPP from nearly all subjects. Exceptions were observed in PPP from subject #10, where a low baseline dPT was observed, and #12, who appeared unresponsive to ARC 19499. Excluding these two individuals, the average clot time in the hemophilia A group for 8 nM ARC19499 was 188 ± 8 seconds. The average clot times for the normal and hemophilia B groups under the same conditions were 204 + 37 seconds and 226 +22 seconds, respectively. Higher ARC19499 concentrations caused only moderate, further decreases in clot times. Average clot times at 500 nM ARC19499 were 179 ± 6 seconds, 200 ± 21 seconds and 161 3 seconds for the normal, hemophilia A (excluding #10 and #12) and hemophilia B groups, respectively. These data indicate that TFPI suppression by ARC 19499 was able to improve clotting activity in a simple, plasma-based clotting assay. EXAMPLE 25 [00569 1 'This example demonstrates that ARC19499 can improve clotting in whole blood samples from hemophilia A and hemophilia B patients, as measured by rotation thromboelastometry (ROTEM). [005701 Blood samples were collected from 39 healthy volunteers (27 male and 12 female) and 40 hemophilia patients (all male). Of the 40 hemophilia patients, 3 hemophilia B (HB) patients and 28 hemophilia A (HA) patients were diagnosed as severe (baseline factor activity <1%), one HA and one IHB patient suffered from moderately severe hemophilia (baseline factor 148 WO 2012/109675 PCT/US2012/024916 activity 1-5%), four HA and two HB patients had mild hemophilia (baseline factor activity >5%). Using a 21-gauge butterfly needle, blood samples were drawn into plastic Vacuette tubes (Greiner Bio-One) containing 3.8% sodium citrate at a volume ratio of 1:9. [005711 Coagulation was analyzed by ROTEM (Pentapharm GmbH), which is based on the original thromboelastography system (TEGT1). In a typical ROTEM experiment, blood is incubated at 37 'C in a heated cup. As fibrin forns between the cup and the pin, the impedance of the rotation of the pin is detected and a trace is generated, indicating clot formation over time. The following parameters may be analyzed front the ROTEM trace: the clotting time (CT), the clot formation time (CFT), the maximum clot firmness (MCF) and the alpha angle (alpha). The clotting time (CT) characterizes the period from analysis start until initiation of the clot. The clot formation time (CFT) describes the subsequent period until an amplitude of 20 mm is reached. The alpha angle is given by the angle between the center line and a tangent to the curve through the 2 mn amplitude point. Both the CFT and the alpha angle denote the speed of clot development. The MCF is calculated from the maximum amplitude of the R-OTEM trace and describes clot stability and strength; the MCF is largely dependent on fibrinogen and platelet function. [00572] In this set of experiments, 300 l- of ARC 19499-spiked whole blood (0, 0.2, 0.6, 2, 6, 20, 60,200 or 600 iM ARCI 9499) was transferred into pre-warmed plastic cups. Blood samples were recalcified using 20 pL 0.2 M CaCI 2 and coagulation was activated by -33 fM tissue factor (TF) (Innovin, Dade Behring, diluted 1:200,000). All analyses were performed at 37 'C. Measurement was performed either without corn trypsin inhibitor (CTI) or with the addition of 100 pg/mL of C'T I (Haematologic Technologies Ie). Comparisons between different concentrations of ARC 19499 in any of the measured parameters were calculated with the Wilcoxon signed rank test with a Bonferroni Correction. To compare healthy controls to patients, a Mann-Whitney U-test was used. To analyze the correlation of hemostatic parameters to Factor Viii (FVIII) activity, the Spearman's rank correlation coefficient was used. A p-value smaller or equal to 0.05 was considered statistically significant. [005731 The baseline whole blood clotting profile of hemophilia patients was characterized by a prolonged initiation phase (as shown by a prolonged CT) and a diminished propagation phase of whole blood clotting (prolonged CFT and a lower alpha angle) compared to healthy controls (p<O.01 for all) in the absence of CTI (Figure 83). 149 WO 2012/109675 PCT/US2012/024916 [005741 Concentrations of ARCI9499 >2nM enhanced whole blood coagulation significantly in both hemophilia patients and healthy controls (p<0.01). The maximum hemostatic effect of ARC19499 was achieved with concentrations >60nM. In hemophilia blood, ARC 19499 decreased the CFT and increased the alpha angle to values equal to those of healthy controls (p>0.4). The clot time was substantially improved by ARC 19499, but not filly normalized. The MCF, though not significantly different between controls and patients, was significantly augmented by ARC 19499 (p<0.05 for 0 nM ARC 19499 compared to >2 nM in hemophilia patients and 200 nM in healthy controls). [005751 A comparison between FVIII coagulant activity (FVlIII:C) activity levels in the hemophilia A patient samples and baseline CT indicated a significant correlation (p<0.01). Therefore, the hemophilia A patient CT data were stratified into three groups (<1% FVIII:C, 1 5% FVIII:C. and >5% F\VIII:C) and replotted next to the healthy control CT data (Figure 84). As previously indicated, concentrations >2nM ARC 19499 significantly shortened the clotting time (p<0.01). The CT values for hemophilia patients and healthy controls remained significantly different (p<0.05), but ARCI 9499 shortened the CT of hemophilia patients by a maximum up to 38%, and those of healthy controls by up to 19%, compared to baseline values, ARC]19499 had the largest effect on the CT in patients with measured FVIII:C <1%. Although the CT of patients with FVIII:C <1% was not entirely normalized, ARC 19499 shortened the CT to the range of healthy controls and to values equal those of patients with an FVIII:C >5%, [005761 Additional ROTEM analyses were performed in blood containing CTI using samples from 28 hemophilia patients and II healthy male controls. Again, the coagulation profiles were significantly different for patients and healthy controls (p<0.01) for all parameters except the MCF (Figure 85). Similar to the measurements in the absence of CTI, addition of ARCi9499 to blood containing CTI significantly shortened the CT and CFT, and raised the alpha angle and MCF (concentrations >20nM, p<;0.01). In blood containing CTI, the pro-haemostatic effect was more pronounced than in blood without CTL Upon addition of 200 nM of ARC! 9499 to blood containing CTI, the CT was not only shortened significantly (p<0.01) but was also no longer different from the baseline CT of healthy controls (p:=0.06). ARC19499 also normalized the CFT and the alpha angle in CTI whole blood, as previously observed in whole blood lacking CTI. ROTEM parameters of hemophilic blood spiked with >60nM of ARC 19499 were equal to baseline values of healthy controls (p>0. 1). 150 WO 2012/109675 PCT/US2012/024916 [005771 One patient with acquired hemophilia A was recruited, This patient showed a FVIII:C activity of 7%, 8.5 BU/mL FVIII inhibitor and an elevated aPTT of 63 seconds. This patient had received two infusions of FV111 bypassing activity (FEIBA), the most recent one within 8 hours of venipuncture. As a consequence, the CT and CFT of this patient were already normal (patient values: CT=496 seconds, CFT=213 seconds; healthy control mean values: CT=607 seconds, CFT=251 seconds). ARC1 9499 shortened the CT and CFT further, and even more than in controls and hereditary hemophilia patients (CT: 47% vs. 19% and 30%, CFT: 45% vs. 38% and 22%). ARC 19499 also increased the alpha angle, as shown in Figure 86. [005781 Since patients with acquired hemophilia are extremely rare, the ROTEM experiment was repeated on normal blood that had been treated with a neutralizing antibody to FVIII. Blood from healthy controls was pre-incubated with a sheep antihuman FVIII polyclonal antibody (specific activity 2300 BU/mg; laematologic Technologies Inc). Figure 87 shows the CT (left panel) and the CFT (right panel) in the same controls; on the left side of each graph, values after inhibition by the FVIII antibody are depicted. The addition of 60 nM ARC19499 normalized both the CT and the CFT in antibody-treated blood. 1005791 These data show that ARCI 9499 had a procoagulant effect on clotting in blood samples from healthy controls and hemophilia patients, as measured by ROTEM. Additionally, ARC1 9499 was able to normalize ROTEM clotting parameters in blood from hemophilia patients. EXAMPLE 26 1005801 This example demonstrates that ARC19499 can improve thrombin generation in plasma samples from hemophilia A and hemophilia B patients, as measured by calibrated automated thrombography (CAT). [005811 Blood samples were collected from 39 healthy volunteers (27 male and 12 female) and 40 hemophilia patients (all male). Of the 40 hemophilia patients, 3 hemophilia B (HB) patients and 28 hemophilia A (HA) patients were diagnosed as severe (baseline factor activity <1%), one HA and one HB patient suffered from moderately severe hemophilia (baseline factor activity 1-5%), and four HA and two HB patients had mild hemophilia (baseline factor activity >5%). Using a 21-gauge butterfly needle, blood samples were drawn into plastic Vacuette tubes (Greiner Bio-One) containing 3.8% sodium citrate at a volume ratio of 1:9. Platelet poor plasma (PPP) was prepared from these samples by two room temperature centrifugation steps, with the 151 WO 2012/109675 PCT/US2012/024916 first spin at 1700xg for 10 minutes followed by a second spin at 18,000xg for 15 minutes. PPP containing 100 g/mL corn trypsin inhibitor (CTI; Haematologic Technologies Inc.) was spiked with different ARC 19499 concentrations (0, 0.2, 0.6. 2. 6, 20, 60, 200 or 600 nM ARC19499) for use in the assay. [005821 CAT assays were performed by adding 80 L of PPP to 20L of a mixture of tissue factor (TF) and phospholipids (PPP-Reagent Low, Thrombinoscope BV) in a 96 well microtiter plate. The final concentrations of TF and phospholipids were I pM and 4 MM, respectively. The reaction was started by adding 20 uL of fluorogenic substrate (FluCa Kit, Thrombinoscope BV) and fluorescence was detected using a Fluoroskan Ascent fluorometer (Thermo Fisher Scientific). Analysis by Thrombinoscope software resulted in thrombin generation curves with thrombin (nM) on the y-axis and time (minutes) on the x-axis. The software determined values for a number of parameters including: lag time (minutes; time till onset of initial thrombin generation); endogenous thrombin potential (ETP; nM; area under the thrombin generation curve); peak thrombin (nM; highest amount of thrombin generated at any one point of the assay); time to peak (minutes; time reach peak thrombin concentration); and start tail (minutes; point in time when the end thrombin generation is reached). Comparisons between different concentrations of ARC 19499 in any of the measured parameters were calculated with the Wilcoxon signed rank test with a Bonferroni Correction. To compare healthy controls to patients, a Mann-Whitney U-test was used. A p-value smaller or equal to 0.05 was considered statistically significant. 1005831 Examples of CAT data are shown in Figure 88 for a representative hemophilia A patient (left panel) and a healthy control (right panel), comparing thrombin generation in the presence and absence of 200 nM of ARC 19499. The addition of ARC 19499 normalized the thrombin generation curve in the hemophilia A patient sample and augmented thrombin generation in the healthy control sample. [005841 Average CAT parameters for all hemophilia patients and healthy male controls are shown in Figure 89. At baseline, the hemophilia patient group displayed a prolonged time to peak, a lower peak thrombin generation and a severely compromised ETP. In the absence of ARC 19499, the following CAT parameters were significantly different between healthy male controls and hemophilia patients: time to peak, peak thrombin generation, ETP and start tail (p<0.001). There was no significant difference in the lag time between healthy controls and 152 WO 2012/109675 PCT/US2012/024916 hemophilia patients (Figure 90). The addition of ARC 19499 significantly enhanced all of the parameters assessed by the CAT assay (Figures 89 and 90). Concentrations above 60 nM of ARC19499 normalized CAT parameters in hemophilia patients. There was no longer a significant difference between baseline values of healthy controls and values measured with addition of>60 nM ARC19499 in hemophilia patients in the following CAT parameters: start tail, time to peak and ETP (p >0.05) (Figure 89). Peak thrombin remained significantly different between patients and controls, but 600 nM of ARC19499 augmented peak thrombin by 185% in hemophilia patients, from 47 nM at baseline to 135 nM, thus reaching the normal range of healthy controls (healthy controls mean peak thrombin 167 nM, minimum 100 nM, maximum 297 nM). ARC19499 shortened the lag time significantly in both groups (p<0.001 for concentrations above 0.6 nM) but differences between the groups continued to be not significant (Figure 90). [00585 Figure 91 shows peak thrombin data stratified into three groups (<1 %/o FVIII coagulant activity (FVIII:C), 1-5% FVII:C., and >5% FVIII:C) and repotted next to the healthy control peak thrombin data. Increasing concentrations of ARC19499 augmented peak thrombin generation in all four groups. ARC19499 did not completely normalize peak thrombin in the <1% FVIII:C plasma relative to the average healthy control value at baseline, but values at 60 nM ARC19499 reached higher than the baseline of patients with >5% FVIII:C concentration and into the range of values observed for healthy controls (hatched region in Figure 91). [005861 CAT assays were also performed on ARC19499-spiked samples from a patient with acquired hemophilia. This patient showed a FVIII:C activity of 7%, 8.5 BU/mL FVIII inhibitor and an elevated aPTT of 63 seconds. This patient had received two infusions of FVIII bypassing activity (FEIBA), the most recent one within 8 hours of venipuncture. As a consequence, ROTEM parameters of clotting in this patient were already normal (patient values: CT:::496 seconds, CFT:::213 seconds; healthy control mean values: CT::607 seconds, CFT:::51 seconds). Thrombin generation curves measured in the presence of 0, 2, 20 and 200 nM ARCi 9499 are shown in Figure 92. Although this patient displayed normal ROTEM values, baseline CAT values were severely compromised. As for patients with inherited hemophilia, increasing concentrations of ARC 19499 normalized thrombin generation in samples from this patient. ARC19499 had the largest influence on the peak thrombin and the ETP of the patient, both of which increased more than 2.5 fold. Peak thrombin was normalized (peak thrombin: baseline 54 153 WO 2012/109675 PCT/US2012/024916 nM -- max 165 nM; mean baseline value of healthy controls: 164 nM). Moreover ARC19499 (200 nM) increased the ETP to values above those of healthy controls (ETP: baseline 973 nM max 2577 nM; mean baseline healthy controls: ETP 1322 nM). [005871 The CAT experiment was repeated on normal plasma that had been treated with a neutralizing antibody to FVIIL. PPP from healthy controls was pre-incubated with a sheep antihum'an FVIII polyclonal antibody (specific activity 2300 BU/mg; Haematologic Technologies Inc). Figure 93 shows the ETP (left panel) and the peak thrombin (right panel) in the same controls; on the left side of each graph, values after inhibition by the FYIII antibody are depicted. The addition of 60 nM ARC 19499 nonnalized both the ETP and the peak thrombin in antibody-treated plasma [005881 These data show that ARC19499 had a procoagulant effect on thrombin generation in blood samples from healthy controls and hemophilia patients, as measured by CAT. Additionally, ARC19499 was able to normalize CAT parameters in plasma from hemophilia patients. EXAMPLE 27 [00589] This example demonstrates that ARC 19499 can improve thrombin generation times (TGT) in plasma samples from severe, moderate and mild hemophilia A patients, and severe hemophilia B patients, as measured by calibrated automated thrombograrn (CAT). 1005901 Blood was collected into 3.2 mnL Vacuette tubes containing 3.2% sodium citrate and 250 Lt 1.3 rng/mL corn trypsin inhibitor (CTI); the final CTI concentration was 100 pg/mL. Samples were collected from patients with severe (<1% FVII; n=1 0), moderate (1-5% FVIII; n:::7) and mild (5-40% FVIII; n:::5) hemophilia A, patients with severe hemophilia B (<1% FIX, n:::5) and healthy volunteers (n:::10). To prepare platelet poor plasma (PPP) for CAT assays, tubes were centrifuged at 2500xg for 15 minutes, the supernatant transferred to fresh Eppendorf tubes, then centrifuged again at I 1000xg for 5 minutes. Plasma was either used immediately or frozen at -80 C for later use. To analyze the effects of ARC19499 on thrombin generation, ARC19499 was added to plasma at concentrations of 10, 100, 300, 1000, 3000 or 10,000 ng/mL (0.9, 9.0, 27.1, 90.3, 271 or 903 nM). 1005911 Thrombin generation time (TGT) assays were performed by calibrated automated thrombography (CAT) on a Thrombinoscope instrument (Thrombinoscope, Maastricht, The Netherlands) consisting of a Thermo Scientific Fluoroskan Ascent Microplate Fluorometer 154 WO 2012/109675 PCT/US2012/024916 (serial no. 374-9003 1C) programmed with Thrombinoscope software version 2.6. 80 L plasma was mixed with 20 L relipidated recombinant tissue factor (TF; final concentration 1 pM) and the assay was initiated by the addition of 20 ML FluCa substrate. Analysis of the data by Thrombinoscope software resulted in thrombin generation curves with thrombin (nM) on the y axis and time (minutes) on the x-axis. The software determined values for a number of parameters including: lag time (minutes; time till onset of initial thrombin generation), endogenous thrombin potential (ETP; nM; area tinder the thrombin generation curve), peak thrombin (nM; highest amount of thrombin generated at any one point of the assay) and time to peak (minutes; time reach peak thrombin concentration). All measurements were performed in duplicate. Differences in means of each group were tested using a student's t-test or ANOVA, as appropriate. [00592] Figures 94-97 show representative CAT data from a healthy volunteer (IV), a patient with severe hemophilia A (SHA), a patient with moderate hemophilia A (MoHA) and a patient with mild hemophilia A (Mi-LA). Median data for healthy volunteers, all three hemophilia A patient groups, and the severe hemophilia B patient group are shown in Figure 98 for experiments performed in freshly processed plasma. Baseline ETP and peak thrombin parameters were decreased in all hemophilia patient groups compared to healthy controls, and the time to peak was increased. The severity of FVIII deficiency had no effect on observed thrombin generation, as baseline parameter values were essentially indistinguishable between the 3 hemophilia A patient groups. FVIII deficiency had little effect on the baseline lag time compared to healthy controls, but FIX deficiency resulted in -2-fold prolongation of the lag time at baseline. Freezing the plasma had little effect on thrombin generation as similar CAT parameter values were observed in plasma samples that had undergone freeze-thaw (Figure 99). [005931 As shown in the individual (Figures 95-97) and median data plots (Figures 98 and 99), the addition of ARC19499 improved thrombin generation in hemophilia plasma. The ETP increased with increasing ARC 19499 concentrations up to 10,000 ng/mL (903 nM) reaching a normal level in all hemophilia groups (Figures 98 and 99). A trend toward improvement was also observed in the peak thrombin. Although normalization of this parameter was not achieved, 3-5-fold improvements were observed in all patient groups. ARC19499 had little effect on the lag time in any of the hemophilia A patient groups, but it did slightly improve the lag time in plasma from hemophilia B. A slight improvement in time to peak was also observed in all of the 155 WO 2012/109675 PCT/US2012/024916 patient groups. In healthy volunteer plasma, the addition of ARC19499 had little effect on any of the CAT parameters. [005941 Median CAT data with interquartile ranges are presented for fresh and frozen plasma in the following tables: Table 3, severe hemophilia A (SHA); Table 4, moderate hemophilia A (MoHA); Table 5, mild hemophilia A (MiHA); Table 6, severe hemophilia B (SHB); and Table 7, normal. Taken together, the data show that ARC1 9499 improved thrombin generation in hemophilia A patient plasma of all severity levels, and in severe hemophilia B plasma. Table 3: TGT on Citrated PPP with CTI - SHA (n=10) AR(19499 ETP[nM4min PeakInM] ng/nL Fresh Frozen Fresh Frozen Median IQR Median IQR Median IQR Median IQR t260- 1244- 10000 1347 i587) 88 63 -I19 107 86-139 1538 1763 1117- 1094- i 3000 1233 1569 176 56-103 103 77-132 1425 1716 1019- 1019-| 1000 1171 1546 1019 50-95 90 67-125 1335 1711 825- 916 300 1078 ~2- 1522 60 44-77' 83 58-117 1206 165 / 741 - 726 100 896 1- 1206 46 36-76 58 42-94 1137 1469 564- 516 10 638 705 34 23-48 31 19-62 978 1169 0 489 2 685 21 13-29 26 10 -35 1 847, ARC19499 Lag time Imin] IT pekinul ng/mL Fresh Frozen Fresh Frozen Med Median IQR Median 1QIR Median IQR 2 9 10000 3.3 3.5 2.9-3.8 10.1 10.6 3 9.6- ,- 3000 3.3 3,5 2.6-4.1 10.1 , 1.5 3 .24-06 9.4 1000 3.3 4 3.6 3.0-3.8 11 9.9 3.8 1+ 3.3 9.5 09 300 3.4 3.7 2.9-4.0 10.1 11.3 38 12.5 11,9 ?5- 9 -1 . 100 .3i 3.6 3.0-4.2 10.5 2q 3 . 12 3.0- 9.3- I 9.2 10 3.3 .3 2.5-4 10.6 11.8 | .5 13.6 14.1 16.2 0 3.6 .1 3.6-5,7 17.8 22.5 156 WO 2012/109675 PCT/US2012/024916 Table 4: TGT on Citrated PPP with CTI -- MoHA (n=7) ARC19499 ETPlnMimin PeaknMI ng/linL Fresh Frozen Fresh Frozen fMedian IQR Median IQR Median IQR Median IQR 1468- 1406 86 10000 1548 706 102.,7 97 87 -133 16* 2062 134 1331 - 1188 -68 3000 1498 637 99.39 88 - 130 1538 | 2054 30 12350 36i 1212 1366 88.92 86 80 - 126 1603 ||205814 965 - | | 099 300 1314.5 | 1349 8125 5 81 68- 108 171i 1762 835 - | | 939 100 919 085 | '8. 48 -85 51- 100 1792 17163 576 -40 10 806 796 49.19 2 60 47 34-87 1102 1308 38'-' -392 0 5179 7 764 22.62 16-42 41 16-64 872 1105 ARC19499 Lag time Imin] TIT peakin]ni ng/ml Fresh Frozen Fresh Frozen Median IQR Median IQR Median IQR Median IQR 1005 10 5- t11.3 1)000 4 $ 5.1 4.0-5.6 12.7 2. 4.8 13.6 14.7 4.0 11.3 30003.3-5.5 12.5 12.6 4.8 13.4 13.1 4.8- 10.- 11.3 1000 4.2 5.8 4.6-6.0 12.3 12.4 5.8 12. 14.1 3.6- 10.3- 05 300 4.1 38 3-5.33 11.2 11.0 4,3 1.3 1 10 42 5 10.8- 10 100 4.2 4.8 33-5.5 11.0 | 11.5 ----------- 4 .7---- ------------- --- 3 .3- 14 .1 26-I i10.5- |11.3 10 4.25 " 4.6 3.3-6.0 13.7 12.2 113 4.6 14.6 14.6 o 4.8 4 5.0 42- 16.4 15-2.3 5.5 5.8.9 | | 16.8 157 WO 2012/109675 PCT/US2012/024916 Table 5: TGT on Citrated PPP with CTI -- Mild HA PPP (n=5) ARC19499 E TP~anM/min] PeakinM] n3g/m1l Fresh Frozen Fresh Frozen Median IQR Median IQR Median IQR Median IQR 1081- 17 - |75 - |8 10000 1366 1081- 1361 1 938 98 1472 1708| 3| 2 i313 1 26 --- -- --- -- -- ------- ------ ----------- 913- 1226- D 67- 2 3000 t1387 " 127 1 3 87 ' 1 | 8 14? 1520 |97 87 1156- 62.- 72.

1000 1097 1 1245 o 82 'o 7 7 398 1330 i02188 804- 1026- 4 59 300 1069 131 1136 120 78 ~ 69 131 12 1 |80 | 6 64 -C 37! - .40-1 100 986 643~ 1045 62 61 1P49 114 76 421- 551 19 - 5 10 709 692 42 39 1068 337- 372- 14 2 16 0 532 493 " 28 2 823 800o 37 | 41 ARC19499 Lag time [min] TT pe k min] ng/ml Fresh__ Frozen Fresh Frozen Median IQR Median IQR Median IQR Median IQR 2.4- 9.3 10000 4 3.6 9.6 8 11 10.5 52 || 56 13., 2.]1 2.3 -8.8 3000 4.1 - | 3|9.8 81 10.3 9-12.4 32 9.6- 1000 4.1 326 10. 11 7' 10.1 1. 5.2 I.Z-126 3.2 3.42 93 10.3 300 4 3.6 10.6 11 1 5.1 -5 3 11.8 13. .1-3.3 - 912 -1 100 4.7 43 10.8 10.6 5.5 1.5 164 14.3 . 2.61 - 10.7 10 4.1 3.6 6 11.6 11.6 6 6. 51t7.1 1. 2.5 -3.9 - 4.0 - 13.1 0 4.8 46 156 136 8.9 10.4 22.1 ' 24.5 158 WO 2012/109675 PCT/US2012/024916 Table 6: TGT on Citrated PPP with CTI -- SHB PPP (n=5) ARC19499 ETP[nM/min] Peak[nMI ng/il Fresh Frozen Fresh Frozen Median 1QR -Median IQR Median IQR Median 1QR 29 - 1177 10000 1484 1736 ~ 74.3 -" 106.1 1592 _________ 1850 '074 - 12 9-7 3000 1360,7 1635 75 "() 97.9 1459 1702 102 [1t 1000 1217.5 4- 1587.5 1200- 68.4 1 94 1435 1667 98.2 1071 893 - 6999- 5- 61 310 0 1 167 893- 1429 99- 62 1 7 ,) 14.3 6 353 1493 972 104 100 1075 71 115 58, 40.7 - 50 1228 1340 85 960. 4.31 - 50-21.7 -- 3 10 787.5 7949 40 41 852 981 46.6 64 186- 189- 92- 9-. 3 0 403 -873 642 14.9 ' 29.3 473 722 25.5 35 ARC19499 Lag time [min] TT per kmin] ng/mi Fresh Frozen Fresh Frozen Median 1QR Median 1QR 1 Median IQR Median IQR 4.7- |4.9 - 11.3 - I 2 10000 6 ' 5.3 12 13.7 1 6.3 64 14.8 | 14.5 4.7- 10.9 - 11.8 3000 6 5.3 5 -5.7 13.3 12.7 6 i 15.7 4.3 4.6 4.5 10.1- 11.4 1000 5.3 ' | 5.5 ' .12 12. 3 | 6.3 6.5 14.6 4.4 4.9 -. 5.3 - 9,8 - I 11 300 5.5 .. 535.712. 12 1 '.9 15.8 14.5 13.8 4.7- 9.9- 10.9) 100 5.3 5.5 5.1-6 123 - , 15 6.2 15.1 3 4.9 - . . 1- 11.2 10 5. . 12. 7 , 13.3 6.81 6.5 19.7 43 0 6.4 - 72 5.8 - 1. 14.2 - 1 14.6 0 7. | 72 - | 19.8 16.7 | 9.6 8.8 | 23.4 8.8 159 WO 2012/109675 PCT/US2012/024916 Table 7: TGT on Citrated PPP with CTI -- Normal PPP (n=10) ARC1 9499 ETP[IMimin] Peak nM] ug/mi Fresh I Frozeui Fresh Frozen Median IQR Median R edian IQR Median IQR 1464- 1455- 227- 197 10000 1696 |M218.S 257 "' 265 266 286 239 | i 286 274 1788- 1746- 20 5- 210 3000 1903 1897 245 261 2424 2356 270 272 1545- | o 1283- 188- 182 100 1680 11901 | 2217 1229 2072 |2374 |3290 263 153- 1548 199- 183- 1 300 1964 1903 225 217 2245 2245 | 245 288 1529 151 200- 189 100 1973 130 230 230 2231 2266 23 8 287 1530- |1611- 204- |204 10 2087 2092 239 220 2 4 2125 | 2189 274 " | 269 | 1517- 2 1745- 161- 2 182 0 1847 | 159 12241 i 232 9 2462 298300 ARC19499 Lag time [mi] TT peak[min] ng/mI Fresh Frozen Fresh Frozen Median IQR Median IQR Median IQR Median IQR .3.7- 6.7 -72 10000 4.3 3.4 3 -- 31 5 9 8 4.6 8&9 9.4 3,6 31 --- , 3000 3.8 79 '" 79 4.5 3 8.8 8.4 1000 4 3.5 8 7.7-9 7.0 4.6 3.9 8 300 4 8.3 8 2 7 9 4.7 3.9 9.3 3.3 3.1 7.9 6 100 3.9 3.5 8 8.5 4.4 3.9 89 10.3 10 3.7 4 3.2-5 9 '' 2 51 10.8 10 3.703 3.2 7.1 -- 7.5 04.,5 3.8 9.3 9.2 4.6 4.7 10.4 9.9 EXAMPLE 28 [00595] This example denonstrates that ARC] 9499 can improve clotting in whole blood and plasma samples from hemophilia A and hemophilia B patients, as measured by thromboelastography (TEG). [00596] Blood was collected into 3.2 mL Vacuette tubes containing 3.2% sodium citrate and 250 L 1.3 mg/mL corn trypsin inhibitor (CTI); the final CTI concentration was 100 pg/mL. Samples were collected from patients with severe (<1% FVIII; n=]0), moderate (1-5% FVIII; 160 WO 2012/109675 PCT/US2012/024916 n=7) and mild (5-40% FVIII; n=5) hemophilia A, patients with severe hemophilia B (<1% FIX, n=5) and healthy volunteers (n=10). To prepare platelet poor plasma (PPP) for CAT assays, tubes were centrifuged at 2500xg for 15 minutes, the supernatant transferred to fresh Eppendorf tubes, then centrifuged again at I 1000xg for 5 minutes. Plasma was either used immediately or frozen at -80'C for later use. To analyze the effects of ARC19499 on thrombin generation, ARC]19499 was added to plasma at concentrations of 10, 100, 300, 1000, '000 or 10,000 ng/mL (0.9, 9.0, 27,1, 90.3, 271 or 903 nM). [005971 Thromboelastography (TEG) assays were performed using a TEG 5000 series instrument from Ilaemoscope. Whole blood TEG assays were performed by adding 300 PL whole blood to 40 pTL 9 pM tissue factor (TF) and 20 pL 0.2 M CaCl2 in a disposable reaction cup. Amplitude versus time traces were analyzed to obtain the R-time (length of time to initiate clot formation, aiplitude:=2 mm), K-value (measure of the speed of clot formation, equal to the tirne required to reach an amplitude of 20 num) and the angle (another measure of the speed of clot formation, calculated frorn the tangent of the amplitude tracing drawn with its origin set to the R-time). Plasma TEG assays were performed similarly, except that supplementary phospholipids (PL) were included. In these assays 300 iL PPP was mixed with 10 pL 38 pM TF, 30 L 48 PaM PL (20% phosphatidyl serine, 20% phosphatidyl ethanolamine, 60% phosphatidyl choline; Avanti Polar Lipids) and 20 L1 0.2 M CaCl2. The final PL concentration in these reactions was 4 pM. [00598] Figures 100-103 show representative whole blood TEG data from a healthy volunteer (HV), a patient with severe hemophilia A (SIIA), a patient with moderate hemophilia A (MoHA) and a patient with mild hemophilia A (MiIA). Median data for healthy volunteers, all three hemophilia A patient groups, and the severe hemophilia B patient group are shown in Figure 104. The baseline R-time values were elevated in all hemophilia patient groups compared to healthy controls, indicating a delay in clot initiation, with the most significant effect observed in the severe hemophilia A and B samples. Additionally, the K-values were increased and angles decreased in hemophilia patient samples compared to healthy controls. Both of these effects indicate less rapid clot formation compared to normal, although the effects were similar in all patient groups regardless of the severity of factor deficiency. [00599] As shown in the individual (Figures 101-103) and median data plots (Figure 104), the addition of ARC1 9499 improved clot formation in whole blood, as measured by TEG. 161 WO 2012/109675 PCT/US2012/024916 Increasing concentrations of ARC 19499 (up to 10,000 ng/mL, 903 nM) substantially normalized all of the TEG parameters in all of the patient groups (Figure 104). ARC19499 restored normal clot initiation (R-time) and development (K value and angle). [006001 Similar results were observed in plasma TEG assays. Individual plasma TEG data from representative patients with severe hemophilia A (SHA), moderate hemophilia A (MoHIA) mild hemophilia A (MiHA) are shown in Figures 105-107, and the median data are presented in Figure 108. Baseline clotting effects in hemophilia plasma samples showed a trend toward correlation with disease severity in all three TEG parameters, with the most substantial effects observed for severe hemophilia A and B samples (Figure 108). The addition of ARC19499 improved all three parameters describing clot formation. Increasing concentrations of ARC19499 appeared to normalize clotting in all of the patient groups, as measured by R-time and K-value. In contrast, while ARC19499 appeared to normalize the angle in both mild and moderate hemophilia A groups, the angle in the severe hemophilia A and B groups did not fully correct even at the highest ARC19499 concentration tested. Nevertheless, substantial improvement was observed in all of the groups. [00601] Median data with interquartile ranges are presented for whole blood TEG measurements in the following tables: Table 8, severe hemophilia A (SIA); Table 9, moderate hemophilia A (MoHA); Table 10, mild hemophilia A (MiiHA); Table 11, severe hemophilia B (SHB); and Table 12, normal. Additional tables show median data with interquartile ranges for plasma TEG measurements: Table 13, severe hemophilia A (SHA); Table 14, moderate hemophilia A (MoliA); 'able 15, mild hemophilia A (MiHA); Table 16, severe hemophilia B (SHB); and Table 17, normal. In all of the tables, the expected normal range for each parameter is shown in italics in the column heading. Taken together, the data show that ARC 19499 improves clot formation in hemophilia A patient plasma of all severity levels, and in severe hemophilia B plasma. 162 WO 2012/109675 PCT/US2012/024916 Table 8: TEG on Citrated Blood with CTI SHA (n=10) __________ AR( R firne K Angle 19499 11nun] rnI de~ (n/m ~10.6 -15,6 121.3271 -56.7 _____Median L IQR Mfedian 1QR Mtedian IQR 10000 .1 10.2 -18.3 ___ 3.-6.6 49.6 ~3.3 - A.2 3000 15.2 2 - ) .4 5.3 3.6__-_T5 38.7 3 1. 2-50. 1000 16.4 1.5 21.8 6.8 4- 9.0 35.7 25.6---47.5 300 19. 164 -29.0 8.2___~ 1 -- h4 30.7 236 -36.3 100 2 :96 3'0 9 63 -- 1t26 25.8 1. .. 10 3.9 2~' 5. 14.7 1I3 253 19.5 11.0 -20. 1 0 5 3___ 43. 730 23__ 15 8 29'9 82O -- 1315 Table 9: TEG on Citrated Blood with CUI --- MoHA (11=7) __________ ARC R thne K kflgie 19499 [uimi [rIMMI [deg] j(g{'I 5.9 -16.3 1.7-5.3 48.5-59 Mledi'rn IQR Median IQR Nfedian 1QR 10000 9 4.5- 24 38 3.2- 4.8 54i 51-59 3000 107 7.7' 16 1 4. 7 3.6 - 61 453 43.6-52 1000 1.6 6.7, 1 -7 6 3.9 --- 10. 43 29.7-50.3 300 4 t 6.0 16.2 6.7 .3.x -- 10 x 42.5 304- 481 100 10. --- 17.8 60 --9 9 29.1 -d0.1 1s 11.2 --21.5 7.4 4.0 -- 10. 277 216.6 -37.8 0 28.1 ~ 1 7 - 75 17.6 6.8 -20.8 1-/1 93 - 6 Table 10: ---TEG-i- on C ita d Blood with CTI --- M illA (n::5) ----------------------------------- ARC R time K Angle 19499 JR3111 [miDIR Ideg] jgI59-16.3 1.7-53 48.5-59 _____ edian IQR NMedian 1: IQR Mediani IQR 0000 7.3 5.5 13. 41, 2.7 6 8 48- KI-- S 3000 7.8 6.5 17 2 83___ 6. 12. 418 30.0 47.6' 1080.6 7.9 t14.5 8 X.0 12.6 4 1, 22,0--48,9 3( 9 7- 21.1 12. 9.7 1 X. 39,1 214--41,0 100.3 10O5 -2-/,( 6.6 9.3 --24 26,8__ 1041 2 10 16.___ 11J8 -- 8, 19 10.0- 32.0 .6.5__ 118 300O 0 22 15.9 -46. 9 1 108 - . 9' 5.5-28.0 T able 11: T-EG on Citrated Blood with (iTI --- SH-B (n::5) ARC I R time K Angle 19499 1IWE3 I 1min [deg] (Inglrl) 5.9 -1 6.3 17-5.3 48.5-6-1.9 Median IQR Median IQR Median 1Q11 10000 13.5 10.3 -16.9 4.9 4.2 -6.4 40,3 36-46.6 3000 15"? '22 -186 5.5 5-6.4 41.9 16,3 -42.6 1 000 20,3 76 2 1.2 6.3 5.4- 10.2 30,7 211L6 - 39.6 300 24. 6 -2.3 -31. 10.3 8 55 243 17-' 18.) 00. 4.9- ~ 34.9 12.5 9.5 14 17 141-~ 35.8 303 -39.2 13.7 1' 1 63 13 1.-1. 0 41.6 39.9 - 57 S 16 14.4 - 3.8 10.04 -6.2 - 12.5 1631 WO 2012/109675 PCT/US2012/024916 Table 12: TEG on Citrated Blood with CTI - HV (n=10) ARC R time K Angle 19499 [mi] [mi] [deg] (ng/nl) 5.9-16.3 1.7- .3 48.5 -679 Median IQR Median IQR Median IQR 10000 9.3 8.7 - 10.3 2. 7 1 .- 3.5 52 51.7 - 61.6 3000 8.5 83 - 11.2 3.2 2.3 - 4.0 5.9 56.7 - 60.9 1000 88 8.4- 10.5 2.8 2.2 -. 2 57.8 54 8 636 300 9 8.4 ---11.2 2.9 12.6 3.4 578 54,2 61,2 100 9.9 7.2 --- 12.2 2.8 2 3 4.5 58.5 500 62,6 10 10'4 9.4 ---11.5 2.8 2.

3 31 57.1 52 0- 62 6 0 1 8,6 12.9 3.2 | 2.

8 42 56 51 6 --57 9 Table 13: TEG on Citrated PPP with CTI - SHA (n=10) ARC R time K A gle 19499 min] [deg] (nI) 10.6 -15.6 12-14.8 32.1 - 56.7 Median IQR Median IQR Median IQR 10000 10.3 8.9-21.5 10 7.5 - 1 323 24.1 38.5 3000 14.2 10.4 - 23.8 15.4 10.9 17.3 22.3 16.5- 25.7 1000 15 15.7 - 25.7 15.1 12.1 19.0 1.2 | 18.8 -23.9 300 15,7 9.8 29,5 19 7.8 21.0 14.9 11.6 16.8 00 | 15.8 14.3 34.1 19.6 18.3 21.0 14 107 14.4 10 23.6 15.7 2-- 19n.8 - 23.0 9.9 8.8--- 10.7 0 326 25.5 - 55.4 24.1 20.4 - 27.3 | .1. 4.4- 7.9 Table 14: TEG on Citrated PPP with CTI MoHA (n=:7) ARC R time K Angle 19499 [mini [min] [degI 10.6-15.6 L2-14.8 32.1 - 567 Median IQR Median IQR Median IQR 10000 1.8 11.5 - 15.2 7.3 4.9 - 9.9 40.4 35.8 - 45.6 3000 16.8 10.6 - 21 8.5 5.9 -8.9 35.6 30.9 - 40.2 1000 17.9 12 -22.1 8.7 5.1 - 10.1 32 27.1 -36.2 300 18.5 12.6 - 22.5 10. . 4 4- 12.3 23 17.3 -29.1 100 19.1 119 23,5 11.5 | 9. 14.4 207 19.2 24.6 10 20.9 13.8 25.6 11.9 | 9,6 14.4 152 13.1 .187 0 23.6 18.2 -28.1 16.5 13 - 18 12 7.9-14 164 WO 2012/109675 PCT/US2012/024916 Table 15: TEG on Citrated PPP with CTI -- MiHA (n=5) ARC R time K Angle 19499 [mini [min] [deg] ngml) __ 10.6 -15.6 1.2 14.8 321 - 56.7 Median IQR Median IQR Median IQR 10000 I18 3.4 - 11.9 3.3 1. 3.8 56.6 53.3 - 73.9 3000 118 5.2 - 13.9 3.4 0 4.0 52.6 51.3 -66.4 1000 12.8 4.0- 14.9 3.4 2.3 - 4.9 50.6 47.8 -- 62.0 300 14 3.7- 18.6 3,7 35 -3.8 46.4 44.1 -- 52.6 100 14.7 5.6- 21.7 5 3.8 6.2 37.9 34.5 41.1 10 15.8 119 2- 22 74 d 2 10.1 29 1 323.5 35.1 0 19.8 17 7 26,9 9,6 6 12.2 211 16.2 35.0 Table 16: TEG on Citrated PPP with CTI --- SHB (n=5) ARC R time K Anole 19499 [mi] [min] [deg] (ng,"ml) 10.6 -15.6 1.2 - 14.8 32.1 -56.7 Median IQR Median IQR Median IQR 10000 102 8.2 - 143 .7 4.8 - 17.7 31. 20.6 - 49.9 3000 123 9.7 - 16.9 14.5 6,5 - 23.2 26.6 17.2 - 24.1 1000 19.2 1 21.8 23 85 - 26.5 22.4 12.1- 30.4 _ 00 _ _ 1 2 - 28 _5 24,2_ 143 28.8 12.5 5.2 -20.4 00 26 13,6- 316 19.6 68 27.4 5.3 4.9-15.3 10 33.1 11-6,9 - 38.3 19.1 17 !,- 28.4 4.8 3.7 14.1 0 36.7 27.9 44.5 22.5 20.5 -59.9 2.6 1.5- 7.6 Table 17: TEG on Citrated PPP with CTI --- [HV (n10) ARC R time K Angle 19499 [mini [mini [deg] (ngml) 10.6 -15.6 12-14.8 32.1-56.7 Median IQR Median IQR Median IQR 10000 9.8 6.7 3.2 4.8 3.5 -- 64 5 .2 50.0 -53.2 3000 8.7 7.0 10.7 7.8 5.2 10.8 -4 5 44.0 -499 1000 11.6 110 - 12.1 10.3 5.8 10.5 40.9 39.9 42,0 300 1 2.'7 1 8 10.4 6.5 11.3 37.7 .36.5 403 100 14.3 13 J 15,3 10.1 | 5.9 11.2 39.3 3 9.6 49 10 15 13- 15,7 11.7 10.1 --11.9 38.8 | 38.2 447 0 12.7 12.1 - 14.3 7.9 . 4. 11.3 40. 38.5-48.3 EXAMPLE 29 1006021 This example demonstrates that the in vitro activity of ARC19499 can be reversed. 1006031 Four reversal agents (ARC23085, ARC23087, ARC23088 and ARC23089) were mixed with ARC 19499 and tested in both the calibrated automated thrombogram (CAT) and the thromboelastography (TEG*) assays in hemophilia A plasma (Figure 109). [00604] For the CAT assay, ARC19499 was incubated with each reversal agent individually for 5 minutes at 37 'C. The mixture was then added to hemophilia A plasma at a final 165 WO 2012/109675 PCT/US2012/024916 concentration of 100 nM ARC19499 and increasing concentrations of ARC23085, ARC23087, ARC23088. or ARC23089 (2.5, 5, 10, 20, 40, 80, 160, and 320 nM). The CAT assay was then performed as previously described using a final TF concentration of 1.0 pM. ARC19499 alone improved the ETP of hemophilia A plasma from ~600 nM to ~900 nM (Figure 109A). All four tested reversal agents blocked this improvement when tested at concentrations >80 nM. ARC23085 and ARC23089 almost completely reversed ARC19499 activity at 160 nM, while ARC23087 and ARC23088 almost completely reversed ARC19499 activity at 320 nM. Looking at peak thrombin (Figure 109B), the four reversal agents showed a similar partial reversal of ARC19499 activity at 80 nM. Again, by 160 nM, ARC23085 and ARC23089 completely reversed ARC 19499 activity, while the other two reversal agents did so by 320 nM. [006051 For the TEG* assay, ARC19499 and one of each of the four reversal agents were mixed with hemophilia A plasma, and clotting was initiated with the addition of TF and CaC1. This was performed with and without a 5 minute preincubation of ARC 19499 and the additional reversal agent at 37 C. When ARCI 9499 was tested alone, the aptamer corrected the prolonged R-value of hemophilia A plasma from 54 minutes to 8.3 minutes (Figure 109C). ARC23085 partially reversed this improvement with R-values of 30 and 24 minutes with and without the 5 iii nute preincubation, respectively. ARC23087 demonstrated no ability to reverse ARC19499 when there was no preincubation, but with the incubation it did reverse ARC19499 activity, resulting in an R-value of 38 minutes. ARC23088 demonstrated little to no ability to reverse ARC 19499 activity in this assay, independent of any preincubation. Similar to ARC23087, ARC23089 showed little ability to reverse ARCi 9499 activity when there was no preincubation. But, when the reversal agent was incubated with ARC19499 prior to the assay, it reversed ARC 19499 activity almost completely, resulting in an R-value of 42 minutes (Figure 109C). [006061 These experiments indicate that the in vitro activity of ARC19499 can be reversed. EXAMPLE 30 [006071 This example demonstrates that ARC19499 does not inhibit the in vitro anti coagulant activity of low molecular weight heparin (LMWH). [006081 In this assay, increasing concentrations of ARC 19499 and increasing concentrations of LMWH were mixed together and added to hemophilia A plasma. The thrombin generation ability of these plasma mixtures was analyzed using the calibrated automated thrombogram (CAT) assay. Figure 110 shows the thrombin generation curves of the increasing aptamer 166 WO 2012/109675 PCT/US2012/024916 concentrations in the presence of each LMWH concentration. ARC19499 was tested at 0.1, 1, 10, 100 and 1000 nM. LMWH was tested at 0 (Figure 110A), 0.156 (Figure 11OB), 0,312 (Figure I10C), 0.625 (Figure IOD), 1.25 (Figure 110E), 2.5 (Figure 1OF) and 5.0 IU (international units)/mL (Figure 110G). [006091 In Figure I IIA, the ETP values for each combination were plotted along the y-aris, with the LMWIT concentrations on the x-axis. The same was true for peak thrombin values in Figure Ii IB. In both cases, therapeutic doses of LMWH (0.5-1.0 IU/mL.) strongly inhibited thrombin generation, and higher concentrations almost completely prevented any thrombin from being generated, even in the presence of up to 1000 niM ARC 19499 (Figure III). ICso values of 381 24.3 nM and 299 ± 18.0 nM fior LMWII were calculated fiom the ETP and peak thrombin data, respectively, in the absence of ARC19499, and these results are consistent with previous measurements (Robert et al., Is thrombin generation the new rapid, reliable and relevant pharmacological tool for the development of anticoagulant drugs?, Pharmacol Res. 2009; 59:160-166). Increasing concentrations of ARC 19499 did not appear to significantly alter the ICsC of LMWII (Figure 112), indicating that ARC19499 does not interfere with the anticoagulant activity of LMWH. [00610] This experiment indicates that even in the presence of 1000 nM ARC 1 9499, therapeutic doses of LMWII still remain anti-coagulant in an in vitro assay. EXAMPLE 31 1006111 This example demonstrates that the TFPI aptamers are stable to serum nucleases. [006121 In this experiment, 50 pM of each aptamer was incubated in 90% pooled human, cynomolgus monkey or rat serum for 72 hours at 37 C. Samples were analyzed by IHIPLC and the percent remaining as a function of incubation time was determined, as shown in Figure 113. [006131 Both ARC19498 and ARC1 9499 were >95% stable over the course of 72 hours in hunan, monkey and rat serum (Figures 113A and 11313). [00614] ARC/i 9500 was >92% stable over the course of 72 hours in human, monkey and rat sernun (Figure 113C), and ARC19501 was >80% stable over the course of 72 hours in human, monkey and rat serum (Figure 113D). 1006151 ARC19881 was >78% stable over the course of 72 hours in human, monkey and rat serum (Figure 1 13E), and ARC19882 was >91% stable over the course of 72 hours in human, monkey and rat serum (Figure 11.3F). 167 WO 2012/109675 PCT/US2012/024916 EXAMPLE 32 [006161 This example demonstrates that the TFPI aptamers have biological activity. [006171 In this experiment, a non-human primate model of hemophilia A was created by inj acting cynonolgus monkeys with a single intravenous (IV) bolus of sheep polyclonal antibody against human Factor VIII (20 mg; 50,000 Bethesda Units). 3.5 hours after the IV injection, the monkeys were treated with either saline (1 mL/kg), recombinant Factor Vila (rFYIla) (NovoSeven ; 90 pg/kg bolus) or ARC19499 (either a 600 gg/kg, 300 ug/kg or 100 gg/kg bolus). Citrated blood samples were acquired before antibody administration (baseline), 2.5 hours after antibody administration, 15 minutes after drug/saline treatment (time=3.75 hours), and 1 and 2 hours after drug/saline treatment (time=4.5 and 5.5 hours, respectively). Blood was processed to generate plasnma, and citrated plasma samples were assayed for prothrombin time (PT), activated partial thromboplastin time (aPTT), Factor VIII function and thromboelastography (TEG*). The saline-treated monkey received a treatment of ARCi 9499 (600 g/kg) after the 5.5 hour time point was drawn. Fifteen minutes after receiving this treatment, another citrated blood sample was drawn and processed for plasma. [00618] In order to ensure that ARC19499 inhibits monkey TFPI, plasma taken from monkeys after antibody treatment were mixed, ex vivo, with increasing concentrations of ARC19499 from I to 1000 nM, and tested in a TEG* assay (Figure 114). The plasma mixed with aptamer was compared to plasma without aptamer, both before and after antibody treatment (solid and dashed lines, respectively). Antibody treatment alone prolonged the R-value. The addition of ARC 19499 to this antibody-treated plasma corrected the R-value to near baseline levels, suggesting the aptamer was cross-reactive with monkey plasma. [00619] In the plasma sample withdrawn 2.5 hours after antibody injection, Factor VIII levels were below 0.6% and remained there for the course of the 5.5 hour assay (Figure 115). As expected, the PT, which is insensitive to FVIII, remained unchanged after antibody administration (Figure I 16A); however, upon injection of rFVIIa, there was a slight dip in PT values from 13.0 0.4 to 10.7 ± 0.4 seconds. aPTT values increased after administration of the Factor VIII antibody (Figure 1 16B). As seen in the PT assay, rFVIIa administration resulted in a decrease in clotting time in the aPT' assay. There was no change to the aPTT values upon saline treatment. ARCi 9499 treatment (at all concentration s) resul ted in a prolong tion in aPTT. The saline-treated animal that received an ARC 19499 bolus after the 5.5 hour time point also 168 WO 2012/109675 PCT/US2012/024916 showed a prolongation in aPTT after aptamer administration. The effect of ARC 19499 treatment on clot development in this hemophilia A-like model was assayed using tissue factor ('IF) activated TEG". In all animals tested, antibody administration resulted in a prolongation in R value (Figure 11 7A). Saline treatment had no further effect on the R-valiue, while both rFVIIa and the 600 sg/kg and 300 ug/kg ARCI19499 treatments resulted in a decrease in R-value to levels close to baseline. The 100 pg/kg dose of ARC19499 was an ineffective dose and did not have a positive effect on the R-value. In the saline-treated monkey, the additional injection of ARC] 9499 at the end of the study had an immediate effect on decreasing the R-value. The angle (Figure i17B) and maximum amplitude (MA) (Figure I I7C) were both reduced after antibody administration. The 600 iag/kg and 300 pg/kg ARCI19499 treatments appeared to have similar increasing effects on the angle as NovoSeven*; whereas, the 100 g/kg ARC19499 treatment behaved similarly to the saline treatment (Figure 11713). All of the treatments, including saline, appeared to have no additional effect on MA (Figure 1I7C). [00620] In a second set of related experiments, monkeys were treated with the same concentration of anti-FVIII antibody followed by 300 pg/kg ARC 19499 or 90 gg/kg NovoSeven* 1 hour later. Citrated blood samples were acquired and processed for plasma at baseline, 60 minutes after antibody administration, and 60, 120, 180, 240, 300 and 420 minutes after drug administration. Plasma samples were tested using the TF-activated TEGR assay as described above. As before, antibody administration resulted in increased R-values (Figure 11 8A), and decreased angle and MA values (Figure 118B-C). ARCi 9499 and NovoSeven" behaved in very similar manners, restoring the R-values to near-baseline levels and improving both the angle and MA values (Figure 118). [00621] 'Taken together, the Factor VIII, aPTT and TEG* all indicate the successful induction of a hemophilia A-like state in these monkeys. The moderate prolongation of the aPTT upon injection of ARC19499 most likely reflects further, non-specific inhibition of the intrinsic cascade, which has been previously observed with other aptamers. However, the clear correction of the clot time (R-value) in TF-activated plasma measured by the TEG* assay suggests that the hemostatic defect due to loss of Factor VIII was successfully bypassed by ARC19499 inhibition of TFPI. [006221 An additional observation of ARC1 9499 treatment was that it appeared to result in an increase in plasma levels of TFPI. In a preliminary experiment, TFPI concentrations in samples 169 WO 2012/109675 PCT/US2012/024916 from these cynomoigus experiments mentioned above, following ARC 19499 treatment, exceeded the upper limit of quantitation of the TFPI ELISA when diluted 1:40. This effect was analyzed more closely by measuring the TFPI levels in EDTA-plasma samples from cynomolgus monkeys that received a very high dose (20 mg/kg) of ARC 19499 either via IV or subcutaneous (SC) administration. Blood samples were drawn periodically over two weeks, which enabled the assessment of changes in TFPI plasma concentrations following a more prolonged exposure to ARC19499. TFPI concentrations were measured in serially diluted plasma samples (diluted up to 1:1600) at the following timepoints: pre-dose, 0.083 (IV only), 0.5, 2, 6, 12, 36, 72, 120, 168, 240 and 336 hours post-dose. In animals treated with either an IV or SC dose of ARC19499, TFPI levels increased immediately from ~0,2 nM (pre-dose) to 5-7 nM at the first time point, then more gradually over time to peak levels of 91 i 12 and 71 + 2 nM, respectively (Figure 119). The pattern of TFPI levels was similar in the rnonkeys treated with either the IV or SC dose, increasing to peak levels at 72-120 hours followed by a gradual decline. However, the ARC1 9499 ta, estimates were 5 minutes and 24 hours for the IV and SC doses, respectively (data not shown). Thus, although ARCI 9499 stimulated an increase in plasma TFPf in vivo, the changes in TFPI and ARC19499 concentrations over time do riot necessarily correlate. The mechanism for the increase in TFPI is unknown, but it is possible that the initial exposure to ARC 19499 caused the rapid release of TFPI front the endothelial surface while subsequent increases were due to slow release of TFPI from intracellular stores, upregulation of TFP1I expression, and/or inhibition of TFPI clearance mechanisms. This increase in plasma TFI levels upon aptamer treatment was specific for ARC! 9499. An aptamer against a target other than TFPI did not appear to increase TFPI levels, especially not to the same level as ARC19499 (data not shown). EXAMPLE 33 [006231 This example demonstrates that ARC 19499 shortens bleeding time in a non-human primate (NHP) model of hemophilia A and that this shortened bleeding time is reflected in most cases by a concomitant corTection in the thromboelastography (TEG*) R-value (a measure of the time to initial clot formation) from the prolonged R-value associated with hemophilia A and present in the NHP model described below. [006241 Bleeding time, with and without ARC]19499 treatment, was assessed in a NHP model of hemophilia A in which cynomolgus monkeys were dosed with an anti-FVIII antibody (FVIII 170 WO 2012/109675 PCT/US2012/024916 Ab) to induce a hemophilia A-like state. As diagramed in Figure 120, bleeding time was measured at the beginning of the experiment (baseline), and then 2.5 hours after FVIII Ab administration. Treatment with 1 mg/kg bolus doses of ARC 19499 were then begun. Bleeding time was measured 1 hour after the first ARC 19499 dose for all groups of monkeys. For those monkeys whose bleeding times had not corrected, bleeding time was again assessed 17 minutes after the second ARC] 9499 dose. For those monkeys whose bleeding times still had not corrected, bleeding time was again assessed 17 minutes after the third ARC19499 dose (groups 3 and 4). [006251 For bleeding time assessment and collection of blood samples, a monkey was anesthetized, and a line was placed in the femoral vein for blood pressure determinations and the saphenous vein on the opposite side from the saphenous vein used for the bleeding time assessment was catheterized with a 22 gauge catheter for dosing and sampling. The schedule for bleeding time assessment and related FVIII Ab and ARCl 9499 dosing and blood sampling are indicated in Figure 120. A pre-treatment baseline blood sample and a bleeding time (pre treatment bleeding time, BTo) were taken 10 and 5 minutes, respectively, before dosing with FVIII Ab. Blood samples were taken by first withdrawing approximately 3-4 ml- of blood mixed with saline, which was then set aside. An undiluted blood sample was then taken using a syringe. The collected blood was immediately injected into citrate-containing tubes and inverted ten times to mix. A sample of the citrated whole blood was analyzed by TEG* and the remaining citrated blood was processed to obtain plasma for analysis. The first blood-saline sample was then injected back into the monkey, followed by a flush of saline equivalent to the volume of blood sample taken. [00626] The saphenous vein was exposed for the bleeding time assessment. A 22 gauge I inch hypodermic needle (Kendall Monoject, Kendall Healthcare, Mansfield, MA) was carefully bent at the bevel to a 90'C angle using a hemostat; the bend was 5 mm from the tip of the needle. The needle was inserted into the exposed vein up to the bend in the needle, puncturing only one wall of the vein. Blood was wicked away from the vein using Surgicutt Bleeding Time Blotting Paper (ITC, Edison, NJ); care was taken to not touch the actual puncture site. Time was measured from the moment bleeding began until cessation of bleeding, as determined by inability to wick away blood. Blood pressure readings were taken one to five minutes before and 171 WO 2012/109675 PCT/US2012/024916 after bleeding time assessment. After bleeding time assessment, the wound was closed with Gluture (Abbott Labs, Abbott Park, IL). [006271 FYIII Ab (sheep anti-human FVIII polyclonal antibody, Lot Y12 17, from Haematologie Technologies Inc (Essex Junction, VT)) was administered to monkeys intravenously (IV) as a single slow bolus dose via a 22 gauge catheter placed in the saphenous vein on the opposite side from the saphenous vein used for the bleeding time assessment. It was kept frozen at -80 'C until use, and was thawed and re-frozen no more than 3 times before use. Each monkey received 12,642 Bethesda units/kg of FVIII Ab. Two hours after the FVIII Ab was given, another blood sample was taken; 30 minutes later the bleeding time was assessed (post FVIII bleeding time, BTFvm). A first dose of ARC 19499 was administered IV as a single slow bolus. In all cases prior to dosing of either the FactorVIlI Ab or ARC] 9499, 0,5-1.0 mL of fluid were removed from the catheter, the FVIII Ab or A RC19499 was administered, and the catheter was then flushed with 2-3 mL warm saline. A blood sample was taken 55 minutes later. One hour after the administration of the first ARC 19499 dose, the bleeding time was assessed (post first ARC] 9499 dose bleeding time, BT1). Determination was then made as to whether the bleeding tirne had been corrected by the first dose of ARC 19499. Correction was considered to have been attained if the difference between the pre-treatment baseline and post first ARC19499 dose bleeding times was less than half the difference between the pre-treatment baseline and post FVIII bleeding times (i.e., (BT 1 BTo) / (BTrvym BTo) < 0.5). If the bleeding time was successfully corrected, the bleeding time study was completed for that animal. If the bleeding time was not corrected, a second dose of 1 mg/kg ARC 19499 was administered ten minutes after the initiation of the prior bleeding time assessment. A blood sample was taken 12 minutes later; a bleeding time assessment was becun 17 minutes after the second ARC19499 injection (post second ARC19499 dose bleeding time, BT2). Determination was then made as to whether the bleeding time had been corrected by the second dose of ARC 19499. If the bleeding time was successfully corrected, the bleeding time study was completed for that animal. If the bleeding time was not corrected, a third dose of 1 mg/kg ARC 19499 was administered ten minutes after the initiation of the prior bleeding time assessment. A blood sample was taken 12 minutes later a bleeding time assessment was begun 17 minutes after the second ARC19499 injection (post third ARC19499 dose bleeding time, BT 3 ). Determination was then made as to whether the bleeding time had been corrected by the third dose of ARC19499. Regardless of whether the 172 WO 2012/109675 PCT/US2012/024916 bleeding time was successfully corrected, the bleeding time study was completed for that animal. If the bleeding time was not corrected, a fourth dose of 3 mg/kg ARC19499 was administered 14 minutes after the initiation of the prior bleeding time assessment; a blood sample was taken for whole blood TEG* analysis but no further bleeding times were assessed due to lack of available vein of type similar to that used in the study. Additional ARC19499 (1 to 3 mg/kg) was administered prior to recovery of each animal as a precaution against bleeding over the next 24 hours. The actual time points for blood sampling, compound dosing and bleeding time assessment for each animal are within 5 minutes of the time point reported. [006281 FVIIIa activity levels in citrated plasma from monkeys in this study were measured using the Coamatic FVIII assay from Chromogenix (Diapharma, Columbus 01H). Samples acquired during the study were compared to a standard curve generated from a pool of the pre treatment baseline plasma samples. All samples and standards were diluted 80x in reaction buffer provided by the kit. The reaction was carried out as per the manufacturer's instructions, reading the change in absorbance at 405 nm over 45 minutes. The FV II activity levels associated with the cynornolgus monkey plasma samples are shown in Table 18 and Figure 121 (Table 18A and Figure 121 A: Group 1: monkeys whose bleeding times were corrected with one dose of 1 trg/kg ARC 19499; Table 18B and Figure 121 B: Group 2: monkeys whose bleeding times were corrected with two doses of 1 mg/kg ARC19499; Table I8C and Figure 121C: Group 3: monkey whose bleeding time was corrected with three doses of 1 mg/kg ARC 19499; Table 18D and Figure 12 1D: Group 4: monkey whose bleeding time was not corrected with three doses of 1 mg/kg ARC 19499). Prior to antibody treatment, the plasma from all monkeys had very high FV I I activity levels, varying from 57.6% to 93.5% (Table 18). After antibody treatment, this activity dropped to below measurable levels (less than 0. 1%) in all animals, and remained low for the course of the assay (Figure 121). Most importantly, there was no difference noted by this assay in the level of FVIII inactivation between the different groups of animals, indicating that the dose of ARC 19499 required to correct the bleeding time was not a related to differing levels of FVIII activity post FVIII Ab administration. Table 18. FVii activity levels 173 WO 2012/109675 PCT/US2012/024916 A. Group 1: Monkeys Whose Bleeding Times were Corrected by One Dose of 1 mg/kg ARC19499 Time NHP NHP NHP NHP NHP NHP point 0703333 0704039 0701565 0604313 0703551 0603477 (min) % % % % %/% Baseline -10 64.4 57.6 74.1 93.5 71.8 82.4 2 hr post 120 -1.6 -2.2 -1.8 -1.5 -1.8 -2.6 FVIHI ab treatment 55 min 210 1.5 -2.1 -1L8 -2.2 -1.6 -2 3 post first ARC19499 treatment B. Group 2: Monkeys Whose Bleeding Tines were Corrected by Two Doses of 1 mg/kg ARC19499 Time NHP NHP NHP NHP point 0610301 0702277 0611655 0511011 (Minl) % % % % Baseline -10 63.0 79.6 59.5 89.7 2 hr post FVIHI ab 120 -2.1 -0.3 -2.3 -2.0 treatment 55 min post first 210 -2.2 -0.1 -22 -1 9 ARC 19499 treatment 12 min post second 237 ns 0.1 -22 -17 ARC 19499 treatment C. Group 3: Monkey Whose Bleed ing Time was Corrected by Three Doses of 1 mg/kg ARC19499 Time NHP0702073 point (min)} Baseline -10 76.8 2 hr post FVI ab treatment 120 0.0 55 min post first ARC19499 treatment 210 -0.2 12 min post second ARC19499 treatment 239 -0.3 12 min post third ARC19499 treatment 278 0.3 D. Group 4: Monkey Whose Bleeding Time was Not Corrected by Three Doses of 1 mg/kgARC19499 Time NHP0607367 point (uin)% Baseline -10 62.5 2 hr post FVIII ab treatment 120 0.8 55 min post first ARC 19499 treatment 210 -1.4 12 min post second ARC19499 treatment 237 -2.0 174 WO 2012/109675 PCT/US2012/024916 _3Ip st third ARC19499 treatment 264 -20 _12 min fourth ARC19499 treatment 296 _ [006291 The mean group bleeding times (+ SEM) for Group I monkeys (whose bleeding times were corrected with one dose of 1 mg/kg ARC19499) are shown in Table 19, The mean group bleeding times for this group are also plotted against the time points of the blood samples in Figure 122 (Figure 122A: in seconds, Figure 122B: in terms of % of baseline bleeding time). Treatment with the anti-FVIII antibody resulted in a prolongation of the group mean bleeding time to 203 15% of the baseline group mean bleeding tirne. Treatment of the rnonkeys with I mg/kg ARC 19499 corrected the group mean bleeding time back to essentially baseline levels (102 +4 11% of the baseline group mean bleeding time). Individual bleeding times for Group 1 monkeys are shown in Table 20; the individual monkey bleeding times at each time point are also plotted in Figure 123 (Figure 123A: in seconds, Figure 12313: in terms of% of baseline bleeding time). All monkeys in this group showed a prolongation of their bleeding times in response to administration of FV III Ab compared to their baseline bleeding times (range: 162 to 252% of the baseline bleeding time). All monkeys in this group also exhibited a correction of their bleeding times in response to administration of I mug/kg ARC19499 compared to their baseline bleeding times (range: 82 to 152% of the baseline bleeding time). Table 19. Mean Bleeding Times in Monkeys Whose Bleeding Times were Corrected by One Dose of I mg/kg ARC 19499 (Group 1) _roui. Mean Bleeding Times Tie-oMe- ian SE N1 (Mi) (see) (see) Baseline -5 41 5.1 2,5 hr post FVII ab treatment 150 84 13 1 hr post ARC 19499 treatment 215 40 3.5 Table 20. Individual Bleeding Times in Monkeys Whose Bleeding Times were Corrected by One Dose of 1 mg/kg ARC 19499 (Group 1) Group 1. Individual Bleedng Times__________ Time NHP NHP NHP NHP NHP NHP point 0703333 _ 0704039 0701565 0604313 0703551 060347 (1Hmi) (see (see) e) - (see) (see) . see) Baseline ~5 36 23 45 42 41 61 2,5 hr post 150 58 85 68 85 145 FVII ab treatment Ihpost 215 3135 37 43854 175 WO 2012/109675 PCT/US2012/024916 ARC19499 treatment 1006301 The mean group bleeding times ( SEM) for Group 2 monkeys (whose bleeding times were corrected with two doses of 1 mg/kg ARC19499) are shown in Table 21. The mean group bleeding times for this group are also plotted against the time points of the blood samples in Figure 124 (Figure 124A: in seconds, Figure 124B: in terms of % of baseline bleeding time). Treatment with the anti-FVIII antibody resulted in a prolongation of the group mean bleeding time to 195 ± 26% of the baseline group mean bleeding time. After treatment of the monkeys with 1 mg/kg ARC19499, the group mean bleeding time did decrease, but only to 175 ± 20% of the baseline group mean bleeding time. An additional dose of 1 mg/kg ARC19499 subsequently reduced the group mean bleeding time to essentially baseline levels (94 ± 17% of the baseline group mean bleeding time). Individual bleeding times for Group 2 monkeys are shown in Table 22; the individual monkey bleeding times at each time point are also plotted in Figure 125 (Figure 125A: in seconds, Figure 125B: in terms of % of baseline bleeding time). All monkeys in this group showed a prolongation of their bleeding times in response to administration of FVIII ab compared to their baseline pre-treatment bleeding times (range: 143 to 263% of the baseline bleeding time). After administration of I mg/kg ARC19499, three of the monkeys exhibited a slight decrease in their bleeding times while one monkey showed a small increase in bleeding time. All monkeys in this group exhibited a correction of their bleeding times in response to a second dose of 1 mg/kg ARC] 9499 (range: 61 to 138% of baseline bleeding time). Table 21. Mean Bleeding Times in Monkeys Whose Bleeding Times Were Corrected by Two Doses of I mg/kg ARC 19499 (Group 2) Group 2: ean Beedin Timei n _s Time point Mean SEM (min) (see) (see) Baseline -5 35 12 2.5 hr poos t FVI II ab treatment 150 62 13 1 hr post first ARC19499 treatment 215 59 60 17 min post second 242 31 8.4 ARC19499 treatment Table 22. Individual Bleeding Times in Monkeys Whose Bleeding Times Were Corrected by Two Doses of 1 mg/kg ARC19499 (Group 2) 176 WO 2012/109675 PCT/US2012/024916 Group 2:_Individnal Bleeding Times Time NP NP NI-IP NHP point 0610301 0702277 0611655 0511011 (min) (see) (sec) (see) (see) Baseline -5 29 23 70 19 2.5 hr post FVIII ab 150 60 38 100 50 treatment 1 hr post first 215 50 33 107 44 ARC19499 treatment 17 min post 242 40 14 50 20 secondARC 19499 treat mend [006311 The bleeding times for the Group 3 monkey are shown in Table 23; the bleeding times are also plotted against the time points of the blood samples in Figure 126 (Figure 126A: in seconds, Figure 126B: in terms of % of baseline bleeding time). This monkey showed a prolongation of its bleeding time in response to administration of FVIII Ab to 220% of the baseline bleeding time. In response to treatment with 1 mg/kg dose of ARC19499, the bleeding time increased to 330% of the baseline. Treatment of this monkey with two doses of 1 mg/kg ARC1 9499 produced a reduction of the bleed time to 210% of the baseline bleeding time. To confirm that this second dose had indeed not corrected the bleeding time, an additional bleeding time assessment was performed 38 minutes after administration of the second ARC19499 dose. This bleeding time of 45 seconds was very close to the 42 seconds measured 12 minutes after the second dose of ARC1 9499 was given, confirming that the bleeding time had not been corrected by the two doses of ARC 19499. An additional dose of 1 mg/kg ARC 19499 corrected the bleeding time to 85% of the baseline bleeding time. Table 23. Bleeding Times in Monkey Whose Bleeding Time was Corrected by Three Doses of 1 mg/kg ARC19499 (Group 3) _Groupg3 Bleeding Times Time NHP0702073 point (mian) (see) Baseline -5 20 2.5 hr post FVIT ab treatment 150 44 1 hr post first ARC 19499 treatment 215 66 17 rmin post second ARC19499 treatment 243 42 37 rmin post second ARC19499 treatment 263 45 _17 minpost third ARC19499 treatment 282 _ 1 177 WO 2012/109675 PCT/US2012/024916 [006321 The bleeding times for the Group 4 monkey are shown in Table 24; the bleeding times are also plotted against the time points of the blood samples in Figure 127 (Figure 127A: in seconds. Figure 127B: in terms of % of baseline bleeding time). This monkey showed a prolongation of its bleeding time in response to administration of FVIII Ab to 143% of the baseline bleeding time. In response to treatment with 1 mg/kg dose of ARCI 9499, the bleeding time increased markedly to 193% of the baseline bleeding time. The bleeding time after treatment of this monkey with two doses of 1 mg/kg ARC1 9499 then decreased to 154% of the baseline bleeding time. An additional dose of 1 mg/kg ARC 19499 failed to significantly change the bleeding time, which was now 159% of the baseline bleeding time. No additional bleeding time assessments could be done on this animal due to a lack sufficient available vein consistent with that used for previous assessments. Table 24. Bleeding Tirnes in Monkey Whose Bleeding Time was Not Corrected by Three Doses of 1 mg/kg ARC1 9499 (Group 4) Group 4: Bleeding Times Time NHP0607367 insec) Baseline 56 2.5 hr post FVIII ab treatment 150 80 1 hr post first ARC19499 treatment 215 108 17 "m.rnost second ARC19499 treatment 243 86 _I7mnppost third ARC19499 treatment_ _ 73 89 [006331 The coagulation status of the cynomolgus whole blood was analyzed using the TEG* assay on citrated whole blood samples. To initiate the clotting reaction, 330 tL citrated whole blood was added to a disposable cup (Haemonetics Corp, cat no, 6211) containing 20 pL 0.2 M [Haemonetics Corporation (Braintree, MA)] and 10 pL tissue factor (TF) (final dilution of 1:200000 at 37 'C). Innovin (Dade-Behring, Newark, DE) was used as a tissue factor (TF) source, reconstituted in water as per manufacturer recommendation, and diluted 1:5555 in 0.9% saline prior to use. Reconstituted stock innovin was stored at 4 0 C for less than 4 weeks. The time to initial clot formation (R-value) was measured using the Iaemoscope TEG* 5000 system (I-aemonetics Corporation, Braintree, MA). [00634] The mean group R-values (i SEM) for Group I monkeys (whose bleeding times were corrected with one dose of 1 rng/kg ARC] 9499) are shown in Table 25. The mean group R values are also plotted against the time points of the blood sample (Figure 128). Treatment with FVIII Ab resulted in a prolongation of the group mean Rvalue to about 4.8 times the group 178 WO 2012/109675 PCT/US2012/024916 mean R-value at baseline. While treatment of the monkeys with 1 mg/kg ARC19499 reduced the group mean R-value from that obtained after FVIll Ab treatment, this R-value was still about 3.2 times the group mean baseline R-value. Individual R-values for Group I monkeys are shown in Table 26; the individual R-values are also plotted against the time points of the blood sample in Figure 129. All monkeys in this group showed a prolongation of their R-values in response to administration of FVIII ab compared to their baseline pre-treatment R-values (range 2.9 to 9.4 fold). All but one of the monkeys in this group also exhibited a reduction of their R-values in response to administration of 1 mg/kg ARC 19499 compared to their baseline pre-treatment R values (range 1.4 to 5.3-fold). One monkey, NHP0701 565, showed a slight increase in the R value after administration of 1 mg/kg ARC 19499. Table 25. Mean Whole Blood TEG" R-values in Monkeys Whose Bleeding Times were Corrected by One Dose of 1 rng/kg ARC1 9499 (Group 1) Group 1: Mean Whole Blood TEG R-values ,Time point kMean SEM Baseline -10 5.6 0.93 2 hr post FV11 ab treatment 120 27 3.3 55 min post ARC 19499 treatment 210 18 4.8 Table 26. Individual Whole Blood TEG* R-values in Monkeys Whose Bleeding Times were Corrected by One Dose of I mg/kg ARC19499 (Group 1) Group 1: Individual Whole Blood TEG- R-values Tme NHP NHP NHP NHP NHP NHP point 0703333 0704039 0701565 0604313 0703551 0603477 (min) (Min) (min) (inf)-- (min) mn) (mi Baseline -10 8. nS 6.0 2.2 5.0 6.2 2 hr post 120 38 6 28.8 34 20.7 22.6 17.8 FVIII ab treatmnet 55 mi 210 294 10.9 35.5 11.6 9.8 8.4 post ARC19499 treatmentL [00635] The mean group R-values (± SEM) for Group 2 monkeys (whose bleeding times were corrected with two doses of 1 mg/kg ARC 19499) are shown in Table 27. The mean group R values are also plotted against the time points of the blood samples in Figure 130, Treatment with the anti-FVIII antibody resulted in a prolongation of the group mean R-value to about 5.9 times the group mean R-value at baseline. In response to treatment with I mg/kg dose of 179 WO 2012/109675 PCT/US2012/024916 ARC19499, which did not correct the bleed time, the group mean R-value was reduced to about 4.0 times the baseline group mean R-value. Treatment of the monkeys with two doses of I mg/kg ARC19499, which did correct the bleed time in this group, further reduced the group mean R-value from that obtained after FViii Ab treatment, although this R-value was still about 3.5 tines the baseline group mean R-value. Individual R-values for Group 2 monkeys are shown in Table 28; the individual R-values are also plotted against the time points of the blood sample in Figure 131. All monkeys in this group showed a prolongation of their R_-values in response to administration of FVIII Ab compared to their baseline pre-treatment R-valies at baseline (range 4.0 to 9.1-fold). All but one of the monkeys in this group also exhibited a reduction of their R values in response to administration of 1 mg/kg ARC 19499 compared to their baseline pre treatment R-values (range 2.4 to 5.0-fold); treatment of these monkeys with two doses of I mg/kg ARC 19499, which did correct all of the bleed times in this group, reduced further the R values in two of these monkeys, and slightly increased the R-value in the third monkey. One monkey, Ni-P0611655, showed a 103% increase in the R-value compared with the R-value post FVJJJ Ab injection after administration of 1 mg/kg AR C19499; treatment of this monkey with an additional 1 mg/kg AR-C19499, which did correct the bleeding tirne, produced a decrease in the R-value to 6.4 times the baseline R-value. Table 27. Mean Whole Blood TEG" R-values in Monkeys Whose Bleeding Times were Corrected by Two Doses of 1 mg/kg ARC 19499 (Group 2) Group 2: Mean Whole Blood TEG R-values Time point Mean SEM (min) (min) (minl) Baseline -10 6,0 1L0 2 hr pos FY11! ab treatment 120 16 4,5 55 min post first ARC19499 treatment 210 24 8.7 12 min post second ARC19499 treatment 237 21 7.1 Table 28. Individual Whole Blood TEG* R-values in Monkeys Whose Bleeding Times were Corrected by Two Doses of 1 mg/kg ARC19499 (Group 2) Group 2: Individual Whole Blood TEFG :R-values Time NHP NHP NHP NHP - point 0610301 0102277_ 0611655 0511011 (min) (min) (min) (min) (min) Baseline -10 ns 8.1 6.0 4.0 2 hr post FV11I ab 120 46.3 5.8 24.1 36.2 treatment 55 min post first 210 8.2 19.8 48.9 19.9 180 WO 2012/109675 PCT/US2012/024916 ARC19499 treatment 12 min post 237 6,7 1 L9 38.3 25.6 ARC19499 treatment [00636] The R-values for the Group 3 monkey are shown in Table 29; the R-values are also plotted against the time points of the blood samples in Figure 132. This monkey showed, in response to administration of FVIII Ab, a prolongation of its R-value to 7.6 times its baseline R value. In response to treatment with a 1 mg/kg dose of ARC 19499, which did not correct the bleed time, the R-value was reduced to 6.1-fold of the baseline R-value. Treatment of this monkey with two doses of 1 mg/kg ARC 19499, which also did not correct the bleed time in this monkey, ffirther reduced the k-value slightly, to 5.8 times that of the baseline. An additional dose of 1 mg/kg ARC 19499, which did correct the bleed time, reduced the R-value to 3 times the baseline R-value. Table 29. Whole Blood TEG* R-values in a Monkey Whose Bleeding Time was Corrected by Three Doses of I mg/kg ARC19499 (Group 3) Group 3: Whole Blood TEG R-values Time NHP0702073 (min) (min) Baseline -10 39 2 hr post FV1 ab treatment 120 29.5 55 min post first ARC19499 treatment 210 23.6 12 miin post second ARC19499 treatment 239 22.5 12 iin _post third A119499 treatment _7_ 1 8 [006371 The R-values for the Group 4 monkey are shown in Table 30; the R-values are also plotted against the time points of the blood samples in Figure 133. This monkey showed, in response to administration of FVIII Ab, a prolongation to 2.9-fold times its baseline R-value. In response to treatment with I rng/kg dose of ARC 19499, which did not correct the bleed time, the R-value was reduced to 1.6 fold times the baseline R-value. Treatment of this monkey with two doses of 1 mg/kg ARC1 9499, which also did not correct the bleed time in this monkey, further reduced the R-value slightly, to 1.5-fold times the baseline R-value. An additional dose of I mg/kg ARC19499, which still did not correct the bleed time, reduced the R-value slightly more to 1.2 times the baseline R-value. A fourth dose of 3 ng/kg ARC19499 had little effect on the R-value, which was now 1.1 times the pre-treatment baseline R-value. No additional bleeding 181 WO 2012/109675 PCT/US2012/024916 time assessments could be done on this animal due to a lack sufficient available vein consistent with that used for previous assessments. Table 30. Whole Blood TEG* R-values in a Monkey Whose Bleeding Time was Not Corrected by Three Doses of 1 mg/kg ARC 19499 (Group 4) Group 4: Whole Blood lEG It-values Time NH1P0607367 point Baseline -10 72 2 hr post FVIH ab treatment 120 21.2 55 min post first ARC19499 treatment 210 11.2 12 min post second ARC19499 treatment 237 10.7 12 min post third ARC19499 treatment 264 8.8 12mm nost fourth AR( 19499 treamtnent 196 _ _ 1006381 The coagulation status of the cynomolgus plasma was also analyzed using the TEG* assay on plasma from the citrated blood sarnples. Citrated whole blood samples were kept at until plasma processing. Samples were centrifuged at 2,000xg for 15 minutes at room temperature. The plasma was removed and immediately stored at -80 'C until shipped for analysis. Prior to analysis, plasma samples were thawed rapidly at 37 0 C. To initiate the clotting reaction, 330 [L citrated plasma was added to a disposable cup (-Iaemonetics Corporation, Braintree, MA, cat no. 6211) containing 20 pL 0.2 M (I-aemonetics Corporation (Braintree, MA)) and 10 pL TF (final dilution of 1:200000 at 37 C). limovin (Dade-Behring, Newark, DE) was used as a tissue factor (TF) source, reconstituted in water as per manufacturer recommendation, and diluted 1:5555 in 0.9% saline prior to use. Reconstituted stock Innovin was stored at 4 'C for less than 4 weeks. The time to initial clot formation (R-value) was measured using the Haemoscope TEG 5000 system (Haemonetics Corporation, Braintree, MA). 1006391 The mean group R-values (i SEM) of plasma samples from Group 1 monkeys (whose bleeding times were corrected with one dose of 1 mg/kg ARC 19499) are shown in Table 31. The mean group R-values are also plotted against the time points of the plasma sample (Figure 134). Treatment with FVIII Ab resulted in a prolongation of the group mean R-value to about 2.8 times the group mean R-value at baseline. While treatment of the monkeys with I mg/kg ARC 19499 reduced the group mean R-value from that obtained after FVIll Ab treatment, this R-value was still about 1.9 times the group mean baseline R-value. Individual R-values for Group 1 monkeys are shown in Table 32; the individual R-values are also plotted against the 182 WO 2012/109675 PCT/US2012/024916 time points of the plasma sample in Figure 135. All monkeys in this group showed a prolongation of their R-values in response to administration of FVIII Ab compared to their baseline pre-treatment R-values (range 1.7 to 5.1-fold). All but one of the monkeys in this group also exhibited a reduction of their R-values in response to administration of 1 mg/kg ARC 19499 compared to their baseline pre-treatment R-valies (range 1,3 to 1.8-fold). One monkey, NIHP0603477, showed a 50% increase in the R-vailue compared with the R-valie post FVIii Ab injection after administration of 1 mg/kg ARCI 9499; the whole blood TEG* analysis had instead shown a 53% decrease in the R-value compared with the R-value post FVIII Ab injection after administration of 1 mg/kg ARC19499. The plasma TEG* analysis of NP0701565, whose whole blood TEG* analysis had shown a slight increase in R-value in response to administration of 1 mg/kg ARC 19499, was actually 66% below the R-value post FV11l Ab injection after administration of 1 mg/kg ARCI 9499. Table 3 1. Mean Plasma TEG*C R-values in Monkeys Whose Bleeding Times were Corrected by one Dose of 1 mg/kg ARC19499 (Group 1) Group 1: Mean Plasma TEG R-values Time point Mean SEM Baselin_(_nin)_ _ (_ in) (nin) baseline -10 5.4 0.37 2 hr post FV1 ab treatment 120 15 1.6 55 min post ARC 19499 treatment 210 10 1.7 Table 32, Individual Plasma TEG k-values in Monkeys Whose BIceding Times were Corrected by One Dose of I mg/kg ARC19499 (Group 1) Group 1: Individual Plasma TEG R-values Time NHP N HP NHP NHP NHP NHP I point 0703333 0704039 0701565 0604313 0703551 0603477_ (min) (min) (min) (min) (min) (min) (min) Baseline -10 5,o 6.0 4.3 4,6 6.3 6.4 2 hr post 120 12 2 16.6 21.9 15.5 14.1 10.8 FVIII ab treatmuent 55 min 210 92 7.8 7.4 ns ns 16.2 post ARC19499 treat end [006401 The mean group R-values (I SEM) of plasma samples from Group 2 monkeys (whose bleeding times were corrected with two doses of 1 ig/kg ARC19499) are shown in Table 33. The mean group R-values are also plotted against the time points of the blood samples 183 WO 2012/109675 PCT/US2012/024916 in Figure 136. Treatment with the anti-FVIII antibody resulted in a prolongation of the group mean R-value to about 4.0-fold of the group mean R-value at baseline. In response to treatment with 1 mg/kg dose of ARC19499, which did not correct the bleed time, the group mean R-valuc was reduced to about 2.2-fold times the baseline group mean R-value. Treatment of the monkeys with two doses of 1 mg/kg ARC 19499, which did correct the bleed time in this group, did not significantly reduce further the group mean R-value from that obtained after FVIII Ab treatment. Individual R-values for Group 2 monkeys are shown in Table 34; the individual R values are also plotted against the time points of the blood sample in Figure 137. All monkeys in this group showed a prolongation of their R-values in response to administration of FVIII Ab compared to their baseline pre-treatment R-values at baseline (range 1.1 to 6.4-fold). In response to treatment with 1 mg/kg dose of ARC19499, which did not correct the bleed times, the plasma R-values of all monkeys in this group were reduced over the baseline group mean R-value (range 0.5 to 4.5-fold). Treatment of the monkeys with two doses of I mg/kg ARC19499, which did correct all of the bleed times in this group, produced unchanged or further reduced R-values, although in two monkeys the R-values were still higher than those at the pre-treatment baseline (range 0.5 to 3.2-fold). Table 33. Mean Plasma TEG I R-values in Monkeys Whose Bleeding Times were Corrected by Two Doses of 1 mg/kg ARC19499 (Group 2) Group 2. Mean Plasma TEC- R-values Time point Mean SEM (min) (min) (min) Baseiti e -10 5.X 0.6 2hr post FY11! ab treatment 120 21 6.5 55 min post first ARC19499 treatment 210 13 4.5 12 min post second ARC 19499 treatment 237 1 3.7 Table 34. Individual Plasma TEG® R-values in Monkeys Whose Bleeding Times were Corrected by Two Doses of I mg/kg ARC 19499 (Group 2) Group 2: Individual Plasma TEG R-values Time NHP NHP NHP NHP - point 0610301 0702277 0611655 0__511011 (min) (min) (min) (min) (min) Baseline -10 4.6 2 5.3 6.0 2 hr post FVIH ab 120 15.4 8.1 33.9 33.7 treatment 55 min post first 210 7.6 315 237 16,8 ARC19499 treatment i po t237 --- ---------- 17--- - 184 WO 2012/109675 PCT/US2012/024916 ARC19499 treatment- _[_______-----_J__________-___________-------------- [006411 The R-values of the plasma samples from the Group 3 monkey are shown in Table 35; the R-values are also plotted against the time points of the blood samples in Figure 138. This monkey showed, in response to administration of FVIII Ab, a prolongation of its R-value to 3.5 fold times its baseline R-value. In response to treatment with 1 mg/kg dose of ARC 9499, which did not correct the bleed tirne, the R-value was reduced to 1.3 times the baseline R-value. Treatment of this monkey with two doses of 1 mg/kg ARC 19499, which also did not correct the bleed time in this monkey, further reduced the R-value to 1.1 times that of the baseline. An additional dose of I mg/kg ARC 19499, which did correct the bleed time, reduced the R-value to 0.9 times the baseline R-value. Table 35. Plasma TEG" R-valucs in a Monkey Whose Bleeding Time was Corrected by Three Doses of 1 mg/kg ARC 19499 (Group 3) Group 3: Plasma TEG- R-vahes Time NHP0702073 point (n__ _ _ _ min) m) Baseline -10 4.7 2 hr post FVIH ab treatment 120 16.4 55 min post first ARC19499 treatment 210 6.2 12 mi post second ARC19499 treatment _ _9 5.2 12m m post third ARC19499 treatment __78___ _____4._____ [006421 The R-values of plasma samples from the Group 4 monkey are shown in Table 36; the R-values are also plotted against the time points of the blood samples in Figure 139. This monkey showed, in response to administration of FVIII Ab, a prolongation to 2.9 times its baseline R-value. In response to treatment with I mg/kg dose of ARC19499, which did not correct the bleed time, the R-value was reduced over the baseline R-value to 1. 1 times the baseline R-value. Plasma from blood taken after treatment of this monkey with two doses of 1 mg/kg ARC] 9499, which also did not correct the bleed time in this rnonkey, exhibited a slightly higher R-value of 1.2 times the baseline R-value. An additional dose of 1 mg/kg A R-C19499, which still did not correct the bleed time, reduced the R-value slightly more to 1.0 times the baseline R-value. A fourth dose of 3 mg/kg ARC 19499 reduced the R-value to below the pre treatment baseline (0.9 times the pre-treatment baseline R-value). No additional bleeding time 185 WO 2012/109675 PCT/US2012/024916 assessments could be done on this animal due to a lack sufficient available vein consistent with that used for previous assessments. Table 36. Plasma TEG" R-values in a Monkey Whose Bleeding Time was Not Corrected by Three Doses of 1 mg/kg ARCI 9499 (Group 4) Group 4: Plasma TEG R-values Tine N11P0607367 point Baseline 10 6,4 2 hr post FVIII ab treatment 120 18.5 55 min post first ARC19499 treatment 210 6.8 12 min post second ARC19499 treatment 237 7.5 12 min post third ARC19499 treatment 264 6.2 12 min post fourth ARC'19499 treatment 296 5.8 [00643] The above example shows that monkeys treated with the FVIII Ab exhibited prolonged bleeding after puncture of the saphenous vein, an observation consistent with the prolonged bleeding that is the hallmark of hemophilia. In a inajority of monkeys tested in this study (II of 12), treatment with up to 3 ng/kg ARCI 9499 corrected this prolonged bleeding time. Six of the monkeys only required one dose of I ng/kg ARC19499 to exhibit this correction; bleeding time in four other monkeys was corrected with two doses of 1 mg/kg ARC 19499. R-values in TEG* analysis of the whole blood from these monkeys exhibited the expected elevation after FVIII Ab administration; this elevation was reduced towards baseline levels after treatment with ARC 19499; a similar pattern was seen in the analysis of the plasma from these blood samples. These data show that ARC 19499 can correct prolonged bleeding in a model of induced hemophilia, supporting the potential clinical utility of ARCi 9499 as a successful therapeutic in the treatment of inhibitor and non-inhibitor hemophilia A patients. EXAMPLE 34 [006441 This example is an evaluation of tolerated and non-tolerated substitutions in ARC 17480 through aptamer medicinal chemistry. [006451 Molecules were generated for testing with modifications at the 2'-position or within the phosphate backbone of residues in ARC17480, as shown in Table 37 below and in Figures 140 and 141. Each individual 2'-deoxy residue in ARC1480 was replaced by the corresponding 186 WO 2012/109675 PCT/US2012/024916 2'-methoxy or 2'-fluoro containing residue, resulting in ARC18538-ARC18541 and ARC19493 ARC19496, respectively (Figure 140). Additionally, some molecules with multiple 2'-deoxy to 2'-methoxy and/or 2'-deoxy to 2'-fluoro residues were generated at the four deoxycytidine residues in ARC17480 at positions 9, 14, 16 and 25, resulting in ARC18545, ARC18546, ARC18549, ARC19476, ARCl9477, ARC19478, ARC19484, ARC19490 and ARC19491 (Figure 140). Each individual 2'-methoxy residue in ARC17480 was replaced by the corresponding 2'-deoxy residue, with 2'-nethoxyuridine residues replaced with both 2' deoxythymidine and 2'-deoxyuridine residues, resulting in ARC] 9448-ARC19475 and ARC33867-ARC33877 (Figure 140). The phosphate between each pair of nucleotides in ARCI 7480 was replaced individually with a phosphorothioate, resulting in ARCI 9416 ARC19447 (Figure 141). 1006461 The modified ARC 17480 molecules were assayed for binding and function. The assays used for this evaluation were the calibrated automated thrombograrn (CAT) assay, a dot blot binding-competition assay and a FXa activity assay. The results of these assays are summarized in Table 37 and depicted in Figure 142. A substitution was considered tolerated for activity if it met the criteria of at least two of the three assays that were carried out. Substitutions that were tolerated ("yes") and not tolerated ("no") are identified in Table 37. The experimental details and the criteria used for each assay are described in the following paragraphs. 1006471 The TFPI-inhibitory activity of each molecule was evaluated in the CAT assay in pooled hemophilia A plasma at 500 nM, 166.67 nM, 55.56 nM, 18.52 nM, 6.17 nM and 2.08 nM aptamer concentration. ARC17480 was included in every experiment as a control. For each molecule, the endogenous thrombin potential (ETP) and peak thrombin values at each aptamer concentration were used for analysis. The ETP or peak thrombin value for hemophilia A plasma alone was subtracted from the corresponding value in the presence of aptamer for each molecule at each concentration. Then. the corrected ETP and peak values were plotted as a function of aptamer concentration and fit to the equation y=(max/(1 + ICso/x)) + int, where y=ETP or peak thrombin, x=concentration of aptamer, max=the maximum ETP or peak thrombin, and int=the y intercept, to generate an IC 50 value for both the ETP and the peak thrombin, The IC 5 0 of each aptamer was compared to the IC 5 o of ARC 17480 that was evaluated in the same experiment. A substitution was considered tolerated in the CAT assay if both the ETP and peak thrombin IC 0 of that molecule were not more than 5-fold greater than that of ARC 17480 evaluated in the same 187 WO 2012/109675 PCT/US2012/024916 experiment. Tolerated substitutions are indicated in Table 37 as meeting the assay criteria ("yes") or not meeting the assay criteria ("no"). [006481 Each molecule was evaluated for binding to tissue factor pathway inhibitor (TFPI) in a binding-competition assay. For these experiments, 10 nM human TFPI (American Diagnostica, Stamford, CT, catalog #450OPC) was incubated with trace amounts of radiolabeled ARC] 7480 and 5000 nM, 1666.67 nM, 555.56 nM, 185.19 nM, 61,73 nM, 20.58 nM, 6.86 nM, 2.9 nM, 0.76 nM or 0,25 nM of unlabeled competitor aptamer. ARC17480 was included as a competitor in every experiment as a control. For each molecule, the percentage of radiolabeled ARC17480 bound at each competitor aptamer concentration was used for analysis. The percentage of radiolabeled ARC 17480 bound was plotted as a function of aptamer concentration and fit to the equation y=(max/(i + x/ICs)) + int, where y=the percentage of radiolabeled ARC 17480 bound, x:::the concentration of aptamer, max:::the maximum radiolabeled ARC 1480 bound, and int=:the y-intercept, to generate an ICo value for binding-competition. The IC.o of each aptamer was compared to the ICo of ARC l?480 that was evaluated in the same experiment. A substitution was considered tolerated in the binding-competition assay if the IC 50 of that molecule was not more than 5-fold greater than that of ARC 1480 evaluated in the same experiment. Tolerated substitutions are indicated in Table 37 as meeting the assay criteria ("yes") or not rneeting the assay criteria ("no"). 1006491 Each molecule was evaluated for inhibition of TFPI in a Factor Xa (FXa) activity assay. The ability of FXa to cleave a chromogenic substrate was measured in the presence and absence of TFI, with or without the addition of aptamer. For these experiments, 2 nM human FXa was incubated with 8 nM human TFPL, Then, 500 pM chromogenic substrate and aptamers were added and FXa cleavage of the substrate was measured by absorbance at 405 nrn (A 405 ) as a function of time. Aptamers were tested at 500 nM, 125 nM, 31.25 nM, 7.81 nM, 1.95 nM and 0.49 nM concentrations. ARC 1480 was included as a control in each experiment. For each aptamer concentration, the A 4 0 5 was plotted as a function of time and the linear region of each curve was fit to the equation v:::mx + b, where y:::A4 05 , x:::the aptamer concentration, m:::the rate of substrate cleavage, and b:=the y-intercept, to generate a rate of FXa substrate cleavage. The rate of FXa substrate cleavage in the presence of TFPI and the absence of aptamer was subtracted from the corresponding value in the presence of both TFPI and aptamer for each molecule at each concentration. Then, the corrected rates were plotted as a function of aptamer 188 WO 2012/109675 PCT/US2012/024916 concentration and fit to the equation y=(Vmax/(1 ± IC 5 /x)), where y=the rate of substrate cleavage, x=concentration of aptamer, and Vmax=the maximum rate of substrate cleavage, to generate an IC 50 and maximum (Vmax) value. The ICj 0 and Vmax values of each aptamer were compared to the IC 50 and Vmax values of ARC 17480 that was evaluated in the same experiment. A substitution was considered tolerated in the FXa activity assay if the IC 0 of that molecule was not more than 5-fold greater than that of ARC 17480 evaluated in the same experiment and the Vmax value was not less than 80% of the Vmax value of the ARC17480 evaluated in the same experiment. Tolerated substitutions are indicated in Table 37 as meeting the assay criteria ("yes") or not meeting the assay criteria ("no"). [00650] This example demonstrates that multiple individual 2'-substitutions in ARC17480 are tolerated for binding and activity, and that some combinations of 2'-substitutions are also tolerated (Table 37 and Figure 142). This example also demonstrates that a phosphorothioate substitution is tolerated between each pair of nucleotides in ARC 1480 (Table 37). Additional combinations of tolerated 2'-substitutions and/or phosphorothioate substitutions in ARCl 7480 will likely be tolerated for binding and activity. Table 37: Tolerated and Non-tolerated Substitutions in ARC 1480 Meets Criteria? Seqluence (5' -+- 3) (3T=inverted dT; s=phosphorothioate; mN=2' SEQ methoxy-containing residue; Binding- FXa Substi ID ARC dN=deoxy residue; fN=2' CAT competition Activity tution(s) NO: # fluoro-containing residue) Assay Assay Assav Tolerated rGrGmrArnArnmmU mAiU mAmUrnrU mU mGnGdCmUdCnGmUnmU mA mGmGmUmGdCmGmUmAmUmA 19 18538 rnUmnA3T NO NO NO NO mGmGmAmAmUmAmUmAdCmU mUiGmGmCrmUdCmGmUnUmA mGmrnGmU mGdCmGnUmAmU mA 20 18539 rnUmA3T YES YES YES YES mGmGmAmAmUmAmUmAdCnU rUmGrmGdCrmUrnCmGrmUrnUrmA mGrnG mUrnGdCmGmU mAnU mA 21 18540 mUmA3T YES YES NO YES mGnGmAmAmUrnAmUmAdCmU rnUrnmGrnGdCrnUdCrn CmGmUrnUmA mrGmmUmGmCmGmUmAmU mA 22 18541 mUrnA3T YES YES NO YES 189 WO 2012/109675 PCT/US2012/024916 rnGrr.G-nArnAmLJ-nArniLrnAfCmLJr.n UmGrnGdCmUdCrn-GmrnUnnArrn GrnGrnUrnGclCmGrn.UmAnUrAm 23 19493 LJrnA3T NO NO NO NO mGrn.GrnA-nArnLrnAmLJ-nAdC-nUJ mU rnGmG'Crn UdCrnGr.n U U mi GrnGrrnUrnGdCriGrUrAriUrrnAr 24__ 19494 UrnA3T YES YES YES YES rnmGrGnA. iArnUrnArnU.iAdC. iU mnU~rnmGdCmUfCmGrn-UmUmArn GrniGrnUrniGdCrrGriUrrnArnUrnArr 25 19495 U~mA3T YES YES YES YES rnGrnG.imAnmU. ArnUmnAdCmnU in U mGmGdCm LJdCmGrn Urn mA rnGrriGm-UrrlGfCm-GrniUr ArnUrniA 26 19496 rnmA3T YES YES YES YES dGrnGrArnAmUrnArUrnAdCrnUr.n UrnGrnGdCmLdCrn.GrnUrn[JrnAmr GGrnnUmGdCmGrn-UmAnUrAm 27 19448 UmA3T YE ~ YES YES YES I rniGdGr mArnArn U rnArnU rnAdCrn Urni UrnG-nGdCrnUdCrrnGmLJ-nLrrArn. GmGmUmGdCnGrrUnAUmArn 28 19449 UrnA3T YES YES YES YES I rnG rriGdArniArr U rnArniU rnAdCrn Urn UrnG. GdCrnUdCrnGrU. UrnArn GGrnnUmGdCmGn-UmArrUrnAm 29 19450 UrnA3T YES YES YES YES rniG rnC3 mAdAm iU rnArnU rnAdCrn Urni UmG-nGdCrnUdCrn.GrnU-nUrn.Arn GmGmL~mGdCnGrnrn~Arn.[JmAr 30_ 19451 UrnA3T'Y YES YES YES I mGrn-,GnA-nAd-,rnAmU-nAdCmUr.n UrnG. GdCrnUdCrnGrU. UrnArn G nGmnirGdCm rp mUmAin U ~mAi ___19452 UmA3T YES YES YES YES I mrnGrr.GnArnAmUdAmUmAdCmUr.n UmG-nGdCrnUdCrn.GrnU-nUrn.Arn GmGmL~mGdCnGrnrn~Arn.[JmAr 32 19453 UrnA3T YES YES YES YES I mGrn-,GnAmAmnUrnAdT-nAdCmUr.n UrnCG rnGdCrniUdCrnGrnU miU rnArTI GrnGmUrnGdCrnGrnUrnAnUmArn 33_ 19454 UmA3T YES YES YES YES rnGrr.G-nAnAmLJ-nArniLdAdCmUJr.n Urrn~t-GdCrnUdCriGrnU mU ~rriArn, GrnGrnUrnGclCmGrn.UmAnUrAm 34__ 19455 UrnA3T YES YES YES YES mGrn.GrnAmArniLrnAmLJ-nAdCdTr.n UnmG-nGdCmnUdCrrnGmU-nUrrArn, GmGmUmGdCmGnUmAnUmArn 35_ 19456 UmA3T NO YES NO NO nmGrrnG-nArnAmLJ-nAnmLrnAdCrnUd TnmGrn.GdCrn UdCrn.Grn Urrn ArnG ___19457 rmGrnUrmGdCrrGmUriArUrmArT!U YES YES YES YES I 90 WO 2012/109675 PCT/US2012/024916 mA3T rnGrrG mAmnArm U mAnUmrnAdCmn U rnLJdGmGdCrm LJdCmGrmUrmU mAm GrmGrmUrmGdCrmGrm!UrmArmUrmArm 37 19458 UrnA3T YES YES YES YES I rGrnGm rAmArnU mAiU mAdCm U mU mGdGdCrm UdCmGrr U m U mAm GmGmUmGdCmGmnUmAmUmAm 38 19459 UmA3T YES YES YES YES rnGrriG mAmArm U mAnU mnAdCm U mUmGmGdCdlTdlCmGrrUmUmArn GmGnUmGdCmGnUmArmUnAm 39 19460 UrnA3T YES YES YES YES iGrGm rAinArnU mAiUminAdCnU mUnGrnGdCmUdCdGrmrnUmUmAm GmGmUmGdCmGrmUrmAmUrmAm 40 19461 UmA3T_ YES YES YES YES mGrmGnAnAmUnAmUnAdCnU mUiGmGdCmUdCmGdTmUmAn GnGmUnGdCmGrmU mAmr U mAm 41 19462 UrnA3T NO YES NO NO mGrGmAmAmUnAmUmAdCmU rUnGrGdCrmUdCrmGrmUdTmArn GmGmUmGdCmGrmUmAmUrmAm 42 19463 UmA3T YES YES YES YES mGmGmAmAmUmAmUmAdCnU nUmGrnmGdCmnUdCnGrUrnUdArm GmGmUmGdCmGmUmArm.UmAm 4_3 19464 UmA3T YES YES YES YES mGrmGnAmAmUmJnAmUmAdCmU iUrnGmGdCrnUdCmGrnUrnmU mAd GrnGmUrnGdCimGmUmAmUmAm 19465 UmA3T YES YES YES YES mGmGmAnAnUmAmUmJnAdCnU mUnGmGdCnUdCmGmUmU mA mGdGmUmGdCnGmUrnAmUmA 4.5 19466 nUmA3T YES YES YES YES nGrmGmAmAmUJmAnUmAdCmU rnUmGrnGdCmUdCrnGnUrnUnA nGmGdTmGdCnGrnUnAmUmA 46 19467 mUJmA3T YES YES YES YES rnGrnGmA-nrnUmArnU-nAdC-nU mUnmGmGdCnUdCmGmUmU mA nGrGmUdGdCmGrmU mAnnUmnA 19468 mUiA3T YES YES YES YES mGGmAiAnUmAmUmAdCmU nLJmGnGdCmUdCnGmUJnUmA mGrmGmnUrGdCdGrmUnAmUmA 48 19469 _mUmA3T NO NO YES NO iG mG mnAmAnU mAiU mAdCm U mU mGmGdCmUJdCmGm UrmUmA nGrGmUrmlGdCmnGdTmnAmUmA 49 19470 mUiA3T YES YES YES YES 191 WO 2012/109675 PCT/US2012/024916 rnGrn.G-nArnAmLJrnAmnJrnAdCrnU mnU nrnmGdCm UdCmrn nUrmU mA rnGmGrnUmGdCrnGrnUdArnUrnA 50__ 19471 mIJmnA3T YES YES YES YES mGrn.GmAmnArnLmAmLJmAdCmUL mU rnGmGdCrniUdCmGm UnmA rn G rriG mU rrGdC miGmrn!iAd-fTmA 51 19472 rnLmA3T YES YES YES YES rnGrn.GrnA. iAr-nUrnArnU.iAdC. iU mnU~rnmGdCmUdCmrnnUrn~A rniGrnGmniUrnGdCmGrni~tmArnUdA 52 19473 mIJmnA3T YES YES YES YES I r-nGrnG. iArnAnU. iAr-nUrnAdCr.nU mLJ-mGmGd~rnLJdCmGmLn Urn mA rn G rrG rn U rrGdC miG rn U miAriU rT.A 53 19474 dTrnA3T- YES YES YES YES rnGrn.GrnA. iAr-nUrnArUrnAdCrniU rniU mGrnGdCrn UdrnGmni rnmA mrnGrr.GnUrrnGdCrnGmrnAmUrnA 54 19475 rnUdA3T YES YES YES YES I rniG rnC3 mAm-Ad U rnArnU rnAdCrn Urni LUmnGdrniUdCrrnGmLJ-nLrrnArn. GmGmUmGdCnGrrUnAUmArn 55 33867 UrnA3T YES YES YES YES I rnG rr.G mAmiArn U mnAdUrmAd~rn Urn Ur-nG. iGdCr-nUdCmrGrnU. iUmpArn. GrnnUmGdCmGm-,UmArrUrnAm 56 33868 UrnA3T YES YES YES YES rniGr mG minmArn U iAri U mAd~d Urni UmG-nGdCrnUdCrn.GrnU-nUrn-Am GmGmLmGd~rnGrnrn~Amp.[mAr 57__ 33869 UrnA3T' NO NO NO NO I mGmvGmnA-nAmUmAmU-nAdC-nUd UrnG. GdCrnUdCmGnU. UmArn Gnimn GdC mGrpmUmAin U ~mAi 58__ 33870 UmA3T YES NO YES YES mrnGm-AmnAmUmAmUmAdCmnU rn U nGrnGdCd UcCmGrn r UmAin GmGmLmGd~rnGrnrn~Arn.[mArn 59__ 33871 UmA3T YES YES YES YES I mGmvGmnA-AmUmAmUmAdCmnU rn U!,rn nGdC rn Ud CmnGd Urn UrmAri GrnGmUrnGdCrnGrnUrAUmArn 60 33872 UmA3T YES YEYS YS miGrrnG-nAmAmLJ-nAmiLJmAdCmnU rn U mGrniGdCrr UdCrniGrn Ud UrmAri GrnGrnUrnGclCrGrn.UrnArnUrAr 61 33873 LrnA3T YES YES YES YES I rnGrn.GmArnAmiL~nmLJrnAdCrnLJ mU rnGrGdCrniUdCrnGrn UnmA mGrn.GdUrnGdCrnGmrnmAmU. A 2) 33874 miLmA3T YES YES YES YES miGrrnG-nAmnrnUJ-nAmiLJmAdCmnU rn U nrnmGdCrnUdCmnGniUrn U mA 63 3875 mrGrnGmiUrnGdCmnGdUrnAmrUm-A YES I YES YES YES 1 92 WO 2012/109675 PCT/US2012/024916 nUmrnA3T mGrnG mAmnArm U mAnU mnAdCm U mUmGmGdCmUdCmGmU mnUmA rn (3 m GmnGUrmGdC mGmU mAdU mA 64 33876 rnUnA3T YES YES YES YES mO mG mAmArnU mAiU mAdCm U mUmGmGdCmUJdCmGmUrmU mA mGmGmUmGdCmGmUmAmUmA 65 33877 dUrnA3T YES YES YES YES mG mGmAmnArm U mAnU mnAdCm U mUmGmGrCmUmCmGmUmUmA mGrmGnUrmnGdCmGmUmAmUmA 66 18545 rnUmA3T YES YES NO YES mO mG mAmArnU mAiU mAdCm U rnUnGrnGmCrnUdCmGmUmUmA mGmGmUmGmCmGmUmAmrU mA 8 18546 rnUmnA3T YES YES YES YES mGrmGnAnAmUnAmUnAdCnU mUmGmGmCmUmCmGmUmUmA mGmGmUmGmCmGmUmAmUmA 67 18549 mUmA3T YES YES NO YES mGmGmAmAmUmAmUmAfCmUm Urn 3mGrmrmCnUdC mGmrnUrnmU mAi GmGmUmGdCmGmUmAmUmAm 68 19476 UmA3T NO YES YES YES mGmGmAmAmUmAmUmAdCmU mnUrmGmmCrnUfC mnGUrnU mA mGmGmUmGdCiGmUiAmUmA 69 19477 mUmA3T YES YES YES YES mGmGnAnAmUmJnAmUnAdCnU mUmGmGmtCmUdCmGmUrnUmA mGmGmUmGfCmGmUmAmUmA 70 19478 muJnA3T NO YES YES YES mGrmGnAnAnUinAmUmJnAdCnU mUnGmGCn-fimUdCmGrn UiUmAm GmGmUmGmCmGmUmAmUnmAm 19484 UmA3T YES NO YES YES nGrmGnAnAmUmJnAnUinAfCmUmJn UmGmGmCmUdCmGnUrmUmAim GmGmUmGmCmGmUmAmUmAm 72 19490 UmA3T YES YES NO YES mnmGmGmnAmUfmmnmUmAdCmU mnGmGmUmGmCmGmUmAUmnA 19491 mUmA3T YES YES YES YES mnUmnGmGd~mUd~rmGmUmUmnA inm mnmUminndOmGminU mAmnU mA 19416__mUmnA3T__________ _ _YES YES__ _YES___YES mGmGsmArnAmUmAnUmnAdCmnU iU minGmGdCmUJdCmGmUiU mA rnmGm GmiiUrmGdCmrGnmU mAinU mA 75 19417 mUmA3T YES YES YES YES 193 WO 2012/109675 PCT/US2012/024916 rnGrn.G-nAsmArniLrnAmLJ-nAdC-nLJ mnU nrnmGdCm UdCmrn nUrmU mA rnG mGmU mGdCrnGmrn-ArU mA 76 19418 mIJmnA3T YES YES YES YES mGrn.GmA-nAsmLJ-nArniLmAdCniU mU rnGmGdCrniUdCmGm UnmA rn G rriG mU rrGdC miGrmU miAriU rriA S 19419 rnLmA3T YES YES YES YES rnGrGrnArArnUs. ArnUrnAdCrnU rnUrnGrnGdCmUdCrnGrnUrnUrnA rniGrnGmniUrnGdCmGrni~tmArUrnA 78 19420 mIJ-nA3T YES YES YES YES mnGmG. AmnAmU. AsmU. AdCmU mLJ-mGmGdCm LJdCmGmL Urn mA rn G rrG rn U rrGdC miG rn U miAriU rT.A /9 19421 mrnmA3T YES YES YES YES rnmGm-A. iiAr-nUmnrUsmnAdCr-nU m U mnGmGdCmLUdCnimL~Jn UmA mGmGmUmGdCmGmUmAmUmA 8) 19422 rnUmnA3T YE~ YES NO YES I rniG rnC3 minmArn U miAri U AsdCmn iU mLJ-mGmGdCmnLJdCmGmL Urn U mA mGmGmUmGdCmGmUmAmUmA 81 19423 mU. A3T YES YES YES YES I mnG rri.G AmiArn U nAin U mniAdCsrtn U mU. GmGdCrnUdCmGmUmU. A mrnGm-UrrnGdCmnGmnUmAmUrA 82 19424 rnUrnA3 YES YES YES YES I rniG rnC3 mi~nArnmU miAriU rnAdC-n Us mnU nGrnGdCm UdCrnGmnUrn U-mA mG r.G inU rnGdC rnGmm nmU mA 83 19425 mnUmnA3Tf Y YES YES YES I mGrn.GmAmAmUmAmUmAdCmnU mUs-nGrnGdCmUdCrnGnUnU-nA miGrmrnL U m.dO mnm inAmU mr.A 84 19426 mnUmA3T YES YES YES YES I mGrnGmAmAmUmAmUmAdCmnU rnU-nGsmGdCrnUdCmGrn-UmUrnA mGrmO in U rnGdC-nmL U nAiUrpmA 85 19427 mUmA3T YES YES YES YES I m~rn.GmAmnAmnUmAmUmnAdCmnU rnUmniimrnsdCrrnUdCrniGrnUrniUrnA 86 14-8 rn.n rndC YEUSi~n~ 86 946 ~mAT ES YES YES YES nmGrnr.GnAinAmLJ-nAmninAdCinU rniUr~nrniGdCsriUdCn~rT!UrrUmniA rnGm m nUmGdCrnG rn U nAnU mA 87 19429 mUJrnA3T YES YES YES YES I mGrn.GinA-nAmninAmLJ-nAdC-nUJ mUmn~mdCrnUsdCmrn.UrnUmnA mG rnGr-n U rnGdC.G im U.AmU rn.A 88 19430 niLmA3T Y ES YES YES YES nmGrnr.GnAmnrnUJ-nAnmLJmAdCmnU rnUrn~rn~dCrnUdCsrnnUrnU-nA 89 19431 rmi-rGimUrdC.m-~rmU.mArnUrnA YES YES YES YES 194 WO 2012/109675 PCT/US2012/024916 nUmrnA3T --------- --------------------------------------------------------------- ------------------------------------ mG mG mAmArm U mAnU mnAdCm U nLJnGnGdCmUdCnGsrmUmLJmA rn (3 m GmnGUrmGdC mGmU mAnU mA 90 19432 rnUmA3T YES _ YES YES YES mO mG mAmArnU mAiU mAdCm U mUmGmGdCmLJdCmGmUsm U mA mGmGmUmGdCmGmUmAmUmA 91 19433 mUmA3T YES YES YES YES mG mGmAmnArm U mAinU mnAdCm U mUmGmGdCmUdCmGmUmUsmA mGmGmUmGdCnGmUrnAmUmA 92 19434 rnUmA3T YES YES YES YES mO mG mAmArnU mAiU mAdCm U mUnGnGdCmUdrnGmU mU mAs mGrmGmUrnGdCmGmUmAnUmA 93 19435 rnUmnA3T YES YES YES YES mGrmGnAmAmUmAmUmAdCnU mUmGmGdCmUdCmGmUmUmA nGsrGnUrGdCnGmJnAnUn 94 19436 AmUmA3T YES YES YES YES mGmGmAmAmUmAmUmAdCmU mU mmGdCmUd Cm (GrnU mU mA mGmGsrUmGdCnGnUinAmUn 95 19437 AmUrnA3T YES YES YES YES mGrmGmAmnAmUmAmUnAdCnU mnUGrnmGdCrnUdCrmGm UrnU mA mGmGmUsmGdCmGmUmAnmUm 96 19438 AmUmA3T YES YES YES YES mGmGnAnAmUmJnAmUinAdCnU mU mmG (dCiUd Cm (GmU mU mA minGmGiUmGsdCiGmUmiiAmUn 97 19439 AinUinA3T YES YES YES YES mGrmGnArnAiUnAmUmJnAdCnU mUnGmGdCm UdCmGmUmU mA mGrmGmUrmGdCsmGmUmAmUm 98 19440 AmrUmA3T YES YES YES YES iGmrGnAnAmUmJnAiUnAdCnU m U mGmGdCmUdCmm UrnU mA minGmGiUmGdCmGsmUiAmUn 99 19441 AinUinA3T YES YES YES YES rnmi~nmAnAmUmU-AdCmnU mUnGmGdCm UdCmGmUmU mA rnGiGmnUminGdC mGrnUsnAmnUrn 19442 AinUinA3T YES YES YES YES mGmnGnAiAmUinAmUiAdCiU iUnGiGdCmUdCmiGmniUmUnA mGrmGmUrmGdCmGmUmAsmUmin 101 19443 AiUinA3T YES IYES YES YES I m Gm GmnAmAmnU mAiU mAdCm U mUnGmGdCmLJdCmGm UmUmA rnGiGmnU mGdC mOrnU mAiUsin 102 19444 AinUinA3T YES YES YES YES 195 WO 2012/109675 PCT/US2012/024916 nGrmGnAnAmLJmAnUnAdCnU mUnGrnGdCmUdCrnGniUrnUmA nGmG mU mGdCnG mUnAmUmA 103 19445 snUmA3T YES YES YES YES mGrmGmAmAnUmAmLJmAdCmU mUnGmGdCm UdCmGmUmUnmA rnrnGm mUrnGdCmrmtrnU mAiU mA 104 19446 nUsmA3T YES YES YES YES mGrnGnAiAnUmAmUmiAdCiU mUnGmGdCmUdCmm UrnU mA rm Grnc mnGUrnGdCmrnGrnU mAnU mA 105 19447__m-rnAs3T__________________ _ YES YES ____YES__ _ YES EXAMPT V [006511 This example is an evaluation of tolerated and non-tolerated deletions in ARC17480. [006521 Molecules were generated for testing with single or multiple residues deleted in the ARC] 7480 sequence, as shown in Table 38 below and in Figure 143. Each individual residue in ARC 1480 was deleted one at a time, resulting in ARC32301, ARC33120-ARC33143, and ARC 18555. In cases where two adjacent nucleotides were identical, the corresponding double deletion was also generated, resulting in AR-C32302 and ARC33144-ARC33148. Additionally, molecules with multiple deletions were generated, resulting in ARC32303, AR-C32305, ARC32306,ARC32307, ARC33889, ARC33890, ARC33891, ARC33895, ARC33900 and ARC33907. 1006531 The ARC17480 molecules with deletions were assayed for binding and function. The assays used for this evaluation were the calibrated automated thrombogram (CAT) assay, a dot blot binding-competition assay and a FXa activity assay. The results of these assays are summarized in Table 38 and Figure 144. A substitution was considered tolerated for activity if it met the criteria of at least two of the three assays that were carried out. Substitutions that were tolerated ("yes") and not tolerated ("no") are identified in Table 38. The experimental details and the criteria used for each assay are described in the following paragraphs. [006541 The TFPi-inhibitory activity of each molecule was evaluated in the CAT assay in pooled hemophilia A plasma at 500 nM, 166.67 nM, 55.56 nM, 18.52 nM, 6.17 nM and 2.08 nM aptamer concentration. ARC 17480 was included in every experiment as a control. For each molecule, the endogenous thrombin potential (ETP) and peak thrombin values at each aptamer concentration were used for analysis. The ETP or peak thrombin value for hemophilia A plasma alone was subtracted from the corresponding value in the presence of aptamner for each molecule 196 WO 2012/109675 PCT/US2012/024916 at each concentration. Then, the corrected ETP and peak values were plotted as a function of aptamer concentration and fit to the equation y=(max/(1 + IC 5 o/x)) + int, where y=ETP or peak thrombin, x=concentration of aptamer, max=the maximum ETP or peak thrombin, and int=the y intercept, to generate an IC 50 value for both the ETP and the peak thrombin. The IC 0 of each aptamer was compared to the IC 50 of ARC 17480 that was evaluated in the same experiment. A substitution was considered tolerated in the CAT assay if both the ETP and peak thrombin IC 50 of that molecule were not more than 5-fold greater than that of ARC1 7480 evaluated in the same experiment. Tolerated substitutions are indicated in Table 38 as meeting the assay criteria ("yes") or not meeting the assay criteria ("no"). [00655] Each molecule was evaluated for binding to tissue factor pathway inhibitor (TFPI) in a binding-competition assay. For these experiments, 10 nM human TFPI (American Diagnostica, Stamford, CT, catalog #450OPC) was incubated with trace amounts of radiolabeled ARC1 7480 and 5000 nM, 1666.67 nM, 555.56 rnM, 185.19 nM, 61.73 nM, 20.58 nM, 6.86 nM, 2.29 nM, 0.76 nM or 0.25 nM of unlabeled competitor aptamer. ARC 17480 was included as a competitor in every experiment as a control. For each molecule, the percentage of radiolabeled ARC 17480 bound at each competitor aptamer concentration was used for analysis. The percentage of radiolabeled ARC17480 bound was plotted as a function of aptamer concentration and fit to the equation y=(max/(1 - x/ICso)) + int, where y::the percentage of radiolabeled ARC 17480 bound, x:=the concentration of aptamer, max:=the maximum radiolabeled ARC 17480 bound, and int:=the y-intercept, to generate an IC 5 value for binding-conpetition. The ICo of each aptamer was compared to the ICso of ARC17480 that was evaluated in the same experiment. A substitution was considered tolerated in the binding-competition assay if the IC 5 0 of that molecule was not more than 5-fold greater than that of ARC 17480 evaluated in the same experiment. Tolerated substitutions are indicated in Table 38 as meeting the assay criteria ("yes") or not meeting the assay criteria ("no"). [006561 Each molecule was evaluated for inhibition of TFPI in a Factor Xa (FXa) activity assay. The ability of FXa to cleave a chromogenic substrate was measured in the presence and absence of TFPI, with or without the addition of aptamer. For these experiments, 2 nM human FXa was incubated with 8 nM human TFPI. Then, 500 pM chromogenic substrate and aptamers were added and FXa cleavage of the substrate was measured by absorbance at 405 nm (As 05 ) as a function of time. Aptamers were tested at 500 nM, 125 nM, 31.25 nM, 7.81 nM, 1.95 nM and 197 WO 2012/109675 PCT/US2012/024916 0.49 nM concentrations. ARC17480 was included as a control in each experiment. For each aptamer concentration, the A 405 was plotted as a function of time and the linear region of each curve was fit to the equation y=mx + b, where y=A 405 , x=the aptamer concentration, m=the rate of substrate cleavage, and b=the y-intercept, to generate a rate of FXa substrate cleavage. The rate of FXa substrate cleavage in the presence of TFPI and the absence of aptamer was subtracted from the corresponding value in the presence of both TFPI and aptamer for each molecule at each concentration. Then, the corrected rates were plotted as a function of aptamer concentration and fit to the equation y=(Vmja/(1 + ICso/x)), where y=the rate of substrate cleavage, x=concentration of aptamer, and Vmax=the maximum rate of substrate cleavage, to generate an IC 0 and maximum (Vmax) value. The IC 5 s and Vmax values of each aptamer were compared to the IC 50 and max values of ARCI '17480 that was evaluated in the same experiment. A substitution was considered tolerated in the FXa activity assay if the IC 5 o of that molecule was not more than 5-fold greater than that of ARC 7480 evaluated in the sarne experitment and the Vmax, value was not less than 80% of the Vmax value of the ARC1 7480 evaluated in the same experiment. Tolerated substitutions are indicated in Table 38 as meeting the assay criteria ("yes") or not rneeting the assay criteria ("no"). [00657] This example demonstrates that multiple individual deletions in ARC 1480 are tolerated for binding and activity, and that some combinations of deletions are also tolerated (Table 38 and Figure 144). ARC33889 and ARC33895 each tolerate a total of seven deletions at their 5'- and 3-ends, resulting in core molecules that are twenty-five nucleotides long. Additional combinations of deletions in ARC 17480 may be tolerated for binding and activity, with or without additional changes in the molecule. Table 38: Tolerated and Non-tolerated Deletions in ARC17480 Meets Criteria? Sequence (5' ---- 3') SEQ (3T=inverted dT; mN=2' Binding- FXa substi ID ARC nethoxey-containing residue; CAT competition Aetivity tuition s) NO: # dN=deoxy residue) Aissay Assay Assay Tolerated nGrmArnArmUrmArmUrnAdCrmU mU rnGmGdCmLJdCmGmUmU mAi GmGmUmGdCmGmUmAmUmA 106 3230 iUmA3T YES YES YES YES 198 WO 2012/109675 PCT/US2012/024916 nGmGmrAmUrmAmUrmAdCmUrm UrnGmGdCnUdCmGrmUmUrnAm GmGmUmGdCnGmUrnAmUmA 107 33120 nUnA3T YES YES YES YES nGmGmAmnAmAmUmnAdCnUn UmGmGdCrm UdCmGnnUmUmAm GrmGrmUnGdCmnGrmUrnArmUnA 108 33121 mrUmA3T YES YES YES YES mGmG mAmAmUmUmAdCmUm UmGmGdCmndrUdCmGrr UmU mAm GrnGrnUmnGdCnGrmUrmArnUmA 109 33122 nUnA3T NO YES YES YES mGmGmAmAnUrm. AmAdCmUm U mGmGdCm UdCmGnUrnmUmAm GrmGrmUnGdCmnGrmUrnArmUnA 110 33123 nUmA3T NO NO NO NO nGrnGmArrAmUmAnUdCmUrn UmrnGmGdCn UdCmGrmUrnUAm GrmrnGmUmGdCmGmUmAmUmA 1 __ 33124_ _mUmA3T__ _ _ _ ___ _ _ _ _NO__ _ _NO __ _NO __ __NO mrnGrGmArmAm UrnAmnU mAmT Urm UmGmGdCm UdCmGnUrnmUmAm GmGmUmGdCmGmUmAmUmA 112 33125 nUrmA3T NO NO NO NO mGmGmAmAm UrnAmUrnAdCrm UmGmGdCmUiCmGrUmUmAm GmGmUmGdCmGmUmAmUmA 113 33126 mUrnA3T NO NO NO NO mGrnGrmArmAm UrnAmUrmAdCr UrnUnGdCmUdCnGnUrnU mAim GrmGnUnGdCmGmUmAnUnA 114 33127 mUrmA3T NO NO NO NO mGrnGmAmnAmnUmAmUmAdCn UmUmGmGmUdCmGmUmUmA mGrGmUmGdCmG mU mArm Um 115 33128 AnUmA3T NO NO NO NO mGmGmArmAnUmAmUmAdCm UrnU mGmGdCdCmGmUmUrnAm GrmGnUnGdCmGmUmAnUnA 116 33129 nUnA3T NO NO NO NO mGrnGmAnAmUmAnUmAdCn UrnUmGrnmGdCm UrnmGrm UrnmU mA mGmGmUmGdCmnGmUmAmUm 117 33130 AnUmA3T NO NO NO NO mGmGmArmAnUnAmUmAdCrm UrnmUrnGrnGdCrnUdCrUrUmAi GmGmUmGdCmGmUmArnmUmA 118 33131 nUnA3T NO NO NO NO nGnGmAmnAm UrnAmU mAdCn UmUnmGmGdCm UdCmGmUmAnm GmGmUmGdCmGmUmrAmUmA 119 33132 mUmA3T NO NO YES NO mGmGmAmAmUmAmU mAdCm UrnUmGrnGdCmUdCrnGmUrnU 120 33133 mGrnGrUrnGdCmGmUrmArmUrm NO NO NO NO 199 WO 2012/109675 PCT/US2012/024916 ArmUmA3T ----------- --------------------------------------------------------- -------------------- -------------- ------------- nGrmGrmArmAmnUrmAnUrmAdCrm LJnUnGnGdCmUdCnGmmLJnU mnArGrmUrmGdCrmGrmUrmArmUrn 121 33134 Arn.UrnA3T NO NO NO NO mnGrnGrmArmAnUrnAmnUrnAdCrm UrmUnGmGdCmLJdCmGmUrmU mAmGmGmGdCmGmUmAmUm 122 33135 AmUmA3T NO NO NO NO nGrmGrmArmAmn UrnAnUrnAdCrm UmUiGmGdCmUdCmGmUmU mArn mGrmUdCmGmnrnUmAmn Urn 123 33136 ArmUrnA3T NO NO NO NO mnGrnGrmArmAnUrnAmnUrmAdCr U mUnGrnGdCmUdCrnGmUrnmU nAmGmGrnU mGmGmnOrnUmAmn Ur 124 33137 ArmUrmA3T NO NO NO NO mGmGmAmAmUmAmUmAdCn UmUiGmGdCmUdCmGmUmU mAnGmGrn U mGdCrnU mAmn Um 125 33138 ArmUrnA3T NO NO NO NO mnGmGmAmAmUmAmUmAdCr UrnmUrnGrnGdCrrUdCrnGrnUrnmU nAmGmGmrUrmGdCrmrGmAnUn 126 33139 ArmrUmA3T NO NO NO NO mnGrnGmAnAnUrmAnUmAdCn UrnUrnGrnGdCrnUdCrnGrnUrnU mnAmGmGmUmGdCmGmUmUm 127 33140 AmUnmA3T NO NO NO NO nGmGmArArnAm U mAmU mAdCrm UrnmUrnGrnGdCrrUdCrnGrnUrnmU iAnGmGmUmGdCrmGnUrmAm 1 128 33141 ArmUrmA3T NO YES YES YES nGnGmArAnUrnAmU nmAdCm UmUnGmGdCnUdCmGmUmU nAmGmGrm. UmGdCmGmUmAm 129 33142 LJnUmmA3T YES YES YES YES nGmGmArArnAm U mAmU mAdCrm UrnUnGrnGdCmUdCrnGnUrnU iAnGmGmUmGdCrmGnUrmAm 1 130 33143 UrmAmA3T YES YES YES YES mGmGrnAmA-nUrrnAmUrnAdCm UmUnGmGdCnUdCmGmUmU mAmmnmrnGrn Urm GdCrGrnU mAi 131 18555 UmAmU3T YES YES YES YES nAmArnUnAmUmAdCmUmUmG nGdCnLJdCnGmnLUmrnUrAnGml GrUrmGdCrmGrmUmrnAmnUrmAnU 132 32302 _mA3T YES YES YES YES rn 3mGmrmU mAiUrnAdCiU inUrI GmGdCnLUdCmGm UrnmU mArmG nGrm UmrnGdCmGrm U mAiU mAn 133 33144 UmA3T YES I YES YES YES 200 WO 2012/109675 PCT/US2012/024916 nGmGmArrAnUmAmUrmAdCrm GrnGdCnUdCrm(GnUrmUnAnG mGmUmGdCrnGmUrnAmUmAm 134 33145 UrmA3T NO NO NO NO nGnGmArmAmLrUrnAnU mAdCr UmUdCnUdCmGnUrmUmAmGm GrmUrmGdCrmGrnUmrnAnUrmAmnU 135 33146 nA3T NO NO NO NO nGrnG mArrAm UmAmUmAdCr UmUmGrnGdCmUdCrnGnAnG mGrn UrmnGdCrnGmn U mAn U mAn 136 33147 UmA3T NO NO NO NO UrnUmrGmnGdCmn LdCmnGmnUrnU mAmmGdCmmUmAmUmAim 137 33148 UmnA3T NO NO NO NO Gdmn UdmGmUrnUmAGmGn mrnUrGdCrGmrnUrArUmnArUr 138 32303_ A3T YE YES YES YES _ mnmAmAnUrAmnUmrAdCmrU mU GmGdCdCCmG U nm U mAmn GmGmUmGdCmGmUmAmUmA 139 32305 mU3T YES YES YES YES mAmAr U mAmn U mAdCm rn U rnU mGdCmUdCmGmUmUmAmm 140 32306 nU3dCmGmUmAmUmA3T YES YES YES YES rnArUmnArnUmnAdCmnUrUrGm (GdCmUdCm(G3rnUrnUrrAmGm 141 32307 rUmnGdCmGmUmArnU3T ______ YES YES YES YES mUrAnUmrAdCmUnUrn3nmGdC mn U!Urn(GmrnUrnmUmrnAmGrUmi 142 33889 GdCmGmCUrnAmU3T YES NO YES YES mAmnUrnAmUmAdCmUinUrnGm GdCmGn UdCrGrUrUmAr Gm3Gn (3 143 33890 mUmGdCGnUmAUmA3T YES YES YES YES nUmAmrnUrAdCmnUmrUnmGrdC mrUdCmrnGmUmUrmAmmrmrn(rnUmrn 144 33891 -3GdCmGmUmAmUA3T YES YES YES YES mrAmUmAdCmUmUrnGmGdCU dCmGmUrUmAm.GnGmUmd 145 33895 CrnmGrnUrAmUmA3T YES YES YES YES nU mUnAdCnUrnUmGmnGdCnU dCmG rUrnmUrAmGmGmUmGd 146 33900 CmGmUmAmUnA3T YES YES YES YES mm UmAdCmU mmnUnrn Ur U GmGdCmUdCmrGnUmUmAmG nGmnUmGdCmGmUmAmUmAm 147 33907 UmA3T I YES YES YES YES EXAMPLE 36 [006581 This example demonstrates that 3'-truneated derivatives of ARC 19499 have functional activity in the CAT assay. 201 WO 2012/109675 PCT/US2012/024916 [006591 ARC21383, ARC21385, ARC21387 and ARC21389 have successive single deletions at their 3'-end relative to ARC19499 (Table 39). These molecules are pegylated at their 5'-ends with a 40 kDa PEG. 'These molecules were added to hemophilia A plasma at different concentrations (300 nM - 1.2 nM) and thrombin generation was measured. ARC19499 was used as a control in the experiment. These 3'-truncated molecules all had activity in the CAT assay that was similar to that observed with ARC19499, with respect to both endogenous thrombin potential (ETP; Figure 145A) and peak thrombin (Figure 145B). [006601 This example demonstrates that ARC19499 can be truncated by removal of the 3'-3T and the three 3'-end core nucleotides and still retain activity in the CAT assay that is similar to that of the parent molecule ARC19499. Table 39: 3'-truncated versions of ARCI 9499 Sequence (5'- +-- ),Y (mN=2'-methoxy-containing residue; dN=deoxy residue; SEQ ID nh=amine linker; PEG40K=40 NO: ARC # kDa PEG) PEG40KnhrmGnGmArmAmUrJmArUrmAd CmUrnUrm GmGdCnUdCmGmUmUnmA nG mGrnUrnGd 3dCm G mU mAm U mAinUrn 148 21383 mA~~~~~mmmmm PEG40KnhmGiGmAmArmUmAmUmAd CmUrnmUnG mGdCmUdCmrnGmUrnUmA 150 21387 mnGmGmUmGdCmGnUmAmUmA PEG40KnhrmGnGmArmArmUmArmUmAd CmUrnUrnGmGdCm UdCmGmUmUnmA 151 21389 mGmGmUmGdCmGmUrmAnU EXAMPLE 37 [006611 'This example describes a strategy for identifying aptamers that bind at least in part to or otherwise interact with one or more portions of tissue factor pathway inhibitor (TFPI). For these experiments, a partial TFPI protein or peptide that comprises the region of TFPI that is to be targeted is used as a selection target, Any portion of the TFPI protein can be used for this type of experiment. For example, a TFPI protein that only contains the K3 and C-terminial domains of full-length TFPI (K3-C TFPI; amino acids 182-276) is the target for selection, A nucleic acid pool is incubated with K3-C TFPI to allow binding to occur, the mixture is 202 WO 2012/109675 PCT/US2012/024916 partitioned to separate bound nucleic acids from unbound nucleic acids, and the bound nucleic acids are eluted from the protein and amplified. This process is optionally repeated for multiple cycles until an aptamer is identified. For some cycles, full-length TFPI is used as the target to ensure that the aptamer binds to the K3-C-terminal region of the protein in the context of the full-length protein. These experiments result in the identification of TFPI-binding aptamers whose binding epitope is contained, for example, within the K3-C-terminal region of the protein. EXAMPLE 38 [006621 This example describes a strategy for identifying aptamers that bind at least in part to or otherwise interact with one or more portions of tissue factor pathway inhibitor (TFPI). For these experiments, full-length TFPI is used as a selection target and a portion of TFPI or a ligand that binds to TFPI is used to elute aptamers that bind to a portion of the TFPI protein. A protein or peptide that contains only a portion of TFPI could also be used as the selection target. For example, a peptide comprised of amino acids 150-190 of TFPI is used for elution. A nucleic acid pool is incubated with full-length TFPI to allow binding to occur, the mixture is partitioned to separate bound nucleic acids from unbound nucleic acids, bound nucleic acids are eluted from the protein by inebation with the TFPI 150-190 peptide, and amplified. This process is repeated for multiple cycles until an aptamer is identified. These experiments result in the identification of TFPI-binding aptamers whose binding epitope contains, for example, all or part of the 150 190 region of TFP. EXAMPLE 39 1006631 This example describes a strategy for identifying aptamers that bind at least in part to or otherwise interact with one or more portions of tissue factor pathway inhibitor (TFPI). For these experiments, full-length TFPI is used as a selection target and a ligand that binds to TFPI is included in the selection to block epitopes from aptamer binding to drive aptamer binding to alternative sites on the protein. A protein or peptide that contains only a portion of TFPI could also be used as the selection target. The blocking ligand is included in the selection step and/or in the partitioning step as a capture method or as a washing reagent. For example, an antibody that binds to the C-terminus of TFPI is included in the selection. A nucleic acid pool is incubated with TFPI in the presence of the antibody to allow binding to occur, the mixture is partitioned to separate bound nucleic acids from unbound nucleic acids, and bound nucleic acids are eluted from the protein and amplified. This process is repeated for multiple cycles until an 203 WO 2012/109675 PCT/US2012/024916 aptamer is identified. Some cycles include the antibody in the washing solution during the partitioning step and some cycles use the antibody as a partitioning method to capture TFPI aptamer complexes, in conjunction with or instead of inclusion of the antibody in the binding step. 'These experiments result in the identification of TFPI-binding aptamers whose binding epitope is not contained, for example, within the antibody-binding region at the C-terminus of TFPL EXAMPLE 40 [006641 This example describes a strategy for identifying aptamers that bind at least in part to or otherwise interact with one or more portions of tissue factor pathway inhibitor (TFPI). For these experiments, full-length TFPI is used as a selection target and a partitioning step is employed that separates aptamers with a desired functional property away from nucleic acids that do not have that functional property. A protein or peptide that contains only a portion of TFPI could also be used as the selection target. For example, the desired aptamer functional property is inhibition of TFPI interaction with Factor Xa (FXa). For these experiments, a nucleic acid pool is incubated with TFPI under conditions that allow for binding and then partitioned to separate unbound nucleic acids from aptamers that are bound to TFPI. The TFPI-bound aptamers are then incubated with FXa that is bound to a hydrophobic plate. Free TFPI and TFPI bound with aptamers that do not interfere with the TFPI-FXa interaction bind to FXa on the plate, while TFPI bound with aptamers that do interfere with the TFPI-FXa interaction do not bind to the plate. Aptamers from the unbound TFPI-aptamer complexes are then amplified. This process is repeated for multiple cycles until an aptamer is identified. These experiments result, for example, in the identification of aptamers that bind to a region of the TFP I protein that mediates inhibition of the functional interaction between TFPI and FXa. EXAMPLE 41 [006651 This example describes a strategy for identifying antibodies that bind at least in part to or otherwise interact with one or more portions of tissue factor pathway inhibitor (TFPI). 'The antigen can be any one or more portions of TFPI that are of interest. For example, the antigen may be a TFPI protein that only contains the K3 and C-terminal domains of full-length TFPI (K3-C TFPI; amino acids 182-276). The antigen is expressed in an expression system or is synthesized on an automated protein synthesizer. Mice are then immunized with the antigen in solution. Antibody producing cells are then isolated from the immunized mice and fused with 204 WO 2012/109675 PCT/US2012/024916 myeloma cells to form monoclonal antibody-producing hybridomas. The hybridomas are then cultured in a selective medium. The resulting cells are then plated by serial dilution and assayed for the production of antibodies that specifically bind to the antigen. Selected monoclonal antibody secreting hybridomas are then cultured, Antibodies are then purified from the culture media supernatants of hybridoma cells. These experiments result in the identification of TFPI binding antibodies whose binding epitope is contained, for example, within the K -C-terminal region of the protein. Exampile42 [006661 This example is an evaluation of tolerated and non-tolerated nucleotide mutations in ARC1 7480. This example is also an evaluation of tolerated and non-tolerated mutations in combination with deletions in ARC17480. [00667] Molecules were generated for testing with mutations at individual or multiple nucleotides within the ARCi 7480 sequences, as shown in Table 40 below and in Figures 146 and 150. For each residue in ARCi 7480, single mutations were sampled. For 2'-methoxy residues, 2'-methoxy-A, 2'-rnethoxy-C, 2'-methoxy-G, and 2'methoxy-T residues were evaluated, for 2'-deoxy residues, 2 -deoxy-A, 2'-deoxy-C., 2'-deoxy-G, and 2'-deoxy-T residues were evaluated. The sequences in Table 40 (ARC33149 -- ARC33180, ARC34856 - ARC34919) encompass the three possible mutations at each nucleotide position. For example, if the nucleotide in ARC 17480 is a 2'-methoxy-A, the corresponding 2'-methoxy-C, 2'-methoxy-G, and 2'methoxy-U mutations are shown in this example. Additionally, some molecules with multiple nucleotide mutations relative to ARC 17480 were evaluated. Some molecules were also tested that had both nucleotide mutations and nucleotide deletions relative to the ARC 17480 sequence. [006681 The substituted ARC 17480 molecules were assayed for binding and function. The assays used for this evaluation were the calibrated automated thrombogram (CAT) assay, a dot blot binding-competition assay and a FXa activity assay. The results of these assays are summarized in Table 40 and depicted in Figure 147. A mutation was considered tolerated for activity if it met the criteria of at least two of the three assays that were carried out. Mutations that are tolerated ("yes") and not tolerated ("no") are identified in Table 40. The experimental details and the criteria used for each assay are described in the following paragraphs. The molecules described in this example were first tested in the CAT assay and the dot-blot binding 205 WO 2012/109675 PCT/US2012/024916 competition assay. In most cases, molecules were only tested in the FXa assay if there was a discrepancy between the results in the CAT assay and the dot-blot binding-competition assay. [006691 The TFPi-inhibitory activity of each molecule was evaluated in the CAT assay in pooled hemophilia A plasma at 500 nM, 166.67 nM, 55,56 nM, 18.52 nM, 6.17 nM and 2.08 nM aptamer concentration. ARC17480 was included in every experiment as a control. For each molecule, the endogenous thrombin potential (ETP) and peak thrombin values at each aptamer concentration were used for analysis. The ETP or peak thrombin value for hemophilia A plasma alone was subtracted from the corresponding value in the presence of aptamer for each molecule at each concentration. Then, the corrected ETP and peak values were plotted as a function of aptamer concentration and fit to the equation y=(max/(. + IC4o/x)) + int, where y=ETP or peak thrombin, x=concentration of aptamer, max=the maximum ETP or peak thrombin, and int=the y intercept, to generate an ICso value for both the ETP and the peak thrombin. The ICo of each aptamer was compared to the ICSo of ARC17480 that was evaluated in the same experiment. A mutation was considered tolerated in the CAT assay if both the ETP and peak thrombin IC 5 o of that molecule were not more than 5-fold greater than that of ARC17480 evaluated in the same experiment. Tolerated mutations are indicated in Table 40 as meeting the assay criteria ("yes") or not meeting the assay criteria ("no"). [00670] Each molecule was evaluated for binding to tissue factor pathway inhibitor (TFPI) in a binding-competition assay. For these experiments, 10 nM human TFPI (American Diagnostica, Stamford, CT, catalog #4900PC) was incubated with trace amounts of radiolabeled ARC17480 and 5000 nM, 1666.67 nM, 555.56 nM, 185.19 nM, 61.73 nM, 20.58 nM, 6.86 nM, 2.29 nM, 0.76 nM or 0.25 nM of unlabeled competitor aptamer. ARC17480 was included as a competitor in every experiment as a control. For each molecule, the percentage of radiolabeled ARC17480 bound at each competitor aptamer concentration was used for analysis. The percentage of radiolabeled ARC 17480 bound was plotted as a function of aptamer concentration and fit to the equation y=(max/(i + x/ICso)) + int, where y=the percentage of radiolabeled ARC 17480 bound, x=the concentration of aptamer, max=the maximum radiolabeled ARC 17480 bound, and int=the y-intercept, to generate an IC5 0 value for binding-competition. The IC 5 o of each aptamer was compared to the IC 50 of ARC 17480 that was evaluated in the same experiment. A mutation was considered tolerated in the binding-competition assay if the IC 0 of that molecule was not more than 5-fold greater than that of ARC 17480 evaluated in the same 206 WO 2012/109675 PCT/US2012/024916 experiment. Tolerated mutations are indicated in Table 40 as meeting the assay criteria ("yes") or not meeting the assay criteria ("no"). [006711 Some molecules were evaluated for inhibition of TFPI in a Factor Xa (FXa) activity assay. The ability of FXa to cleave a chromogenic substrate was measured in the presence and absence of TFPI, with or without the addition of aptamer. For these experiments, 2 nM human FXa was incubated with 8 nM human TFPI. Then, 500 pM chromogenic substrate and aptamers were added and FXa cleavage of the substrate was measured by absorbance at 405 nm (As 05 ) as a function of time. Aptamers were tested at 500 nM, 125 nM, 31.25 nM, 7.81 nM, 1.95 nM and 0.49 nM concentrations. ARC17480 was included as a control in each experiment. For each aptamer concentration, the A 4 05 was plotted as a function of time and the linear region of each curve was fit to the equation y=mx + b, where y=A 405 , x=the aptamer concentration, m=the rate of substrate cleavage, and b=the y-intercept, to generate a rate of FXa substrate cleavage. The rate of FXa substrate cleavage in the presence of TFPI and the absence of aptamer was subtracted from the corresponding value in the presence of both TFPI and aptamer for each molecule at each concentration. Then, the corrected rates were plotted as a function of aptamer concentration and fit to the equation y=(Vmax/( 1 + IC 50 /x)), where y=the rate of substrate cleavage, x=concentration of aptamer, and Vmax=the maximum rate of substrate cleavage, to generate an IC 50 and maximum (Vmax) value. The IC 5 o and Vmax values of each aptamer were compared to the IC 5 o and Vmax values of ARC] 7480 that was evaluated in the same experiment. A mutation was considered tolerated in the FXa activity assay if the IC 50 of that molecule was not more than 5-fold greater than that of ARCl 7480 evaluated in the same experiment and the Vmax value was not less than 80% of the Vmax value of the ARC 17480 evaluated in the same experiment. Tolerated mutations are indicated in Table 40 as meeting the assay criteria ("yes") or not meeting the assay criteria ("no"). [00672] Some residues in ARC17480 tolerate single mutation while others do not (Figures 146 and 147, Table 40). Single mutations at the 5'- and 3'-ends of the molecule are well tolerated, as evidenced by the activity of molecules with any of the four possible nucleotides at residues 1-6 and residues 30-32 (ARC33149-33154, ARC34856-34867, ARC33178-33180, ARC34914-34919). Single mutations at position 19 (ARC33167, 34892, 34893) are also well tolerated. Residues 8, 10, 11, 15, 21, 28, and 29 (ARC33156, ARC34870, ARC34871, ARC33158, ARC34874, ARC34875, ARC33159, ARC34876, ARC34877, ARC33163, 207 WO 2012/109675 PCT/US2012/024916 ARC34884, ARC34885, ARC33169, ARC34896, ARC34897, ARC33176, ARC34910, ARC34911, ARC33177, ARC34912, ARC34913) are somewhat tolerant to single mutation, while the remaining residues are largely intolerant to single mutation (residues 7, 9, 12-14, 16 18, 20, and 22-27; ARC33 155. ARC34868, ARC34869, ARC33157. ARC34872, ARC34873, ARC- 160, ARC34878, ARC34879, ARC3 3161, ARC34880, ARC3488 1, ARC33162, ARC34882, ARC34883, ARC33164, ARC34886, ARC34887, ARC- N165, ARC34888, ARC34889, ARC33166, ARC34890, ARC34891, ARC33168, ARC34894, ARC34895, ARC33 170, ARC34898, ARC34899, ARC33171, ARC34900, ARC34901, ARC33172, ARC34902, ARC34903, ARC33173, ARC34904, ARC34905, ARC33174, ARC34906, ARC34907, ARC33 175, ARC34908, and ARC34909). [006731 The tolerance of mutations at the 5'- and 3'-ends suggests that these residues are not important for ARCi 7480 function. Examination of the activity of several molecules adds data that supports this observation (Figure 148, Table 40). Multiple combined mutations at positions 1-5 and 30-32 result in a molecule that meets the activity criteria (ARC34854), supporting the conclusion that these nucleotides may not be necessary for activity. However, ARC33893, which has 5 nucleotides at the 5'-end (residues 1-5) and 3 nucleotides at the 3'-end (residues 30-32) of ARC 1480 deleted, does not meet the activity criteria in the CAT or the FXa assay. ARC33929 is similar to ARC33893 in that it lacks residues 1-5 and residues 30-32, but it is substituted at residue 6 with 2'-methoxy-G and at residue 29 with 2'-methoxy-C (numbering refers to residues in ARCl?480). ARC33929 meets the activity criteria in the CAT, binding, and FXa assays, indicating that residues 1-5 and 30-32 can be removed from the ARC17480 sequence if mutations in the molecule are also made. These same mutations at positions 6 and 29 are also tolerated in the context of mutations at residues 1-5 and residues 30-32, as evidenced by the activity of ARC34855. These experiments suggest that residues 6-29 may form the core sequence of ARC 17480. [006741 Results from testing of a variety of molecules indicate that some nucleotides in the sequence are likely form base pairs with one another (Table 40. Figure 149). Molecules that contain mutations at the mA at position 6 and the mU at position 29 (6mA:29mU) but maintain base pairing between these two residues meet the activity criteria. Namely, ARC33183 (6mU:29mU), ARC34920 (6mG:29mC), and ARC34921 (6mC:29mG). As stated above, single mutations at position 6 are tolerated while those at position 29 are partially tolerated. Taken 208 WO 2012/109675 PCT/US2012/024916 together, these results demonstrate that residues 6 and 29 are tolerant to mutation if they are substituted together following Watson-Crick base-pairing rules and strongly suggest that residues 6 and 29 are base paired. Molecules that contain mutations at the mU at position 7 and the mA at position 28 (7mU:28mA) but maintain base pairing between these two residues meet the activity criteria. Namely, ARC331 84 (7mA:28mU), ARC34922 (7mG:28mC), and ARC34923 (7mC:28mG). As stated above, single mutations at position 7 are not tolerated while those at position 28 are partially tolerated. Taken together, these results demonstrate that residues 7 and 28 are tolerant to mutation if they are substituted together following Watson-Crick base-pairing rules and strongly suggest that residues 8 and 27 are base paired. Molecules that contain mutations at the nA at position 8 and the mU at position 27 (8inA:27mJ) but maintain base pairing between these two residues meet the activity criteria. Namely, ARC33185 (8mU:27mA), ARC34924 (8mG:27mC), and ARC34925 (8mC:27mG). As stated above, single mutations at position 8 are partially tolerated while those at position 27 are not tolerated. Taken together, these results demonstrate that residues 8 and 27 are tolerant to mutation if they are substituted together following Watson-Crick base-pairing rules and strongly suggest that residues 8 and 27 are base paired. The conclusion that residues 6 and 29, 7 and 28, and 8 and 27 form base pairs is also supported by toleration of multiple mutations at these positions within the sarne molecule that retain the proper base-pairing interactions (ARC35173-35180). However, when these same multiple mutations are made in molecules with residues 1-5 and 30-32 deleted, only some of the mutations are tolerated (AR C35181-35189), suggesting that residues 1-5 and 30-32 make some positive contribution to binding and activity when these mutations are made in the context of the fill-length molecule. [00675] Molecules with combinations of single tolerated mutations were also tested and are shown in Figure 150 (ARC35190, ARC35191. ARC35193-35195, ARC35220, ARC3522, ARC35223-35229, ARC3523 1. ARC35232). These molecules did not meet the criteria of the binding-competition or the FXa assays. These experiments indicate that although single mutations in the molecule may be tolerated, they may not be tolerated in combination with other mutations in the molecule. [006761 This example demonstrates that a number of single and multiple mutations are tolerated within the ARC 17480 sequence. Evaluation of molecules with mutations suggest that the core aptamer sequence is contained within nucleotides 6-29 and that residues 6 and 29, 7 and 209 WO 2012/109675 PCT/US2012/024916 28, and 8 and 27 likely form base pairs within the sequence. Additionally, although many mutations are tolerated individually, these are not always tolerated in combination with one another. Table 40: Tolerated and Non-tolerated Nucleotide Mutations in ARC17480 Sequence (5' -+--- 3') SEQ (3T=inverted dT; mN=2' ID ARC methoxy residue; dN=deoxy CAT Biedin A CT competition Activity Substitntion( NO: # residue) Assay Assay Assay s) Tolerated mnU mGrn0rAmUrnAmUrAd CmUmUmGmGdCmUdCmGm UmUmAmGmGmUmGdCmG not 15i 33149 mUmAmUmAmUrnA3T Yes Yes tested Yes mGnUmAmAmUmAmUmAd CniUmLmGmGdCmU dCmGni LJmUmAmGmGmUmGdCmG not 154 33150 nUrnAmUmAmUmA3T Yes Yes tested Yes mGrnGm UAmUmAn Ad CmmLmi nG nGdCmIdCmGm UnUmAmGmGmUmGdCnG not 155 33151 mUrnAmmAmUmA3T Yes Yes tested Yes mGmGmAmU mU mAmUmAd CmUmUmGmGdCmiUdCmGm UmUmAmGmGmUmGdCmG not 156 3315Z2 mUmAmUmAmU mA3T Yes Yes tested Yes miGmGmiAmAmCmAmU mAdC n'tifmUimGm(3idCmLd(mn(iiiU mUmAmtGm~imUmGdCmGm not 157 33153 UmAmUmAmUnA3T Yes Yes tested Yes mGmGn-0mAmUmUmUmAd CnimUnUmGmGdCmUdCmGm UnUmAmGmGmUmGdCmG 158 33154 nUnmUmAmUmA3T No Yes Yes Yes mGrnmGmAmAmIJmAmCmAdC mUnUmGmGdCmUdCmGmU mUmAmGmGmUmGdCmGm 159 33155 LJmAmUmAmUmA3T No Yes No No mGrnGnAnAmUmAniUniUd CmmmTinJG GdCmUdCmGm UmUmAmGmGmUmGdCmG 160 33156 mUmAmUnAmUmA3T No Yes No No 210 WO 2012/109675 PCT/US2012/024916 mirn(inAiriUmAmbmA dT rmUmlJGmGdCmUdCmGmU rnUmAmm(im(iUmGdCmm(ii not 161 33157 UmnAmUmAmUmA3T No No tested No mGmGmAmAmU mAmUmAd CmCmUmGmGdCmUdCmGm UmUm-AmGmGmUmGdCmG not 162 33158 mUmAmLInAmUmA3T Yes Yes tested Yes mGmGmAmAmUmAniUnAd CmUmCmGmGdCmUdCmGn UmUmArnGmGmLjmGdCmG not 163 33159 mUmAmUmAmUmA3T No No tested No mGmGniA mAmUmAm JAd CmUm-UniimUmGdCmUdCmGn UmUmAmGmG uinmGdCniG not 164 33160 mUmAmUmAmUmA3T No No tested No mGmG mAmAmUmAmUimAd CniUmUmGmUdCmUdCmGm UmUmAmGmGmUmGdCmG not 165 33161 niUmAnmUmAmUmAk3T No No tested No mciirn(imAmnAmiUmAmUmAd CmLJmILmGmGdTm UdCmGm riUInAmGmGmUrGdCmG 166 33162 mUmA mUAmUmA3T No Yes No No mGmGmAmAmU mAmUmAd CmUmUmGmGdCmCdCmGm UmUm-AmGmGmUmGdCmG not 167 33163 mUmAmUmAniUmA3T No No tested No mGmGmAmAmU mAmnimAd CmUmUmGmiGdCnLUdTmGm UmUmAmGmGmLJmGdCmG not 168 33164 mUmAmUmAmUmA3T No No tested No mGmGniAmUmAmUnmJnAd CnUmUmniiGdCrnldCmUmn UmUmAm GmGmuUnmGdCmG not 169 33165 mUmAmUmAm)mA3T No No tested No mGmGmAnmAmUmAmUmAd CmniU mLmGmGdCmU dCmGm CmiUmAmGmGmUmGdCmGm not 170 33166 UmAmUmAmUmA3T No No tested No 1imrn(imnAinmAmihin 'mUmAd Cm J nGmGdCmU dCmG UimCmAmGmGmUmGdCmGm not 171 <3167 mnAmUmAnmA'2T Yes Yes tested 211 WO 2012/109675 PCT/US2012/024916 mciirn(imAimAmiUiAmimAd Cm mmGnGdCmUdC3rnm UiriUmnUmGmGmrnGdCmG not 172 33168 mrnAmUmAmUmA3T No No tested No mGmGmnAmAmU rmAmUmAd CmUmUmGmGdCmUdCmGm UmiUmAmUmGmUmGdCmG 173 33169 mJmAmUmAmUmA3T Yes No No No mGmGmAmAmU mAniUntAd CmUmUmGmGdCrnLUdCmGm UmUmArnGnLUmLJmGdCmG not 174 33170 mUmAmUmAmUmA3T No No tested No mGmGnA mAmUmA mAd CmUm UniiGdCmdCmGmn UiUmAnmGmGnCmGdCnGm not 175 33171 UiAmnLmAmIjnmA3T No No tested No mGmGnAmAmUmAmUimAd CniUmUmGmGdCmUdCmGm UmUmAmGinGmUniUdCmG not 176 33172 niUmAmnUmAmUmA3T No No tested No nkirn(inAimAmUiAiUniAd CmmJmGjnGdCmUdCmGm Uir~imAmGmGmdrnGdlmGm 177 33173 UmAmUmAm~mA3T No No No No mGmGmAimAmiU mAmUmAd CmUinUmGmGdCmUdCmGm UmUm-AmGmGmUmGdCmU not 178 33174 mUmAinUmAniULmA3T No No tested No mGmGmAmAnU mAmiUntAd CmUmUmnGmiGdCnmUdCmGm UmUmAmGmGmLimGdCmG 179 33175 mCmAmUmAmUmnA3T No No No No mGmGniAmUmAmU mA J nAd CnUmUn-iiGdCmldCmGmn UiUnAm GmGmuUmGdCiG not 180 33176 m -UUmUmAmUnA3T No No tested No mGmGmAmAmUmAmU mAd Cmni UmimGmGdCmUdCmGn UmUmAmGGmUmGdCmG 181 33177 niUmiAmCmAmUniUA3T No Yes Yes Yes rinGinGnAiAm m~miAimAd CmLmUmmnGdCiUdCm3rn UiriUAmGnIGmUrnGdCmG not 182 33178 mUmAimiJunmUnA3T Yes Yes tested Yes 212 WO 2012/109675 PCT/US2012/024916 mciirn(imAimAmiiUmAmdinmiAd Cmm nmGjnGdCmUdC3rnm UiriUmAmGmGUrnGdC nG 183 33179 rnUmAmnAmCmA3T Yes Yes Yes Yes mGmGmAmAmU rmAniUmAd CmUnUrmGmGdCniUdCmGm UnUm-AmGmGnUmGdCmG not 184 33180 mUmAmLjmAmUmU3T Yes Yes tested Yes mAmGmAmAmU mAniUmAd CmUmUmGmiGdCrnUdCrmGm UmUmArnGmGmLjmGdCmG not 185 34856 mUmAmUmAmrnUmA3T Yes Yes tested Yes mCmG nAnAmUmniAmlUmAdC mUmUniGmGdCmbdCinGnU mUmAmGmGmUmGdCmGm not 186 34857 UmAmLrmAmLjmrA3T Yes Yes tested Yes mGmAmAmAmUmAmU mAd CniUmUmGmGdCmUdCmGm UmUmAmGmGmUmGdCmG not 187 34858 mUmAmUrmAmUmA3T Yes Yes tested Yes rnGmCmmAmArnrb nAimAdC nmmnGmrGdCmUdCmGmI rnfmAn(imm(iiUmGdCm(mii not 188 34859 UmAmUmAniUmA3T Yes Yes tested Yes mGmGmGmAmU mAmUniAd CmUmUmGmGdCmUdCmGm UmUm-AmGmGmUmGdCmG not 189 34860 mUmAmUmAmU mA3T Yes Yes tested Yes mGmGmCmAmUmAmUmAdC mUmUmGnmGdCnUdCmGmU mUmAmGmGmUrmGdCmGm not 190 34861 UmAmUmAmUmA3T Yes Yes tested Yes mGmGniAmGmUmAmUnAd CmUmUiiGmidCrnUdC nGm UmUmAmnGmGnUJmGdCmG not 191 34862 mUmAmUmAmUmA3T Yes Yes tested Yes mGmGmAmCnUmAmLimAdC mUrnUmGmGdCmUdCmGmU niULmAmGmGmUmGdCmGm not 192 34863 UmAmUmAmUmA3T Yes Yes tested Yes rnirn(imAAmAmAAi'mUmAd CmLmJGmnGdC m dCmGrn U~iriUmAmG mmrnGdCmG 193 34864 _minJAmimAnAmiUm'A3T No Yes Yes Yes 213 WO 2012/109675 PCT/US2012/024916 mmGmrn(iiAmimininAmiimAd Cm nJ nmGGdCmUdCmGm ImUmAmGmGmUmGdCmnG not 194 34865 mUmAmnlmAmUmA3T Yes Yes tested Yes mGmGmAimmU rmGniUmAd CmUnUrmGmGdCniUdCmGm UnUm-AmGmGnUmGdCmG not 195 34866 mUmAmLIrmnAmUmA3T Yes Yes tested Yes mGmGmiAmUrnCmUmAdC mnUmnUmn~mGdCmnUdCmn~imU mUmAmGmGmUrnGdCrmGm 196 34867 UmAmUmAniUmA3T No Yes Yes Yes mGmGnAmA mUmAmAmAd CmUmUmniiGdCmUdCmGm UiUmAm GmGmuUmnGdCniG not 197 34868 mUrmAmUiAmLJmA3T No No tested No mGrn-mAmAmmUmArmGmAd CniUniUmGmGdCmUdCmGm UmUmAmGmGmU mGdCmG not 198 34869 niUmAnmUmAmUmA3T No No tested No mimm(inAimAmUmAmmnG d Cm mmGjnGdCmUdCmGm U IUmAmGmGmUmGdCmG 199 34870 mUmA nAmUmA3T No Yes Yes Yes mGmGmAmAmU mAmUmCdC mUmUmGmGdCniUdCmGmU mUmAimGmGmUmGdCmGm not 200 34871 UmAniUmAmUmA3T No No tested No mGmGmAmAmU mAnUmAd AmUmUhGmGdCmUdCmGm UmUmArnGmGmLJmGdCmG not 201 34872 mUmAmUmAmUmA3T No No tested No mGmGniAmUmAmUmA J nAd GmUmm GmGdCiUdCnGm UiUmAm GmGmnUJmGdCniG not 202 34873 mUmAmUiAmLrmA3T No No tested No mGrnGmAinAmUmAmU mAd CmAfnUmGmGdCmUdCmGm UmUmAmGmGmU mGdCmG not 203 34874 niUmAnmUmAmUmA3T No No tested No mGmGnAiAmUmAnimimAd CmGmlmnGGdCmUdCmGm UmiJiAmGmniGmUmGdCm(i not 204 34875 _mimAmtlmAmUmA3T No No tested No 214 WO 2012/109675 PCT/US2012/024916 iirn(imnAiriUmAmbmAd CminAmGmnGdCmUdCrnm UiriUmnAmGmGrniGdCm(i not 205 34876 rnAmUmAmUmA3T No No tested No mGmGmnAmAmU rmAmUmAd CmUmGmGmGdCmUdCmGm UmUmAmGmGmUmGdCmG 206 34877 mLImAmUinAmUmA3T No Yes Yes Yes mGmGmAmAmU mAniUmAd CmUmUmAmGdCrnLUdCmGm UmUmArnGmGmLjmGdCmG not 207 34878 mUmAmUmAmUrnmA3T No No tested No mGmGnA mAmUmA mAd CmnUmiUmCmGdCniUdCmGm UmUmAm GmGnJmUGdCniG not 208 34879 mUmAmUmAmL)mA3T No No tested No mGmG mAmAmUmAmlUimAd CniUmUmGnAdCmUdCmGm UmUmAmGmGmUmGdCmG not 209 34880 niUniAmUmAmUmA3T No No tested No nkirn(inAmAmiiiUmAiUmiAd Cm nmGjnCdC nUdCimGm JiriUmAmGmGmI rnGdCnG not 210 34881 rnUmAnlnAmUmA3T No No tested No mGmGmAimAmi mAmUmAd CmUmUImGmGdAmUdCmGm UmUm-AmGmGmUmGdCmG not 211 34882 mUmAmUmAniULmA3T No No tested No mGmGmAnAmUmAmnimAd CmUmUmGmGdGmLdCmGm UmUmAmGmGmLjmGdCmG not 212 34883 mUmAmUmAmUrnmA3T No No tested No mGmGniAmUmAmUmnmnAd CmnUmnUiGmGdCrnAdCinGin UmUmAm GmGnUJmGdCniG not 21' 34884 mUmAmUmAmUmA3T Yes Yes tested Yes mGmGmAnAmLJmAmU mAd Cmni UmLmGmGdCmGdCmGn UmUmAimGmGmUmGdCmG 214 34885 niUmAnYUmAmUmA3T No Yes Yes Yes rnGmGnAimAmUmAmUmAd CmITmJmmGdCmUdAmG JirJimAmGmGmUrnGdCnG not 215 34886 _mAmlTJnAmUmA3T No No tested No 215 WO 2012/109675 PCT/US2012/024916 mirn(imAiAmiiUmAmdimAd CrImTmimGmGdCmUdGimGm IjUimAmGmGrniGdCm(i not 216 34887 rnAmUmAmUmA3T No No tested No mGmGmnAmAmU rmAmUmAd CmUmUmGmGdCmUdCmAm UmUm-AmGmGmUmGdCmG not 217 34888 mLmAmUmAmUmA3T No No tested No mGmGmAnAmUmAniUmAd CmUmUmGmiGdCrniUdCmCn UmUmArnGmGmLjmGdCmG not 218 34889 mUmAmUmAmUmhA3T No No tested No mGmGnAmAmU mA J nAd CmUmUmni iGdCmlidCm uGin AiUmAm GmGmuUnmGdCniG not 219 34890 mUmAmUmiALmA3T No No tested No mGmGnAimAmUmAimUimAd CniUmUmGmGdCmUdCmGm GmUmAmGmGmU mGdCmG not 220 34891 niUmAnmUmAmUmA3T No No tested No nkirn(inAimAmUmAniUniAd CmmmGjnGdCmUdCmGm UmIAmAmGmGmrnGdCmG not 221 34892 mUmAm mAmUmA3T Yes Yes tested Yes mGmGmAmAmU mAmUmAd CmUinUimGmGdCmUdCmGm UmGmAmGmGmUmGdCmG not 222 34893 mUmAmnUmAmU mA3T Yes Yes tested Yes mGmGmAniAmUmAnimAd CmUmUmGmGidCnmUdCmGm UmUtmGmGmGmLJmGdCmG not 223 34894 mUmAmUmAmUmhA3T No No tested No mGmGniA mAmUnAm JmAd CnUmUn-iuiGdCrnidCmGmn UmUmCiGmGmbmnGdC nGm not 224 34895 UmAmUnAmLlnA3T No No tested No mGmGmAmAmLimAmUmAd Cmni UmLmGmGdCmUdCmGrn UmUmAmAmGmU mGdCmG not 225 34896 mUmA nvmmUmA3T Yes Yes tested Yes rtinG nAmAmiUiAnimmAd Cm m nmGnGdCiUdCm3rn UmhmAmCmGmUmGdCmGm not _226 _ 34897 _l-1 UmAmUliAniTmAT Yes Yes___ tested ___Yes 216 WO 2012/109675 PCT/US2012/024916 mirn(imAiAmiiUmAmdimAd Cm mmGnGdCmUdC3rnm UirjinmAmGmAmrnGdCm(i not 227 34898 mrnmAmUmAmUmA3T No No tested No mGmGmnAmAmU rmAmUmAd CmUmUmGmGdCmUdCmGm Umi UmAmGmCniUmGdCmGm not 228 34899 UmJnAmUmAmUmA3T No No tested No mGmGmAmAmU mAniUmAd CmUmUmGmiGdCrnLUdCmGm UmUmArnGmGmAmGdCmG not 229 34900 mUmAmUmAmUmhA3T No No tested No mGmGnAmAmU mA J nAd CmUmUmn-iiGdCmldCm uGin UiUmAm GmG nGmGdCniG 230 34901 mUmAmUmALmA3T No Yes No No mGmG nAmAmUmAmU mAd CniUmUmGmGdCmUdCmGm UmUmAmGmGmUmAdCmG not 231 34902 niUmAnmUmAmUmA3T No No tested No rciirn(imAmnAmiUmAmUniAd CmmJm GmGdCiUdCGm UirimAIGmGmnrnCdCmGm not 232 3 4 9 03 UmAmUmA mUimA3T No No tested No mGmGmAmAmU mAmUmAd CmUinUimGmGdCmUdCmGm UmUmAmGmGmUmGdAmG not 233 34904 mUmAinUmAniULmA3T No No tested No mGmGmAiA niUmnAminAd CmUmUmnGniGdCnmUdCmGm UiUmAmGmGmLimGdGmG not 234 34905 mUmAmUmAmUmhA3T No No tested No mGmGnAmAmUmAmnlinAd CnUm Un-iiGdCrnldCmGmn UmUmAm GmGmuUmGdCmA not 235 34906 mUmAmUmAmUnA3T No No tested No mGmGmAnAmUmAmU mAd Cmni UmLmGmGdCmUdCmGrn UmUmAmGmGmU mGdCmCm not 236 34907 UmAmUmAmUmA3T No No tested No nkirn(inAinAmUin AiUmiAd CnmmIGd mldCmGrn JiimAimGmi(mi rnGdCmG not 237 34908 rnAmAmimAimA3T No No_ _ tested No 217 WO 2012/109675 PCT/US2012/024916 mirn(imAiAmiiUmAmdimAd Cm mmGnGdCmUdCmrn JiriIJmAmGmGnmrnGdCm(i not 238 34909 rnGAmUmAmUmA3T No No tested No mGmGmnAmAmU nAmUnimAd CmUmUmGmGdCmUdCmGm UmUm-AmGmGmUmGdCmG not 239 34910 mLmGmUmAmUmA3T No No tested No mGmGmAmAmU mAnLiUmAd CmUmUmGmGdC niUdCmGm UmUmAnGmGmLimGdCmG 240 34911 mUmCmIJmAmUmA3T No Yes Yes Yes mGmGnAmAmU mA J nAd CmUmniUn-iuiGdCmlidCm uGin UmiUmAmmGmUnmGdCniG 241 34912 mUmAmAmALmA3T No Yes No No mGmGnAimAmUmAimUimAd CniUnmUmGmGdCmUdCmGm UnUmAmGmGmUmGdCmG 242 34913 niUniAmGmAmUmA3T No Yes No No tiirnm(inAiAmUmAiUniAd CmmmGjnGdCmUdCmGrn JiriIJmAmGmiGmdrnGdCm(i not 243 34914 mUmAm nimmmA3T Yes Yes tested Yes mGmGmAmAmU mAmUnimAd CmUmUmGmGdCmUdCmGm UmUm-AmGmGmUmGdCmG not 244 34915 mUmAmUmCmUmA3T Yes Yes tested Yes mGmGmAmAmU mAnUmAd CmUmUmnGmGdC niUdCmGm UmUmAnGmGmLimGdCmG not 245 34916 mUmAmUmAmnAmA3T Yes Yes tested Yes mGmGnAmAUmAm JAd CnUninUmiGmGdCrnidCmnGin UmUmAmGmGmUnmGdCmG not 246 34917 mUmAnUiAmGnA3T Yes Yes tested Yes mGiGmAynAmUmAmU mAd Cmni mUmGmGdCmU dCmGm UmUnimAmGmGmUmGdCmG not 247 34918 mUmAmUnimAmUmG3T Yes Yes tested Yes nkirn(inAimAmUiAmUmA d Cmm mGmGdCiUdCm3rn UhiriUmAmGmGmrnGdCmG not 248 34919 _nUi'AmimAnAiUmC3T Yes Yes tested Yes 218 WO 2012/109675 PCT/US2012/024916 mi-rn J4 J iar rijrimAd1 mrni Jrnm ~ dCmGirI rn .rnAin(ijy(imLJniyi-d(iirni not 249 34854 UijnAmUmUmCmL3T Yes Yes tested Yes mArn Ji n XdCrnUmUm~Gd 250 33893 md-'drnm3 No Yes N..o No rnGrnJi n XdCrnUmUmnGd rtdi nGi nUrUmAmGmGrn 251 3392 9 UrnGdCrnGmiI~rnAmC3T Yes Yes Yes Yes 4 rnUrU mU niU mCmGmU mAdC mUrnAmGmGrnU rn dCrnIrn not 252 34855 UniAm ~mUmCmU31T Yes Yes tested Yes mGmGmAmiA m niiJmAd CinmU_' ni3m C(Ii nt d~ld("m~i Umnl-rmAmnin(jimI-rd(-mG(' 253 33183 mUmAmAmAmnUmA3IT Yes Yes Yes Yes mGmnimAmAmUrnArnAiuAd CniiLUniGrnGdCrnUdCmGm UrnUrnArGmGnU mGdCrnG 254- 33184 _miUniUnUmAmUrnAYT No Ys Yes Yes mti~rn(i nAiniAinLJmAndltjmiitd Crni~~~mljmnGdCjiUd~~ UmjiLJmAmii'mii(mntimi~dC 'nG 255 3'318 5 mAmrnLfnynmLrn~A3T Yes Yes Yes Yes mGrnGmAjnAjumrGnilnAd Cr~n~nGi~nGdCnd~dmi UmUrnAmnGmluimGdCmG not 256 '49')0 mUrnAmCmArn J nA3T Yes Yes tested Yes rnGrGmAniAmU rn ~mU mAdC rnUrAmGmGmU rn idC mGrn not 257 3 4-92 1 UihiAmGnrAniUmA3T Yes Yes tested Yes mGrnGn iAAmUrnAm Gi nAd CniUn iUn miGidCrn Jd~ni u 258 34922 mUmCmjyAmAmUniA3T Yes No Yes Yes mGmn-hAmAmUrnArnCmAdC mL~mLmniGrnGd.CrnUdCmGmnU miLnArGrnGmUinGdCmGm not 259 134923 1 UmGmUinm A3T Yes Yes tested -Yes----- 219 WO 2012/109675 PCT/US2012/024916 iirn(imnAiriUmAmi3d Cm 1 mJmhGnG(dCmUdC3rnm jiriUmAmGmGrniGdCnG not 260 34924 mCmAmrnUmAm"nA3T Yes Yes tested Yes mGmGmnAmAmU rmAmUmCdC mUmUmGmGdCniUdCmGmU mUimAmGmGmUmGdCmGm not 261 34925 GnAmUmAmUmA3T Yes Yes tested Yes mGmGmAmAmnUmrn 3mnAmCdC mUmUm~imGdCmnUdCm~imU mUmAmGmGmUrnGdCmGm not 262 35173 GmUmCmAmUnmA3T Yes Yes tested Yes mGmGmAmmUmnLmGmUd CmUmUmn-iiGdCrnldCm uGin UiUmArnGmGinJmUGdCniG not 26' 35174 mAmCmAmAmUmA3T Yes Yes tested Yes mGmGnAimAmUmAimCmUdC niUMUmGmGdCmUdCmGmU niUmL-hAmGmGmUmGdCmGm not 264 35175 AmGmUmAimUiA3T Yes Yes tested Yes rnGimGnAnAmUmCiriLmiGdC rinU GmnGdCmUdCiGml n.rmAn(imGimUmGdCinGmC not 265 35176 mAnGnAmUmA3T Yes Yes tested Yes mGmGmAimAmU ni mCmU dC mUmUmGmGdCniUdCmGmU mUmAimGmGmUmGdCmGm not 266 35177 AmGmAnAmUnmA3T Yes Yes tested Yes mGmGmAmAmU rnCmUmUdC mnUmUmn~imGdCmnUdCm~imU mUmAmGmGmUrnGdCmGm not 267 35178 AmAmGrnAnLUmA3T Yes Yes tested Yes mGmGniA mAmUm GmAmAd CnUm Un-iiGdCrnIdCmnGin UmUmAm GmGm UJmGdCmG not 268 35179 mUmUmCmAmIUmA3T Yes Yes tested Yes mGrnGmAmAmLimAmAmGd Cmni UmLmGmGdCmUdCmGrn UmUmAmGmGmU mGdCmG not 269 35180 mCmUmUmAniUmA3T Yes Yes tested Yes mGrmAmCdCnimUfmGmGdC mUdCmGmUmUmAmGmGm 270 35181 LJmGdCmGmGmUmC3T No Yes Yes Yes 220 WO 2012/109675 PCT/US2012/024916 mUmGnLUdCmUmUmGmGdC mUdCmGmmLJUmAmJmGm 271 35182 LmGdCmGmAmCmA3T No No No No mAmCmUdCmUUmGmGdC mUdCmGmmLimUmAmGmGm 272 35183 UrmGdCmGmAmGmU3T No No Yes No mCmmGdC mUmUmGdC mUdC mGmUmUmAmGmGm 273 35184 UmGdCmGmCmAmG3T No No Yes No mLmCmUdCmLmUnGmGdC mLTdCmGnUmnUmAmGmGm 274 35185 UmJnGdCmnGmAmGmA3T No No Yes No mArnGmAdCmU niUmGmGdC mLTdCinGiUmUmAmGmGm 275 35186 UmJnGdCmGmIUmCmU3T No Yes Yes Yes mCnUnUdCmUmUnGmGdC mUdCmGmUmnUmAmGmGm 276 35187 UnGdCmGmAmAnmG3T No No No No mGmAmAdCmtrnUimGmGdC mUdCmGmUmnUmAmGmGm 277 35188 UnGdCmGrnUrnUmC3T No No Yes No mAmAmGdCmU nUimGmGdC mUdCmGmUmU mArmGmGm not 278 35189 UmGdCmGmCmUnU3T No No tested No mGmGAmAmUmAmUnGd CmC nGmGmGdCniAdCmGm UmAmAmCmGm)mGdCnGm not 279 35190 UmAmL)mAmUiA3T No No tested No mGmnGmAmAmUmArmUmGd CmCmGmGmGdCmGd.CmGm UmAmArmCmGmUmGdCnGm not 280 35191 UmAmUmAmUmA3T No No tested No mimGnmAAmiiUmAiiUmdiid C(im(im(irmm(idCnAdCm(iim UmrmAm(mimLmGdCm~m not 281 35193 Um-AmUm-AmUmA3T No No tested No mGnGmuAiuAmU mArnUrnGd. CmCmGmGmGdCmAdCmGm UmCmAmCmGmUmGdCmGm not 282 35194 UnAmUmAmIUmA3T No No tested No 221 WO 2012/109675 PCT/US2012/024916 iirn(imnAiriUmAmmdiid (Crn(iiGrnGdCnAdCmGm JiAmAmAmGmrnGdCnG not 283 35195 rnnAmUmAmUmA3T No No tested No mGmGAmiAmUmhAniUmGd CmUmUmGmGdCnmAdCmGm UnAmAnmCmGniUmGdCmGm not 284 35220 UmJnAmUmAmIUmA3T No No tested No mGmGmAmAmUrnAnLUmGd CmUmUmGmiGdCrnGdCmGm UmAmArnCmGmUrnGdCmGm not 285 35221 UiAmUmAniUmA3T No No tested No mGmGniAmAmUrnAmnUmGd CniUniUniGmGdCrnAdCnGin UmGmArnCmGmLrnGdCmGm not 286 35223 UmAmLJmAnUmA3T No No tested No mGmGnArmAmUmAmUmGd CniUmumGmGdCrmAdCmGm UmCnAmCmGmUmGd.CmGm not 287 35224 UnAmUmAmUmA3T No No tested No mciirn(imnAiriAmUmAiUmiG d Cm JmmGmGdCmAdCmGm JiAmAmAmGmrntGdCm(i not 288 35225 rnmAmnUmAmUmA3T No No tested No mGmGmnAmAmU mAmUmGd CmCmGmGmGdCmUdCmGm UnUm-AmCmGniUmGdCmGm not 289 35226 UnAniUmniAmUmA3T No No tested No mGmGmAnAmUrnAnUmGd CmCmGmGmGdCmUdCrmGn UmUArnAmGmLimGdCmG not 290 35227 mUmAmUmAmUmhA3T No No tested No mGmGniA mAmUrnAmUGd CmCmUJmGmGdCmA dCmGm UmAmAnGmGmUJmGdCmG not 291 35228 mUmAmUiAmnUmA3T No No tested No mGrnGmAinAmLmAmUmGd Cm 'mnUmGmGdCmGdCmGm UiAmAmGmGmUmGdCmG not 292 35229 nUmAmUnimAmUmA3T No No tested No mtirn(imnAiriAmUiAmUmdiid CrnrnirCnGdCnAdCmGmi Ji(hmAmGmiGmUrnGdC mG not 293 35231 minAmimA m3T No No tested _ ___No 222 WO 2012/109675 PCT/US2012/024916 rtiirn(h AmAmjUmiAmnmjiii3d CmCrnUrnGrnGdCmAdCimGm UmCmAmGmGmUmGdCrnGm not 294 3522 UnAmUmiAi mA _T No No tested No EXAMPLE 43 [006771 This example demonstrates that FXa partially competes with radiolabeled ARC 17480 for binding to TFPL [006781 For these experiments, human TFPI at a final concentration of 10 nM (American Diagnostica, Stamford, CT, catalog #4900PC) was incubated with trace amounts of radiolabeled ARC!17480 and different concentrations of human FXa (Haem'atologic Technologies, Essex Junction, VT catalog #HCXA-0060) or ARCI 9499 (2000 nM - 0.10 nM). A control was also carried out in the absence of TFPI in which trace amounts of radiolabeled ARC] 7480 was incubated with different concentrations of FXa (2000 nM - 0.10 nM). The percentage of radiolabeled ARC 17480 bound was plotted as a function of competitor concentration and fit to the equation Y:= (max/(i 4 x/ICco)) - int, where y:= the percentage of radiolabeled ARC17480 bound, x:= the concentration of competitor, max:= the maximum radiolabeled ARC17480 bound, and int = the y-intercept, to generate an IC 50 value for binding-competition. Figure 151 shows a graph of FXa and ARC 19499 competition with radiolabeled ARC 17480 for binding to TFP. FXa partially competes with ARC17480 for binding to TFPI, as evidenced by a plateau in the competition-binding curve at concentrations above 75 nM. In contrast, ARC 19499 completely competes with radiolabeled ARC17480 for binding to TFPL. ARC 17480 shows no detectable binding to FXa in the control experiment that lacks TFP. 1006791 These results demonstrate that FXa partially competes with ARC 17480 for binding to TFPI and suggest that FXa and ARC 17480 may be able to bind to TFPI at the same time. EXAMPLE 44 [006801 This example evaluates the potential for drug-drug interactions between ARC 19499 and replacement factor VIII. 'This example demonstrates that ARC19499 can improve coagulation in a spatial model of clot formation, in hemophilia plasma activated with immobilized tissue factor, in the presence or absence of replacement factor VIII (FVI1). [006811 The key property of the spatial experimental model is that blood plasma clotting is activated by a surface covered with immobilized tissue factor (TF). The fibrin gel then 223 WO 2012/109675 PCT/US2012/024916 propagates into the bulk of plasma. From measurements of clot size versus time, the following parameters may be calculated: lag time (delay between contact of plasma with activator and beginning of clot formation), initial velocity of clot growth (Vinitia, mean slope of the clot size versus time curve over the first 10 min after the lag time), spatial velocity of clot growth (Vstationary, mean slope over the next 30 min), clot size after 60 min of the experiment. [00682] In order to predict the likely effects of ARC19499 on spatial clotting propagation based on our current knowledge of the coagulation network regulation, we first carried out computer simulations of clotting for four different cases: normal plasma, normal plasma with completely inactivated TFPI, hemophilia A plasma (no fVIII) and hemophilia A plasma with completely inactivated TFPI activity. Computer simulations of blood clotting were carried out using a detailed mechanism-driven mathematical model of clotting [Panteleev et al (2006) Biophys J 90: 1489-1500] with minor modifications. Reaction kinetics in the model were described by a set of differential equations. The mathematical model was biphasic: reactions occurred both on a surface with activator (TF) and in the volume of plasma. The following model constants were different from the original model: since blood was collected into corn trypsin inhibitor (CTI) to inhibit contact activation, kcomact (constant of contact activation) was set to 0 m in-; association rate constants of factors Vla and VII with TF (kLIa,"r and A ),"" ) were set to 0.0094 nM-'min-'; dissociation rate constants of factors VIIa and VII with TF (a * and kJ"/-) were set to 0.0342 min-; the dissociation rate constant of factors Xa, Vlla and TF was set to 385 min; the catalytic constant of factor X activation by the complex VIla-TF (kca V/tP) was set to 20 min'; the Michaelis constant (K a-) was set to 390 nM; since experiments were performed at platelet-free plasma, effective constants kgJ' and k " were set to 0 nM 2 min-. [006831 The results of computer simulation at a tissue factor (TF) density of 5 pmole/n 2 showed that complete inactivation of TFPI leads to significantly shortened lag time, while the effect on clot propagation is small in both normal and hemophilia plasmas (Figure 152A). Increasing the TF density to 100 pmole/m led to a substantially shorter lag time in both normal and hemophilia plasmas when TFPI was present (Figure 152B). In contrast to the 5 pmole/m2 TF density simulation, complete inactivation of TFPI at 100 pmole/m TF density led to only minor improvements in velocity and clot size while lag time was not affected. These simulation1s predicted that TFPI blocking should affect the initial clot formation phase, predominantly 224 WO 2012/109675 PCT/US2012/024916 changing lag time and initial clot growth velocity. As shown in Figure 152C for the lag time, the effects of TFPI blocking should be greater at lower TF densities. [006841 To understand the relative roles of TFPI and fVIII in spatial clot formation and to predict possible drug-drug interaction effects for TFPI antagonism and fVIII supplementation, computer simulations were also performed for a range of concentrations of fVIII from 0 to 0.7 nM, and TFPI from 0 to 2.5 nM (ie. from 0 to 100% activity for both proteins). Clotting in the model was initiated by TF surface density of 10 pmole/mi 2 . (Figure 153). TFPI antagonism influenced clot initiation time by shortening lag time (Figure 153A), while the effect of flIiI was predominantly on the clot propagation velocity (Figure 153C). The integral parameter, clot size (Figure 153D), depended on TFPI in the whole range of fV7III concentration. However, the absolute efficiency (with regard to overall clot size increase) of TFPI antagonism remained constant with fVIII level increase, while its relative efficiency decreased. [006851 Experimental measurements of spatial clotting were performed in a specially designed chamber (Figure 154A). Plasma samples were loaded into the well of the chamber that was subsequently placed in the thermostat. All experiments were performed at 37 0 C. Clotting was initiated by immersion of an insert with TF immobilized on its end face into the chamber. Clot formation was registered by light scattering from the fibrin gel using a CCD camera (Figure 154B). The chamber was uniformly illuminated with monochromatic light. Scattered light passes through the optical filter that reduces external noise. Images were captured every 15 seconds. The acquired series of images was then processed by computer and parameters of spatial dynamics of blood clotting were calculated. The acquired series of images was then processed by computer and parameters of spatial dynamics of blood clotting were calculated. [006861 For the purposes of this set of experiments, surfaces were derivatized with a TF density of 1-2 pmole/m'. Thromboplastin was immobilized on a polystyrene surface by chemical sorption method essentially as described [Fadeeva et al (2010) Biochemistry (Mosc) 75: 734-7431. The surface was covered by a thin layer of PEI as follows. Albumin at 4 mg/n, 2% PEI, and 1% glutaraldehyde were mixed at a 2:1:1 volume ratio and spread over the polystyrene surface at 13 qL/cm t . The polystyrene surface was dried for 24 hours at room temperature (RT). 'Then it was incubated with 0.5% glutaraldehyde for 1 hour at RT. and washed four times for 15 minutes with distilled water. The obtained surface was incubated in 12 nM thromboplastin (0.2 mL/cm) for 1 hour at RT, and washed twice with distilled water for 15 225 WO 2012/109675 PCT/US2012/024916 minutes. Free glutaraidehyde on the surface was blocked by incubating for 1 hour with 0.1 M glycine (0.2 mL/ci2). Finally, the activator was washed four times with 20 mL of distilled water for 15 minutes, dried at 37 "C for 30 minutes and sealed in a polyethylene bag. The activators were stored at 4 'C. [006871 The TF activity on the surface was characterized by the ability to activate factor X in the presence of excess factor Vila. Activators (1 x4 min) or calibrator TF solution prepared with a calibrator TF from Actichrome TF assay were placed into the wells of a 96-well plate, Buffer A (I M Tris, 150 mM NaCl, 0.1% PEG-6000, p1H 8.7) was then added at 20 tL. This was followed by the addition of factor VIla (20 pL, 60 nM) in buffer A containing calcium chloride (15 mM) and incubation for 5 min at RT. Finally, 20 pL of factor X at 1,5 pM in buffer A was added. Activators were incubated at 37 'C for 30 minutes, Activators were removed from the wells. To stop the activation and begin the detection, 40 pL of solution containing EDTA at 25 mM and chromogenic substrate S-2765 at 1.05 rnM was added to the wells. The rate of chromogenic substrate cleavage was determined by light absorption at 405 nm wavelength using a Thermomax microplate reader in the kinetic mode. TF concentration was determined frorn the calibration curve. [00688] Blood was collected from hemophilia A patients with their informed consent. All patients were diagnosed with severe hemophilia A (FVIII: :C<1i%), were on factor VIII prophylaxis receiving 30 or 50 IU/kg (except for patient 8), and reported not using factor VIII concentrates for 3 days before the experiment. Blood samples from most of the patients were collected before the administration of factor VIII (0 h), and at 1, 4, 24, and 48 h after the administration (for patients 7-9, only samples at 0, 1 and 24 hr were collected). Only the samples collected at 0, 1 and 24 hr were used for the spatial clotting experiments, while APTTs and factor VIII levels (with the clotting activity-based assay) were determined for all samples. Figure 155 shows patient characteristics and factor VIII and APTT values determined before factor VIII administration. Figure 156 shows factor VIII levels and APTT values determined for each patient at different timepoints, all of which are in good agreement with known parameters of factor VIII pharmacokinetics. Blood for spatial clotting experiments was collected at a 9:1 v/v ratio into a solution containing 3.8% sodium citrate (pH 5.5) plus corn trypsin inhibitor (CTI) at 0.1 mg/iL (final concentration). Then blood was processed by centrifugation at 2,500 g for 15 min to obtain platelet-poor plasma, and then additionally centrifuged at 11,000 g for 5 min to obtain platelet 226 WO 2012/109675 PCT/US2012/024916 free plasma and frozen at -80 'C for later use. Before experiments, frozen plasma was thawed for 10 min under flowing RT water, and the plasma pH was stabilized at 7.2- 7. by lactic acid treatment as described [Sin'auridze et al (1998) Biochim Biophys Acta 1425: 607-616]. ARC 19499 aliquots were thawed at RT for 30 min before the first experiment of the day. ARC19499 was dissolved in phosphate buffered saline (PBS) to achieve the necessary final concentration. At 15 min before each experiment, 300 pL of plasma was supplemented with 18 pL of AR-C19499 solution. In control experiments with a final concentration of 0 nM of ARC1 9499, plasma was supplemented with the same volume of vehicle PBS. Activator was placed into buffer (20 nM Hepes, 150 mM of NaCl, pH=7.2-7.4) to reduce bubble formation near the activator during the experiment. The solution of 1 M CaC 2 , buffer with activator and prepared plasma were incubated separately at 37 'C for 15 minutes. The experimental chamber was placed into the thermostat of the experimental device at 37 C.. Plasma was subsequently recalcified by addition of 6 tL I M CaCl2, quickly mixed and 300 pL of plasma was placed into the experimental chamber. The activator was taken out of the buffer, buffer excess was removed by blotter, and the activator was placed into the experimental chamber to start clotting. [00689] For each experiment, the parameters of clot growth were determined on the basis of image series. First, the background image was subtracted from each image of the series, and the resulting images were analyzed. A perpendicular to the activator was drawn and clot profiles (plots of mean light scattering [based on pixel intensity] versus distance from the activator) were calculated. The clot size was determined for each profile as a coordinate where the light scattering intensity was 50% of the maximal one, which corresponds to half-maximal fibrinogen conversion into fibrin Las described in Ovanesov et al (2005) J Thromb Haemost 3: 321-331]. Spatial clotting parameters including the lag time, initial velocity of clot growth (c or Vtjtiai), spatial velocity of clot growth (f or Vstationary) and clot size after 60 rnin were calculated from the clot size versus time plots. For each experiment, four perpendiculars to the activator surface were drawn at different areas of activator. Profiles of clot growing were analyzed and four values for each clotting parameter were obtained and then averaged to obtain means. [00690] The experimental effect of ARC19499 on spatial clotting in hemophilia A plasma was tested using plasma from 9 patients. Figure 157 illustrates the effects of 100 nM ARC19499 added in vitro to hemophilia A plasma collected at different timepoints after fVIII infusion. Figure 157A shows typical light-scattering time-lapse images of clot growth initiated by 227 WO 2012/109675 PCT/US2012/024916 immobilized TF at surface density of 2 pmoie/nt in hemophilia A plasma before factor VIII administration (0 hr) and at I and 24 hours after factor VIII administration, in the presence and absence of ARC19499 (100 nM). In these images, the TF-coated activator is seen as a vertical black strip on the left side of each image. Panels B, C and D of Figure 157 are clot size versus time plots derived from the associated images in Panel A. Panel C also illustrates the parameters used for experimental analysis throughout the study. [006911 As a control, a series of experiments was performed comparing the effects of ARC1 9499 on freshly prepared plasma and on the same plasma that had undergone a cycle of freezing and thawing. Since the plasma preparation method used in this study included freezing/thawing plasma before the experiment, this could have shifted clotting parameters to hypercoagulation values and led to the appearance of spontaneous clotting in the bulk of plasma during the experiment. Figure 158 shows the ratios of spatial clotting parameters with or without 300 nM ARC] 9499 measured in freshly prepared plasma collected into CTI and the same frozen/thawed plasma. This figure shows that there were no significant differences in the clotting parameters or their response to ARC] 9499 between freshly prepared plasma and the same frozen/thawed plasma. [006921 Figures 159 to 167 show the dependence of clotting parameters on ARC] 9499 concentration before (0 h), and at 1 and 24 h after factor VIII had been administered to patients I to 9, respectively. Panels A, B, C and D of each figure display lag time, initial velocity, stationary velocity and clot size, respectively. Panel E shows the clot size dependence on factor VIII and ARC 19499 concentrations. In all patients, ARC 19499 improved spatial clotting by shortening the lag time and increasing the clot size. ARC19499 caused significant effects for a whole range of factor VIII concentrations, although the extent of these effects varied from patient to patient. The effects on Via. and Vstationary were more mixed, but increases in these parameters were observed in several of the samples. [006931 The drug-drug interaction outcome was similar in the majority of patients to that predicted in simulations. As shown in Panel E of Figures 159 to 167, ARC 19499 had its greatest effects on clot size at low factor VIII concentrations, while factor VIII was most effective at low ARC 19499 concentrations. This was observed for most plasmas except for those from patients 2 and 3, where the relative effect of ARC 19499 was similar for all factor VIII concentrations (and vice versa). In addition to this, we could not study low factor VIII concentrations in patient 9, 228 WO 2012/109675 PCT/US2012/024916 who had very high initial factor VIII level. Still, we were able to observe the lack of significant ARC 19499 effects at high factor VIII concentration. Patient 7 plasma had significant spontaneous clotting in the 24 h points, which did not allow estimating clot size. However, data from the 0 and 1 h points allowed inclusion of this patient's plasma into the same category. [006941 The results obtained using plasmas from different patients were averaged for further analysis. Figure 168 shows the effect of 100 nM ARC19499 addition for three different concentration ranges of factor VIII in the blood of patients: less then 5'%, from 5% to 30%, and more than 30%. Means and S.EM. for all clotting parameters are shown. Note that the numbers of experiments in different panels of Figure 168 are somewhat different. This is caused by the fact that not all clotting parameters could always be calculated. For example, the lag time cannot be calculated for experiments where there is no clotting. Conversely, only lag time and not clot size at 60 min can be calculated for experiments in plasma with spontaneous activator independent clotting. [00695] Panel A of Figure 168 shows a comparison of average lag tires with and without 100 nM ARC 19499 addition for the indicated factor VIII concentration ranges. ARC1 9499 induced a statistically significant decrease in lag time for all three ranges of factor VIII concentration (the means are significantly different with P<0.05 using paired t-test). However, the values achieved with AR C19499 were not significantly different from each other (P>0.05 using unpaired t-test). There was also some decrease in the ARC19499 efficiency with factor VIII concentration increase (lag time shortened by 50% for FVIII<5%, but the effect was only 25% for FVIII>30%). 1006961 Panels B and C of Figure 168 display the effect of ARC19499 on the clot growth velocities for the same concentration ranges. The effect of ARC 19499 on the initial velocity was statistically significant (except for FVIII>30%) and that on the stationary velocity was not (except for 5%<FVIII<30%), but they were rather small (18% to 30% increase except for initial velocity at FVIII<5%, where the effect exceeded 40%). [006971 As shown in Panel D of Figure 168, ARC19499 caused a statistically significant increase in clot size for all three ranges of factor VIII concentration (P<0.01). The effect of ARC 19499 decreased from 200% down to 40% as factor VIII concentration increased (from less than 5% to more than 30%). In other words, addition of ARC 19499 led to the achievement of a 229 WO 2012/109675 PCT/US2012/024916 plateau that is the same for all factor VIII levels (values of clot size with ARC 19499 were not significantly different for these three concentration ranges at P=0.05 using the unpaired t-test). [006981 Finally, the averaged spatial clotting parameters obtained at 0, 1 and 24 hours after factor VIII infusion are shown as a function of ARC 19499 concentration in Figure 169. Vertical error bars indicate standard errors and the number of experiments equals that of patients, i.e. n=9, The absolute dose-dependence of the ARC 19499, with respect to the magnitude of the increase on clot size (Panel D), appeared to be similar ffor all three tinepoints. The relative effect of ARC19499 is maximal for the 0 h tinepoint (squares), and smallest for the 1 hr (circles) and 24 h (triangles) timepoints. [006991 The mechanisins of action for TFPI and ARC19499 based upon data from the spatial coagulation model are schematically represented in Figure 170. In normal plasma, extrinsic tenase is inhibited by TFPI in fXa-dependent manner after some fXa has been produced, thus preventing further activation of factors X and IX. This, however, is normally compensated by intrinsic tenase: in contrast to fXa, fIXa is inhibited by antithrombin fiI slowly and can efficiently diffuse in space, and binds fVIIIa to produce fXa far from the activator (Figure I 70A). This pathway does not function in hemophilia A plasma (Figure 17013): clotting initiation is not affected, but spatial propagation is impaired. Blocking TFPI by ARCI 9499 in hemophilia A plasma prevents inhibition of mernbrane-bound complex VIa-TF. Consequently higher production of factors Xa and IXa leads to increase of thrombin level and accelerates the initial phase of clot formation (Figure 1 70C), but not the defective spatial propagation phase. To summarize, inactivation of TFPI with ARC19499 does not normalize impaired spatial clot propagation in hemophilia A, but rather accelerates clotting initiation ultimately making final clot size similar to normal one. [007001 Similarly, in Figure 171 we used mathematical modeling to show factor Xa produced by intrinsic and. extrinsic tenases as function of space and time, in the presence or absence of tVIII and/or TFPI. Panel A shows a typical clot size versus time plot for normal and hemophilia A plasmas in the presence and absence of TFPI. Panel B shows the effects on factor Xa generation by the intrinsic tenase (right panels) and the extrinsic tease (left panels) under the same conditions. As shown in these panels, hemophila A essentially eliminates intrinsic factor Xa generation. TFPI inhibition has little effect on intrinsic factor Xa generation, but substantially boosts extrinsic factor Xa generation. Notably, the boost in factor Xa production is localized near 230 WO 2012/109675 PCT/US2012/024916 the activator; factor Xa cannot get far from the activator because of its rapid inhibition in plasma. Therefore, the effect of TFPI antagonism is localized: clots get larger, but only so much. This gives advantages when there is no factor VIII, and. extrinsic factor Xa is the critical component of coagulation; in contrast, when factor VIII is present and provides clot propagation far from the activator, TFPI inhibition can do little to improve clotting. [00701] In conclusion, the data from this study indicate that spatial clot formation is significantly improved by ARC 19499 addition for the whole range of factor ViII concentrations in hemophilia A plasma, mainly by shortening lag time and increasing clot size. In contrast, factor VIII has little effect on lag time, but increases spatial clot growth velocity. The effect of ARC19499 on the lag time is only slightly affected by increasing levels of factor ViII. With respect to clot size, the relative effect of ARC 19499 was large at low factor VIII concentrations and small at high factor VIII concentrations in the majority of the studied patients (seven out of nine). And, vice versa, the effect of factor VIII was large at low ARC 19499 concentrations. In the remaining two patients, the relative effect of A R-C9499 was similar for all factor VIII levels, although the available data do not give any indication that this difference in the response may be due to initial factor VIII level or factor VI1I concentrate used (IHaemoctin, Kogenate, or Octanate). [00702] The overall conclusion from the studies using the spatial in vitro experimental model of coagulation is that the effect of ARC 19499 in hemophilia A plasma gets smaller with increasing factor VIl concentration. The same is true for the factor VIII effect as a function of ARC 19499 concentration. This suggests that patients can be safely treated with ARC1 9499 without concern for their current factor Vmi level, and, vice versa, patients taking ARC19499 during clinical trials can be safely treated with factor VIII concentrates. EXAMPLE 45 [007031 This example demonstrates that ARC 19499 protects Factor Xa (FXa) from inhibition by TFPI in a chromogenic assay with purified components. TFPI is a slow-acting, potent FXa inhibitor [Huang et al (1993) J Biol Chem 268(36):26950-269551, requiring the Kunitz-2 domain of TFPI [Girard et al (1989) Nature 338:518-520; Petersen et al (1996) Eur J Biochem 235:310 316], but other TFPI domains also contribute to strengthening this interaction (Huang et al 1993). The initial encounter with FXa involves the rapid formation of a weak collision complex between TFPI and FXa that slowly isomerizes to a tight-binding complex. In the presence of a 231 WO 2012/109675 PCT/US2012/024916 FXa substrate, this results in a biphasic velocity curve with an initial burst of FXa activity (u 0 ) followed by a slower steady-state velocity (us). In this example we explored the effect of ARC 19499 on TFPI inhibition of FXa, as it is reflected in u, and u,. [007041 The ability of FXa to cleave a chromogenic substrate (S-2765, Chromogenix) was measured in the presence of various concentrations of TFPI and/or ARC 19499, with alterations in the order in which the components were added to the mixture, and the length of time they were incubated together prior to starting the reaction. In the first experiment, increasing concentrations of TFPI (from 0-32 nM) were mixed with FXa (1 nM final) and S-2765 (0.5 mlM final), with either FXa added last (Figure 172A) or S-7265 added last (Figure 172B). When FXa was added last (Figure 1 7 2A) the progress curve for the cleavage of S-7265 began with an initial burst of product generation characterized by u 0 that transitioned into a steady-state velocity (is) as the FXa/TFPI complex stabilized. Increasing concentrations of TFPI up to 32 nM caused incremental decreases in both o 0 and u. When substrate was added last (Figure 172B), FXa and TFPI were rnixed together just prior to substrate addition. Although these steps were completed within only a few minutes, the equilibration of FXa and TFPI was sufficiently rapid that, by the time substrate was added, formation of the tight complex appeared to be largely complete. The progress curve in this case exhibited less of a biphasic character and the rate of product formation primarily reflected the steady-state velocity i. In the presence of > 8 nM TFPI, inhibition is nearly complete under these conditions and little product is formed. [00705] In order to examine the effects of ARC 19499 on these reactions, 8 nM TFPl was mixed with increasing concentrations of ARC19499 (1, 10, or 100 nM final) with I nM FXa and 0.5 mMn1 S-2765. Either FXa was added last (Figure 172C) or S-2765 was added last (Figure 172D). ARC19499 appeared to have a significant effect on the steady-state velocity. This is most clearly evident in Figure 172D, where little FXa activity was observed in the presence of 0 or 1 nM ARC19499, but substantial activity was restored with higher ARC19499 concentrations. A similar effect on steady-state velocity is evident in Figure 172C, while the effect on the initial velocity of the reaction is less clear, but appears to be small. [007061 Further experiments were performed to examine the effects of ARC 19499 on ius Similar to the experiments in Figures 1 72B and 172D, substrate was added last, but to ensure that inhibitory complexes were completely equilibrated, FXa, TFPI, and aptamer were incubated for 30 minutes at room temperature prior to substrate addition (Figure 173). Final concentrations of 232 WO 2012/109675 PCT/US2012/024916 FXa (1 nM) and S-2765 (0.5 nlM!) were the same as in the previous experiment, but TFPI was varied from 1 to 40 nM and ARC19499 was varied from 1.25 to Z2560 nM. In Figure 173A, the FXa reaction velocity is plotted as a function of aptamer concentration on the x-axis, and in Figure 173B the same data are plotted as a function of TFPI concentration on the x-axis. The observed velocity was 40 - 45 mOD/min in the absence of TFPI or ARC19499 and substantial inhibition was observed even at the lowest TFPI concentration (1 nM). As previously observed, nearly complete inhibition occurs with TFPI concentrations > 8 nM. Increasing concentrations of ARC19499 were able to increase FXa activity in the presence of TFPI. At all TFPI concentrations, the highest concentrations of aptamer were unable to completely restore FXa activity to levels seen in the absence of TFPI (filled diamonds, Figure 173A). Figure 173B shows that the effect of ARC19499 was saturated by 160 nM aptamer. This figure demonstrates that even saturating concentrations of ARC 19499 did not fully reverse TFPI inhibition in this assay; TFPJ remained a weak inhibitor of FXa. [00707] Taken together, these two figures suggest that AR-C19499 may play its most significant role in inhibiting TFPI after it has corne in contact with FXa. This would explain the small effect on initial velocity of the reaction, and the much larger effect on the steady-state velocity. These data also suggest that, although ARCI 9499 inhibits TFPJ, the resulting TFPI:ARC 19499 complex can still interact with and inhibit FXa, explaining why in Figure 173, there is not complete inhibition of TFPIL. EXAMPLE 46 1007081 This example examines the regions on TFPI where ARC17480 and ARC 19499 bind. For these experiments, antibodies that bind to different regions on TFP3I were used to compete for binding to TFPI with ARC 19499 in a plate-based binding assay, or to compete for binding to TFPI with ARC17480 in a dot-blot binding assay. The antibodies used for competition are shown in Table 41 below. [007091 This example demonstrates that antibody M105273 (monoclonal antibody from Fitzgerald Industries) competed for binding of ARC19499 to TFPI in a plate-based binding assay and competed for binding of ARC 17480 to TFPI in a dot blot-binding assay (Figure 174 and Figure 175A). Antibody M105273 was raised against a fragment of TFPI containing K3 domain amino acid residues 160-250, and binds to TFPI somewhere in this region (Table 41). This example also demonstrates that antibody M105271, M1 05272, and AIITFPI-5138 all compete 233 WO 2012/109675 PCT/US2012/024916 with ARC 19499 for binding to TFPI in the plate-based binding assay (Figure 174) but not in the dot blot-binding assay (Figure 175A), M105271 is a monoclonal antibody raised against a fragment of TFPI containing KI domain amino acid residues 17-87. M105272 is a monoclonal antibody raised against a fragment of TFPI containing K2 domain amino acid residues 88-160. AHTFPI-5 138 is a monoclonal antibody raised against the N-terminus of TFPI (residues 1-16). Meanwhile, M105274 which was raised against the C-terminus of TFPI (amino acid residues 251 -276), only demonstrated weak competitor activity in both assays (Figure 174 and 175A). PAHTFPI-S, a polyclonal antibody raised against a truncated version of TFPI containing only the K-1 and K2 domains, did not demonstrate any competition in the dot blot-binding assay, similar to ARC32603, the negative control oligonucleotide (Figure 1715B), but did compete as well as ARC19498 in the plate-based binding assay (Figure 174). The antibodies used for the competition experiments are shown in Table 41 below. [00710] For plate-based binding experiments, 400 ng/well of TFPI (American Diagnostics, cat# 4900PC) in 100 plL Dulbecco's Phosphate-buffered Saline (DP3S) was used to coat a 96 well Maxisorb plate at 4 'C. The TFPI solution was then removed and the plate was subsequently washed 3 times with 200 L wash buffer (DPBS + 0.05% Tween 20) at room temperature. The plate was then blocked with 200 pL of 10 mg/mL bovine serum albumin (BSA) in DPBS for 30 minutes at room temperature. The BSA blocking solution was then removed and the plate was washed 3 times with 200 pL wash buffer. The competing antibodies were serially diluted and mixed with ARC] 9499 at the final concentration of 25 nM ARC 19499 and 0.1% BSA in DPBS, and the mixture was then added to the assay plate and incubated for 3 hours at room temperature, ARCI 9498 was similarly mixed with ARC 19499 and used as a positive control in the antibody competition assay. Wells were then washed, as described above, For experiments using PAHTFPI-S for competition, 100 pL. of 0.5 pg/mL rabbit monoclonal anti-PEG antibody (Epitomics, cat #0 2061-1) in assay buffer was added to the plate and incubated for 3 hours at room temperature. Anti-PEG antibody solution was then removed and the plate was washed as described above, followed by addition of 100 pL of 1:1000-diluted anti rabbit IcG-HRP secondary antibody in assay buffer to each well (Cell Signaling Technology, cat # 7074) and incubated for 30 minutes. The secondary antibody solution was removed and the plate was washed, as described above, For the remaining antibodies, 0.5 pg/mL of 100 L biotinylated rabbit monoclonal anti-PEG antibody (Epitomics, cat # 2173) in assay buffer was 234 WO 2012/109675 PCT/US2012/024916 added to the plate and incubated for 3 hours at room temperature. Anti-PEG antibody solution was then removed and the plate was washed as described above, followed by addition of 100 piL of streptavidin-B-tRiP (4800-30-06) from R&D Systems (Minneapolis, MN) diluted 200-fold in DPBS and incubated for an additional 1 hour at room temperature. The streptavidin-HRP was then removed and the plate was washed as described above. Then 100 pL of TMB solution (Pierce, #34028) solution was added to each well and incubated for 2 minutes, followed by addition of 100 pL stop solution (2N H 2 SO4) to each well to stop the reaction. The assay plate was then read at 450 nm using a Victor 3 V 1420 multilabel counter (Perkin Elmer). Percent inhibition of binding was calculated using 0 nM antibody in 25 nM ARCI19499 as 0% inhibition, and 0 nM antibody and 0 nM ARC19499 as 100% inhibition. The IC 50 was calculated based on 4-parameter logistics using Prism 4 Graphpad software. [007111 As shown in Figure 174, antibodies M1 05271, which binds within the KI domain of TFPI, M105272, which binds within the K2 domain of TFPI, M105273, which binds within the K3 domain of TFPI and A[ITFPI-5 138 which binds to the N-terminal region of TFPI all competed with ARC19499 for binding to recombinant TFPI in the plate-based binding assay. Of these, M105273 against the K3 domain of TFPI competed the best, blocking nearly 100% of the interaction between ARCi 9499 and TFPI, while the others block between 60% and 85% of the interaction. PAIITFPI-S, which is a polyclonal antibody directed against the K1I and K2 domains of TFPI, inhibited nearly 50% of the interaction at the highest concentration that could be tested (300 nM); however, further inhibition is possible at higher concentrations of this antibody since a plateau was not achieved. M 105274, a monoclonal antibody that binds within the C-terminal portion of TFPI, only partially competed (< 50%) with ARC19499 for binding to TFPI in this assay (Figure 174). 1007121 The antibodies in Table 41 were also tested in a dot blot-based competition binding assay. In these experiments, trace amounts of radiolabeled ARC 17480 were incubated with 10 nM recombinant TFPI with or without the addition of antibody. Antibodies were tested at 1000 nM, 333 nM, 11I nM. 37.0 nM, 12.4 nM, 4.12 nM. 1.37 nM, 0.46 nM, 0.15 nM and 0.051 nM. ARC 17480 was included as a competitor in every experiment as a control. For each molecule, the percentage of radiolabeled ARC 17480 bound at each competitor aptamer concentration was used for analysis. The percentage of radiolabeled ARC 17480 bound was plotted as a function of aptamer concentration and fit to the equation y=(max/(1 + x/ICo)) + int, where y=the percentage 235 WO 2012/109675 PCT/US2012/024916 of radiolabeled ARC 17480 bound, x=the concentration of aptamer, max=the maximum radiolabeled ARC 17480 bound, and int=the y-intercept, to generate an IC 50 value for binding competition. Figure 175 shows the binding-competition experiments carried out with M105271. M105272, M105z73, M105274, PAHTFPI-S, and ARC32603. These experiments demonstrate that antibody MI 05273 competes for ARC1 7480 binding to TFPI in the dot blot competition assay (Figure 175A). M1 05274 partially competes for binding in this assay (Figure 175A), while M105271, M 105272, and PAHTFPI-S show no significant competition for binding with ARC] 7480 (Figure 175A-B). [007131 The main differences between the two sets of experiments described above may be summarized as follows: 1) the plate assay utilized immobilized TFPI while the dot-blot assay utilized 10 nM TFPI in solution: 2) the competed aptamer in the plate-based assay (ARC19499) had a high-molecular weight PEG at the 5'-terminus, while the competed aptamer in the dot-blot assay (ARC 17480) only had a radiolabeled phosphate group; and 3) the competed aptamer in the plate-based assay was added at a concentration of 25 nM while the competed aptamer in the dot blot assay was added only in trace (picomolar) amounts. Any of these factors could have contributed to discrepancies in the results, specifically the apparent competition by MI 05271, M105722 and PAHTFPI-S that was evident in the plate-based assay but not the dot-blot based assay. The two assays were in agreement showing nearly complete competition by MI 05273 (against the K3 domain of TFPi) and partial competition by MI 05274 (against the C-terminal region of TFPI). Taken together, these experiments suggest that the K3 domain plays an important role in the interaction between ARC17480/ARC19499 and TFPI, and the C-terminal domain may play a role as well. However, as the plate assay data indicates, multiple regions of TFPI may contribute to this interaction, either directly or indirectly, and the data do not preclude involvement of any particular domain or region of TFPI in aptamer binding. Table 41: Antibodies used in ARC 19499 competition assays Region of mature TFPI Type used as an antigen for Antibody target region Antibody I antibody generation in TFPI MII05721 mouse monoclonal 17-87 K1 region _4 05 72 2 _nmuse nonocloi_ 88-160 _ 2 region M 05723 mouse monoclonal 160-250 K3 region M105724 mouse monoclonal 251-276 C-termal peptide A fHTFPI- mouse monocIonI 5138 1-16 N-terminal peptide 236 WO 2012/109675 PCT/US2012/024916 Truncated version of sheep polyclonal TFPI containing the K1 PAHTPI-S and K2 domains K1 and K-2 domains EXAMPLE 47 [007141 This example evaluates ARC 19499 concentrations in plasma samples obtained in a non-human primate model of hemophilia A. This example expands on Example 32 describing the effectiveness of ARC19499 in correcting the clotting defect in a monkey model of Hemophilia A. [007151 In this experiment, a non-human primate model of Hemophilia A was created by injecting cynomolgus monkeys with a single intravenous (IV) bolus of sheep polyclonal antibody against human Factor VIii (20 mg; 50,000 Bethesda Units). 3.5 hours after the IV injection, the monkeys were treated with either saline (1 rnL/kg), recombinant Factor VIla (rFVlla) (NovoSeven*; 90 ,g/kg bolus) or ARC19499 (either a 600 pg/kg, 300 gg/kg or 100 g/kg bohls). Citrated blood samples were acquired before antibody administration (baseline, time:::0), 2.5 hours after antibody administration, 15 minutes after drug/saline treatment (time::::3.75 hours), and I and 2 hours after drug/saline treatment (time = 4.5 and 5.5 hours, respectively). Blood was processed to generate plasma, and citrated plasma samples were analyzed using a number of assay methodologies including prothrombin time (PT), activated partial thromboplastin time (aPTT), Factor VIII function and thromboelastography (TEG ). Notably, the 300 qg/kg and 600 pg/kg doses of ARC19499 appeared to be effective in correcting the clotting defect in these samples as measured by TEG (Example 32). In order to verify that ARC19499 was present in these samples, plasma concentrations of ARCi19499 were measured by high performance liquid chromatography with ultraviolet absorbance detection (HIPLC-UV). [007161 In order to prepare plasma samples for HPLC analysis, a small aliquot (50 pL) of test plasma was nixed with 25 pl of digestion buffer (60 rnM Tris HCL, pi 8.0, 100 mM EDTA and 0.5% SDS) and 75 pL of proteinase solution (1. 0 mg/nL. proteinase K in 10 mM Tris HCl, pH 7.5, 20 niM CaCi2, 10% glycerol v/v). The 150 pL samples were incubated overnight, shaking, at 55 C. Following incubation, samples were centrifuged (14,000 pin; 4 C; 15 minutes), and 100 pL of the supernatant was withdrawn and transferred to HPLC injection vials. The assay injection volume was approximately 25 jpL. The instrumental conditions used for HPLC analysis are shown in 'Table 42. The lower limit of quantitation (LLOQ) was 0.2 pg/mL 237 WO 2012/109675 PCT/US2012/024916 with a linear concentration range of 0.2 to 500 ptg/mL. The HPLC method was calibrated relative to concentration reference standards of ARCI 9499 prepared in blank monkey plasma and extracted by the same proteinase method used to prepare in vivo samples. All reported concentrations of ARC 19499 are based on the mass of aptamer, excluding the mass of PEG. Table 42: Instrumental conditions for lIPLC-UV Equipment: IPLC system equipped with an autosampler, column temperature-controller and UV detector Column: Dionex DNAPAK PA-100 (4 x 250mm) with Guard (4 x 50nun) Column Temperat-ure: 80 'C Flow Rate: 0.9 mL/min Injection Volume: 25 p[L UV Detector: 260 nm Run Time: 30 minutes RT Approximately 15.8 min A: 75% 25 mM sodium phosphate dibasic buffer (pH 7.0) and 25% Acetonitrile. Mobile Phase: B: 75% 25 mM sodium phosphate dibasic in water (pH 7.0) and 25% Acetonitrile containing 400 mM NaClO4 Time %A %B 0.00 75 25 2.00 75 25 20.0 50 50 Gradient Table: 22.0 50 50 24.0 0 100 26.0 0 100 26.5 75 25 30,0 75 25 [007171 Concentrations of ARC 9499 measured in individual plasma samples can be found in Table 43 (300 pg/kg) and Table 44 (600 tg/kg). Monkeys that were treated with 300 ptg/kg had measured mean levels of ARCI 9499 between 6.80 and 8.05 ,g/mL (0.61-0.73 pM) over the course of the study. Monkeys that were treated with 600 pg/kg had measured mean levels of ARC19499 between 14.93 and 15.73 pg/mL (1.35 -- 1.42 pM) over the course of the study. 238 WO 2012/109675 PCT/US2012/024916 Table 43: ARC19499 plasma concentrations (pg/ni) in cynomolgus monkeys treated with 300 gg/kg ARC 19499 DR9N DV35 DT24 DP4F DT2L FA2P Mean SEM 0.25 hr post ND* ND ND 7.56 7.70 8.91 8.05 0.430 treatment 1 hr post 800 735 7.32 6.39 6.36 7.72 7.19 0.277 treatment 2 hr post 7.87 6.72 7.03 6.07 6.07 706 6,80 0.279 treatment * ND, not determined Table 44: ARC19499 plasma concentrations(pg/mL) in cynomolgus monkeys treated with 600 pg/kg ARC 19499 DP4F DV35 Mean SEM 0.25 hr post 15.98 15.48 15.73 0.252 treatment 1 hr post 15.87 14.57 15,22 0.647 treatment 2 hr post 15.52 1435 14.93 0586 EXAMPLE 48 1007181 This example demonstrates that ARC19499 protects Factor Xa (FXa) from inhibition by TFPI in an assay of FXa activity using purified components and a chromogenic FXa substrate. 1007191 To study TFPI-mediated inhibition of FXa, cleavage of the FXa substrate was monitored as described previously [Huang et al (1993) J Biol Chem 268(36):26950-51 with modification. Factor Xa was from Enzyme Research Laboratories. Recombinant human TFPI was purified from a cell line from Novo Nordisk (Copenhagen, Denmark) as described [Pedersen et al (1990) J Biol Chem 265(28):16786-931. Either 80 pL of FXa (0.Z2 nM, final concentration) or FXa with TFPI (2 nM, final concentration) was incubated in the assay buffer (20 mM Hepes, pH 7.4, 150 mM NaCi, and I mg/mL bovine serum albumin) with 3 mM CaCl2 at room temperature for 20 minutes, after which 20 pL of the chromogenic substrate (0.3 mM final concentration Pefachrome FXa 5523, Centerchem Inc.) or 20 pL of the chromogenic substrate 239 WO 2012/109675 PCT/US2012/024916 with TFPI (2 nM) was added to monitor the FXa cleavage of substrate. When investigating the influence of ARC 19499 on TFPI inhibition of FXa, varying concentrations up to 4 FM (final concentration) of aptamer were also included in the assay. FXa cleavage of substrate was monitored in a 96-well microtiter plate of a ThermoMaxTM plate reader (Molecular Devices, Sunnyvale, CA). [007201 In order to characterize the effect of ARCI 9499 on TFPI-m ediated inhibition of FXa, we first determined the apparent affinity between the aptamer and human TFPI. In this assay, varying concentrations of aptamer were mixed with either I or 2 nM TFPI and added to FXa to allow the FXa-TFPI complex to form. Uninhibited FXa was assayed by the cleavage of the specific chromogenic substrate. The data were normalized to relative FXa activity in which FXa activity in the absence of TFPI is I and FXa activity in the presence of TFPI but absence of the ARC] 9499 is zero. These normalized results are shown in Figure 176. It can be seen that the more aptamer present with TFPI, the more FXa activity rernained in the assay. The mid-point in the curves between the origin and the plateau estimates the apparent binding constant for the ARC 19499 and TFPI, which is at a concentration of approximately 50 nM ARC19499. Figure 178 illustrates that, even at a saturating concentration of aptamer, the relative FXa activity does not reach 100%. A possible explanation for these data is that ARC19499 does not bind to the second Kunitz domain of TFPI that is in direct contact with FXa; it therefore fails to completely block the interaction between FXa and TFPI. In other words, while the aptamer-TFPI complex retains FXa inhibitory activity, it is not as potent as TFPI alone. 1007211 We next investigated the interaction between FXa and TFPI. Similar to previous reports, we tested the interaction of FXa and TFPI by monitoring the cleavage of Xa substrate in the presence/absence of TFPI [Huang et al, 1993; Franssen et al (1997) Biochem J 323:33-7; Baugh et al (1998) J Biol Chem 273(8):4378-86]. FXa cleaves the chromogenic substrate with pseudo-first order conditions as shown in Figure 177A (curve labeled #1). When TFPI and the chromogenic substrate were added to FXa at the same time, the rate of substrate cleavage decreased gradually as FXa was inhibited by TFPI (curve 2 in figure 177A). When FXa was pre incubated with TFPI before the addition of substrate, the substrate cleavage rate at equilibrium was as shown in figure 177A (curve 3). When the aptamer was included in the assay, FXa activity in the absence of TFPI was not affected, as shown by curve 4 in figure 177B. However, when the aptamer was added to FXa simultaneously with TFPI, the rate of substrate cleavage 240 WO 2012/109675 PCT/US2012/024916 was dramatically increased as shown by curve 5 in figure 177B compared to curve 1 in figure 177A. As shown by curve 6 in figure 177B, when FXa and TFPI were pre-incubated to allow formation of the FXa-TFPI complex, addition of the aptamer enhanced the rate of Xa cleavage of substrate at equilibrium as compared to curve 3 of figure 177A. The comparison between curves #3 and 6 indicates that the aptamer was able to remove TFPI from the pre-formed Xa-TFPi complex, thereby allowing significant recovery of FXa activity. [007221 The slope of lines 1 and 4 in figure 177 represent the initial FXa concentration in the assay. Because TFPI is a slow-inhibitor of FXa, the slopes of curves #2, #3, #5, and #6 at the latter phase of the reaction represent FXa activity of each experiment at equilibrium. In other words, these slopes represent the amount of FXa that was uninhibited by TFPI under each condition. Since the uninhibited FXa concentration can be derived from the slopes, the FXa TFPI complex concentration at equilibrium represents the difference between the initial FXa concentration and the FXa concentration at equilibrium. Likewise, the TFPI concentration at equilibrium can also be derived. Thus, the FXa-TFPI equilibrium constants, Ki, in each assay were calculated as 0.24 nM, 0.15 nM, 4.3 nM, and 3.4 nM for curves #2, 3, 5, and 6, respectively. It is apparent that the presence of ARC 19499 caused a 20-fold increase in the Ki of TFPI for FXa. These data indicate that the aptainer interferes with the interaction of TFPI with FXa thereby rendering TFPI a less potent factor inhibitor of FXa. 1007231 In summary, the results indicate that ARC 19499 does not directly affect FXa activity, but it is able to interfere with TFPI binding to factor Xa and can also dissociate TFPI from the factor Xa-TFPI complex. EXAMPLE 49 [00724] This example demonstrates that ARC 19499 protects the extrinsic Xase complex from inhibition by TFPL. [007251 In this assay, 1 pM of re-lipidated recombinant human tissue factor (Innovin'T Baxter), 1 nM factor Vila (Enzyme Research Laboratories), 150 nM Factor X (FX), 3 mM calcium chloride, 2 nM TFPI, and varying concentrations of ARC 19499 were incubated at 37 'C with the chromogenic substrate for Factor Xa (Pefachrome FXa 5523, Centerchem Inc.). Recombinant human TFPI for use in these assays was purified from a cell line from Novo Nordisk (Copenhagen, Denmark) as described [Pedersen et al (1990) J Biol Chemn 265(28):16786-93]. 241 WO 2012/109675 PCT/US2012/024916 [007261 Factor Xa (FXa) cleavage of the substrate is shown in Figure 178A. Curve #1 shows FXa generation by the extrinsic Xase complex in the absence of TFPI; curves #2. to 5 show FXa generation in the presence of 2 nM TFPI with 4000, 1000, 100, and 10 nM ARC19499; curve #6 shows FXa generation in the presence of 2 nM TFPI but no ARC 19499. The data in figure 178A were transformed [as described by Baugh et al (1998) J Biol Chem 273(8):4378-86] into the amount of active FXa and are shown in figure 178B. Curve #1 indicates that the extrinsic Xase resulted in persistent FXa activity when TFPI is absent; curve #6 shows that TFPI shuts down Xa generation in the absence of ARCi 9499; curves #2 to 5 show that TFPI inhibition of factor Xa generation was delayed in the presence of ARC19499. It is also clear that to a certain extent, the more ARC 19499 that is present, the longer factor Xa generation can persist. There is a section in curves #2 to 6 where the active factor Xa is at equilibrium (where the curves turn horizontal). The point where each of the horizontal lines is extended toward the origin and intercepts with curve #1 may be defined as the point where TFPI turns off the extrinsic Xase. The turn-off time was plotted against the concentration of ARC 19499 and is shown in figure 178C. It is apparent that 4000 nM ARCi 9499 is at saturation in this assay. From figures 178A to 178C, it is also clear that ARCi 9499 prolongs factor Xa generation by the extrinsic Xase, but does not completely block TFPI regulation. The mid point of the curve in figure 178C is approximately 50 nM, consistent with the data shown in figure 176. EXAMPLE 50 1007271 This example demonstrates that ARC19499 prevents TFPI inhibition of FXa mediated activation of prothrombin within the prothrombinase complex. 1007281 TFPI inhibition of prothrombinase activity was performed as described previously [Franssen et al (1997) Biochem J 323:33-7] with some modification. 1,2-Dioleoyl-sn-Glycerol 3-phosphoethanolamine (DOPE), L-a-PhosphatidyIcholine (PC), and L-a- Phosphatidylserine (PS) for use in preparing phospholipid vesicles were from Avanti Polar Lipids, Inc. (Alabaster, AL). Unilamellar phospholipid vesicles (15% PS, 41% PC and 44% PE) were prepared as described [Mayer et al (1986) Biochim Biophys Acta 858(1):161-81. Recombinant human TFPI was purified from a cell line from Novo Nordisk (Copenhagen, Denmark) as described [Pedersen et al (1990) J Biol Chem 265(28):16786-93]. Factor Xa (FXa) was from Enzyme Research Laboratories and Factor Va (FVa) was from Haematologic Technologies. Prothrombin was prepared from prothrombin complex concentrates by chromatography on Q-Sepharose binding 242 WO 2012/109675 PCT/US2012/024916 (salt elution), heparin affinity chromatography, metal chelate chromatography, and calcium elution from HiTrap Q. The prothrombinase complex (0.1 pM factor Xa, 1 nM factor Va, and 20 pM phospholipids membrane) was incubated with or without TFPI (1 nM) at 37 9 C for 20 minutes before prothrombin (300 nM) and the chromogenic substrate for thrombin (0.3 mMTos Gly-Pro-Arg-pNA-AcOH, Centerchem, Inc.) were added. Thrombin cleavage of substrate was continuously monitored at 37 'C for 30 minutes. [007291 Curves #1 and #4 in figures 179A and 1719B (respectively) represent the thrombin generation from prothrombin by the Xa/Va complex in the absence (figure 179A) or presence (figure 179B) of aptamer. As can be seen, the presence of aptamer did not affect the prothrombinase activity in the absence of TFPl. When FVa was omitted from the assay, no thrombin generation could be detected as shown by lines #3 and #6. Thrombin generation froni the prothrombinase decreased when 1 nM of TFPI was included in the assay as shown by curve #2 in figure 179A. However, addition of ARCI 9499 to the assay restored thrombin generation to baseline levels as shown by curve #5 in figure 179B. Since this assay was designed to monitor the second-order reaction, the observed data can be fitted to the following equation: [00730] y:= A + Bx + Cx [00731] where C, the second differentiation constant, is proportional to the prothrombinase concentration. The fitted second differentiation constant from curves #1, 4, and 5 are the same, whereas the fitted constant in curve #2 is about 67% of the others. Since the second differentiation constant is proportional to the prothrombinase concentration, the data in figure 179 indicate that although prothrombin competes with TFPI on factors Xa/Va complex on the membrane, under the figure 179A's condition. TFPI inhibits about 1/3 of prothrombinase activity. However, ARC19499 blocks TFPI-mediated inhibition of prothrombinase, leading to restoration of activity as shown in figure 179B. [007321 'These results indicate that the aptamer interferes with TEPI inhibition of FXa. This interference applies not only to free FXa, but also to FXa within the prothrombinase complex. EXAMPLE 51 [007331 This example demonstrates that ARC19499 has biological activity in FVIII-deficient plasma and whole blood. ARC 19499 shortened the clot time in the plasma dilute prothrombin time (dPT) assay and the tissue-factor activated whole blood clotting assay. 243 WO 2012/109675 PCT/US2012/024916 [007341 For clot time measurements in plasma, Factor VIII (FVIII)-deficient plasma pooled from donors with hemophilia A was from HRF. Inc. (Raleigh, NC) and normal plasma was from Thermo Scientific (Middletown, VA). The dilute prothrombin time (dPT) was performed by mixing 70 pL of plasma with 70 pL of lipidated recombinant tissue factor (InnovinTM, either 0.5 or 1 pM) solution and incubating at 37 'C for 3 minutes before adding 70 pL of 25 mN CaCl2 to the plasma/Innovin mixture. Clotting time was recorded on a Start4 coagulometer. When testing the efficacy of ARCl 9499 in shortening the dPT in FVIII-deficient plasma, the aptamer was diluted into either 0,5 or I pM of tissue factor (TF) solution and incubated with FVIII-deficient plasma at 37 'C for 3 minutes before CaCl2 was added to initiate the clotting reactions. [007351 As shown in figure 180A, in normal pooled plasma, a final concentration of 0.25 pM tissue factor resulted in a dPT of 160 seconds in the absence of ARC]9499, whereas 0.5 pM of tissue factor resulted in a dPT of 140 seconds. Progressive prolongation of the dPT occurred as the FVIII level decreased, as shown in figure 180A. In the absence of FVIII, 0.25 pM TF failed to produce a measurable clot within 360 seconds (the pre-set parameter) whereas 0.5 pM of TF resulted in a clotting time of 270 seconds. When ARC 19499 was included in the dPT assay in factor VIII-deficient plasma, clotting times were shortened. As shown in figure 18013, in the presence of 0.25 pM TF, 30 nM ARC19499 shortened the dPT to 240 seconds. Similarly, 30 nM of ARC 19499 shortened the clot tirne in the presence of 0.5 pM TF to 180 seconds. 1007361 For measurements in whole blood, samples from individual patients were used. Outpatients with severe hemophilia A (<1 IU/dL FVIII activity, with or without FVIII inhibitor) were recruited from the Comprehensive Hemophilia Clinic at the University of North Carolina. All recruited patients had not been treated with FVIII or any bypassing product for at least 7 days. Blood samples were obtained by clean venipuncture using a protocol that was approved by the Institutional Review Board for human subjects committee at the University of North Carolina and transferred to tubes containing EDTA (final 5 mM) before testing. Similar to the dPT test in factor VIII-deficient plasma, 10 pL of the Innovin solution with varying amounts of ARC19499 was added to 150 gL of the EDTA-treated blood to achieve 0.5 pM tissue factor and the designated ARC 19499 concentrations. The whole blood/Innovin/ARC19499 mixture was incubated at 37 'C for 3 minutes before 70 pL of 25 mM CaCl2 was added to initiate clotting. In the absence of ARC19499, the hemophilia A blood samples did not clot in 360 seconds as initiated by 0.5 pM lipidated recombinant tissue factor. However, the clotting time shortened 244 WO 2012/109675 PCT/US2012/024916 dramatically when ARC 19499 was included in the assay as shown in figure 181. Although each individual responds to ARC19499 differently, the general trend, 30 nM of ARC19499 reaches the optimal effect, is the same. EXAMPLE 52 [007371 Pharmacologic abrogation of TFPI activity may be a viable approach to the management and/or prevention of bleeding in hemophilia. The interaction between aptamer BAX499 (formerly ARCI 9499, provided by Arche mix Corp.) and its target molecule, human TFPI was investigated. The apparent binding constant (Ki) between BAX499 and TFPI was 50 nM. In the presence of BAX499, the Ki between TFPI and factor Xa increased 20-fold, indicating that BAX499 reduced the affinity between TFPI and factor Xa. In a system using purified components, BAX499 delayed TFPI-mediated inhibition of extrinsic Xase activity, and completely prevented TFPI inhibition of the prothronbinase complex. In a plasma system, BAX499 shortened the dilute prothrombin time (initiated by 0.25 pM TF) in factor VIll-deficient plasma. Specifically, 10 nM aptamer produced a clotting time similar to plasma containing 30 IU/dL FVIII. In addition, when added to freshly drawn severe hemophilia A blood, BAX499 also shortened the whole blood clotting time significantly. Overall, these results indicate that BAX499 attenuates TFPI activity and support the concept of targeting TFPI for inhibition in the treatment of hemophilia. EXAMPLE 53 1007381 This example examines the regions on TFPI where ARC 19498 binds. In this experiment, the hydrogen-deuterium exchange (HDX) profile of TFPl in the presence and absence of ARC 19498 was evaluated to determine sites of protection by the aptamer. HDX was performed on TFPI in the presence and absence of the aptamer, the protein was digested by pepsin-catalyzed proteolysis, and the mass of the resulting peptides measured by mass spectrometry. Regions of TFPI that exchange more slowly in the presence of the aptamer than in its absence are likely sites of interaction. [007391 The experimental approach used to evaluate the TFPI/ARC 19498 interaction by HDX is illustrated in Figure 182. This figure shows, in general, how the method could be used to determine the epitope of an antibody for its antigen. The same general approach can be applied to the specific case of the TFPI/ARC 19498 interaction by substituting TFPI for "antigen" and ARC19498 for "antibody". The method is based on the premise that protein amide hydrogens 245 WO 2012/109675 PCT/US2012/024916 exchange with protons in aqueous solvent at a measurable rate. When the protein is transferred to a deuterium oxide (D 2 0) based solvent, amide hydrogens exchange with deuterium atoms, leading to an increase in the protein's molecular mass. The rate of HDX can be measured by freezing the reaction at appropriate time points and measuring the increase in mass over time by mass spectrometry. By digesting the protein with the appropriate enzyme and evaluating the masses of its component peptides by liquid chromatography mass spectrometry (LC-MS), one can further probe exchange rates of particular regions of the protein. The addition of a ligand, such as an antibody or aptamer, can slow HDX at sites of interaction by reducing the accessibility of amide hydrogens within those regions to solvent. The site of ligand interaction can be mapped on the protein sequence by identifying component peptides whose HDX profile is perturbed by the addition of ligand. [007401 A preliminary experiment evaluated the exchange properties of TFPI in the absence of aptamer at two pus in order to determine the tirne window for subsequent exchange experiments. HDX was performed by diluting full-length TFPI (Arnerican Diagnostica) into phosphate buffered saline (PBS), pH 7.0, in D2O, or 50 mM citrate buffer, pH 5.0 containing I nM CaCl 2 , 1 rnM MgCL, 2 m[M Foscholine-12 in D 2 0. The final concentration of TFPI in these solutions was 4.7 uM and the concentration of D0 was 85%. These were incubated at 23 'C for 30, 100, 300, 1000 or 3000 seconds and then 40 p.L was mixed with 20 pL of a chilled quenching solution. For pH 7 exchange, a quench solution of 8 M urea, I M tris(hydroxymethy)phosphine (THP), 5 mM Foscholine- 12, pH 4 was used. For pH 5 exchange, a quench solution of 8 M urea, I M THP, 100 mM citrate, 2 mM Foscholine- 12., pH 4 was used. Each sample was passed over an immobilized pepsin column at 200 pL/min in "buffer A" [0.05% trifluoroacetic acid (TFA) in 1-1201. Peptic fragments were loaded onto a reverse-phase trap column and desalted with "buffer A" at 200 pL/min for 3 min. Peptic peptides were then separated by a C1 8 column with a linear gradient of 13% - 40% "buffer B" (95% acetonitrile, 5% 1, 1 2 0 , 0.0025% TFA) and analyzed by mass spectrometry. As a control, a fully deuterated sample was prepared by diluting TFPI into 85% D 2 0 containing 100 mM tris(2-carboxvethy l)phosphine (TCEP) and incubating for 3 hours at 60 C. [00741] The HDX results for TFPI in solution at pH 5 and 7, 23 C, are shown in Figures 183 through 185. The data indicate that TFPI is a highly dynamic protein. The majority of the protein exchanged completely (>70% deuteration) before 30 seconds at 23 "C at pH-1 7 (Figures 183 and 246 WO 2012/109675 PCT/US2012/024916 184). Most of the segments are significantly deuterated even at 30 seconds at 23 'C at pH 5, which is equivalent of 0.3 seconds. 23 'C at pH 7 (Figures 183 and 184). As shown in Figure 185. the build-up in deuterium content is continuous with time between timepoints measured at pH 5 (squares) and pH 7 (diamonds). For the purposes of this figure, the pH 5 timepoints were converted to pIH 7 equivalents (e.g. 30 seconds at p1H 5 is equal to 0.3 seconds at p1H 7). This figure indicates that the change in pH does not alter the dynamic properties of TFPI. [007421 The HDX experiment in the presence of aptamer was performed using ARC19498 immobilized on POROS" AL chromatography resin (Invitrogen). The amine-reactive aldehyde groups on the POROS* AL resin are able to react with the 5'-terminal amine of ARC19498. Conjugation to the resin was carried out essentially according to the manufacturer's instructions. First, 0.1 g POROS* AL resin was incubated with 4 mg NaCNBH 3 (63.7 mol) dissolved in 400 pL of PBS, pH 7.0 (1 hour, roorn temperature) containing -40 nrnoles ARCI 9498. 2.8 M Na2SO4 was added stepwise in five 80 pL portions, and the final mixture [50 pM ARC 9598, 5 mg/mL (80 mM) NaCNBH 3 , 1.4 M Na2SO 4 ] was incubated overnight at room temperature. The conjugated resin was washed with PBS and blocked with a solution of NaCNBH 3 (8 mg/miL) and ethanolamine (~1 M) in PBS (pIT 7.2). The resin was then washed again with PBS, dried and resuspended in PBS, pH 7.0. [007431 HDX with TFPI in the presence of ARC19498 was first evaluated using the "on solution/off-column exchange" procedure depicted in the top panel of Figure 183. In this procedure, the deuterium on-exchange reaction is carried out in solution, and the off-exchange reaction (re-equilibration with H20) is carried out with TFPI bound to the AR-C 19498 column. The deuterium on-exchange reaction was performed in "exchange D" buffer appropriate for the desired pH 1 as indicated in Figure 186. A chromatography column (~100 ml- bed volume) prepared with the ARC19498 conjugated resin was cleaned with 0.8% formic acid, then equilibrated with "exchange ID" buffer (3 parts PBS, pH 7.0, 17 parts "exchange D" buffer) containing 85'% D 2 0. To initiate the on-exchange step, 6 pL of a concentrated TFPT solution (1 mg/mL; 31 LtM) was diluted in 34 pl. "exchange D" buffer to give final concentrations of 4.7 pM TFPI and 85% D 2 0. The reaction was incubated for either 150 or 500 seconds at 3 'C during which time it was injected onto the ARC 19498 column and the column was washed with 100 uL of "exchange HD" buffer at 3 C. The column was then washed with 200 piL of "exchange 11" buffer at the appropriate pH (Figure 185) at 3 'C to terminate the on-exchange reaction and 247 WO 2012/109675 PCT/US2012/024916 initiate the off-exchange reaction. The column was then incubated for either 75 or 250 seconds at 3 'C. The off-exchange reaction was then terminated by adding 110 pL chilled "buffer E" (8 M urea, I MT HP, 50 mM citrate, 2 mM Foscholine-12 pH1 2.5 in H2-O) to the column. TFPI was eluted from the column with an additional 40 pL chilled "buffer E". [00744] To initiate analysis of the eluted TFPI, the 40 pL fraction was mixed with 20 PL chilled "buffer Q" (200 mM citrate, pH 4 in 1120) and 55 iL applied to an immobilized pepsin column which was run at 200 pL/min in "buffer A". The solution of peptic fragments was collected and desalted before loading onto a CI 8 column for fractionation using a linear gradient of 13-40% "buffer B". The peptides were then analyzed by mass spectrometry. [00745] A second experiment was performed using the "on-colum/off-colunn exchange" procedure depicted in the bottom panel of Figure 183. In this procedure, TFPI was loaded onto the ARC 19498 column before the deuterium on-exchange reaction, and both the on-exchange and off-exchange reactions were carried out on the column. The purpose of this experiment is to control for regions of TFPI that exchange slowly, independent of ARC19498. In this case, the TFPI stock solution was diluted in "exchange H" buffer and applied to the ARC 19498 column that had also been equilibrated in "exchange H" buffer. The on-exchange reaction was initiated by passing 200 pL of "exchange IHID" at 3 0 C over the column, and incubating for either 150 or 500 seconds. Subsequent steps and analysis were carried out as described for the "on solution/off-column exchange" experiment. [00746] The results of HDX experiments with TFPI are shown in Figures 187-189. Overall, the data indicate that two general regions of TFP were protected upon binding to ARC] 9498, residues 15-69 and 191-272. Figure 187 shows the deuterium content in terms of % deuteration plotted as a function of time for each segment of TFPI after on/off exchange at pH 5, 6, and 7 at 3 0 C Peptides were obtained covering approximately 70% of the protein sequence. Blue triangles represent data from on-solution/off-column experiments and purple diamonds represent data from on-column/off-column control experiments. All exchange times were converted into pH 7 at 23 0 C equivalent (e.g., 150 s at p- 1 6 at 3 0 C is equal to 1.85 s at pl 7 at 23 'C). Differences in deuteration level are shown in tabular format in Figure 188. The average deuteration levels across all ps and exchange times of segments 191-205, 208-236, and 255 272 after on-solution/on-column experiments were more than 5% higher than after on column/off-column experiments. For pH 5 in particular, the average differences in deuteration 248 WO 2012/109675 PCT/US2012/024916 levels were 10% or more for all three segments. Similarly, the average deuteration levels of segments 15-25, 31-47, and 56-69 after on-solution/on-column experiments were more than 5% higher than that after on-column/off-colunm experiments. [007471 Differences in deuteration levels for each segment are illustrated in graphic format, juxtaposed against the TFPI amino acid sequence, in Figure 189. In this figure, each block represents a peptide followed. In Panel A, each block contains six time points corresponding to the two exchange times (150 and 500 s) at pH 5, 6, and 7. Dark blue indicates no protection upon aptamer binding. Lighter colors indicate more deuteriums after on-solution/off-column exchange than after on-column/off-column exchange as shown in the color key. Panel B shows the average difference in deuteration levels for each segment of TFPI across all exchange times and pls. Again, lighter colors correspond to regions of protection (>5%). These data indicate that residues 15-69 and 191-272 are protected upon binding of ARC 19498 to TFIPL Residues 15-69 roughly correspond with the K1 domain of TFPI (residues 26-76), while 191 -272 correspond to the K1 domain (189-239) and C-terminal segment (240-276), suggesting that these are potential sites of interaction for ARC 19498. EXAMPLE 54 [00748] Tissue factor pathway inhibitor (TFPI) is the principal regulator of clotting initiation and a promising target for pro- and anti-coagulation therapy. The aptarner ARCi 9499 is a high affinity specific TFPI antagonist designed to improve hemostasis by inactivating TF'PI. However, it is not immediately obvious to what extent and in what manner stimulation of coagulation onset by targeting TFPI will affect spatial and temporal characteristics of clot propagation. The ARC 19499 effect on clotting was examined in a spatital, reaction-dissusion experimental system in comparison with that of recombinant activated factor VII (rVIIa). [007491 Clotting propagation in recalcified plasma activated by a surface with immobilized tissue factor (TF) was monitored by videomicroscopy. [007501 ARC 19499 dose-dependently improved coagulation in normal and hemophilia A plasma activated with tissue factor (TF) at 2 pmoie/mn by shortening lag time and increasing clot size up to ~2-fold. The effect was TFPi-specific as confirmed by experiments in TFPI-depleted plasma with or without TFPI supplementation. Clotting improvement was half-maximal at 0,7 nM of ARC 19499 and reached plateau at 10-1000 nM. The ARC 19499 effect decreased with TF surface density increase. In contrast to this, rViia improved clotting in hemophilia A plasma 249 WO 2012/109675 PCT/US2012/024916 activated with TF at 2 or 2.0 pmole/m 2 , both by shortening lag time and increasing spatial velocity of clot propagation. The effect of rVIIa was increased with its concentration until there was activator-independent clotting throughout the reaction chamber at rVIIa>30 nM. [007511 'These results indicate that ARC 19499 significantly improves coagulation in a spatial experimental model of coagulation by specifically inhibiting TFPI in a manner qualitatively different from that of rVIla. [007521 The invention having now been described by way of written description and example, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the description and examples above are for purposes of illustration and not limitation of the following claims. 250

Claims (19)

1. An aptamer that binds to tissue factor pathway inhibitor (TFPI), wherein the aptamer comprises a nucleic acid sequence of residues 6-29 of SEQ ID NO: 2.

2. An aptamer that binds to tissue factor pathway inhibitor (TFPI), wherein the aptarer comprises a nucleic acid sequence of SEQ ID NO: 2, and wherein the aptamer further comprises a single nucleotide substitution in residues 1-6 or 30-32 of SEQ ID NO:2.

3. An aptamer that binds to tissue factor pathway inhibitor (TFPI), wherein the aptaner comprises a nucleic acid sequence of SEQ ID NO: 2, and wherein the aptamer further comprises a single niucleotide substitution chosen from Figure 147.

4. An aptamer that binds to tissue factor pathway inhibitor (TFPI), wherein the aptaner comprises a nucleic acid sequence of SEQ ID NO: 2, and wherein the aptamer further comprises multiple mutations that maintain base pairing interactions at positions 6 and 29, 7 and 28, or 8 and 27.

5. An aptamer that binds to tissue factor pathway inhibitor (TEPI), comprising a structure according to formula 1: X(I)-X(2)-X(3)-X(4)-X(5)-X(6)-U-X(7)-C-X(8)-X(9)-X(10)-X(i1)-X(12)-C-X(i4)-C-X( 1 5)-U X(16)-X(1 7)-X(1 8)-X(19)-U-X(20)-C-X(2 1)-U-X(2)-X(23)-X(24), wherein X(l). X('), X(3), X(4), X(5),X(6). X(14), X(22). X(23) and X(24) are each independently selected from A, C, G, or U; X(7) is A or G; 251 WO 2012/109675 PCT/US2012/024916 X(8), X(2 1) are each independently selected from C or U: X(9) is G or U; X(l0), X(i1), X(13), X(17),X(i8), X(19) are each G; X(12) is A, G or U; X(15) is A; X(16) is A, C or G; and X(20) is A or C.

6. The aptamer of claim 5, wherein one or more of X(i)-X(24) are methylated nucleotides.

7. A pharmaceutical composition comprising a therapeutically effective amount of the aptamer of claim I or 5.

8. A method for treating, preventing, delaying the progression or ameliorating a bleeding disorder mediated by TFPI, comprising administering to a subject the composition of claim I or

9. The method of claim 8, wherein the subject is a mammal.

10. The method of claim 8, wherein the wherein the bleeding disorder is selected from the group consisting of: congenital or acquired coagulation factor deficiencies; hemophilia A; hemophilia B; hemophilia C; hemophilia A or B with inhibitors; other factor deficiencies; deficiency of a2-plasmin inhibitor; deficiency of plasminogen activator inhibitor 1; multiple factor deficiency; functional factor abnormalities; joint hemorrhage; spontaneous bleeding in other locations; hemorrhagic stroke; intracranial hemorrhage; lacerations; hemorrhage associated 252 WO 2012/109675 PCT/US2012/024916 with trauma: acute traumatic coagulopathy; coagulopathy associated with cancer; von Willebrand's Disease; disseminated intravascular coagulation; liver disease; menorrhagia; thrombocytopenia and hemorrhage associated with the use of anticoagulants.

11. A kit comprising the aptamer of claim I or 5.

12. The aptamer of claim I or 5, wherein the aptamer further comprises at least one chemical modification selected from the group consisting of: a chemical substitution at a sugar position, a chemical substitution at an internucleotide linkage, and a chemical substitution at a base position.wherein the modification is selected from the group consisting of: a chemical substitution at a sugar position, a chemical substitution at an internucleotide linkage, and a chemical substitution at a base position.

13. The aptainer of claim 12, wherein the high molecular weight., non-imimunogenic, compound is polyethylene glycol.

14. An aptamer that binds to human tissue factor pathway inhibitor (TFPI), wherein the aptamer comprises the primary nucleotide sequence: ggaauauacu uggcucguua ggugeguaua ua and wherein the aptamer competes for binding to TFPI with a reference aptamer selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.

15. The aptamer of claim 14, wherein the aptamer has at least one chemical modification.

16. The aptamer of claim 15, wherein the modification is selected from the group consisting of: chemical substitution at a sugar position, a chemical substitution at an internucleotide linkage, and a chemical substitution at a base position. 253 WO 2012/109675 PCT/US2012/024916

17. The aptamer of claim 16, wherein the modification is selected from the group consisting of: incorporation of a modified nucleotide; a nucleotide cap; conjugation to a high molecular weight, non-immunogenic compound; conjugation to a lipophilic compound; incorporation of a CpG motif; and incorporation of a phosphorothioate or phosphorodithioate into the phosphate backbone,

18. The aptamer of claim 17, wherein the high molecular weight, non-immunogenic, compound is polyethylene glycol.

19. An aptamer that binds to tissue factor pathway inhibitor (TFPI) and has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the primary nucleotide sequence of SEQ ID NO: I or 7. 254