WO2010078469A2 - Sand fly salivary proteins as novel factor xa inhibitors and methods of use - Google Patents

Abstract

It has been surprisingly discovered that a sand fly salivary gland polypeptide, or a nucleic acid sequence encoding the polypeptide, can function as an anticoagulant. The present disclosure provides methods of treatment of disorders associated with abnormal coagulation of the blood utilizing compositions that include the sand fly salivary gland polypeptides of the disclosure.

Description

SAND FLY SALIVARY PROTEINS AS NOVEL FACTOR Xa INHIBITORS

AND METHODS OF USE

REFERENCE TO RELATED APPLICATION(S)

This application claims the benefit of U.S. Provisional Application No. 61/142,107, filed December 31, 2008, which is incorporated herein by reference.

FIELD OF THE DISCLOSURE The disclosure relates to methods of using sand fly salivary proteins, or nucleic acid sequences encoding these proteins, to inhibit blood coagulation in a subject. More specifically, this disclosure relates to sand fly salivary proteins that act as inhibitors of components of the coagulation cascade, such as Factor Xa.

BACKGROUND

The classic model of blood coagulation involves a cascade of events (the coagulation cascade), which involves the interaction of numerous plasma derived enzymes and cof actors. The cascade can be initiated by two separate mechanisms, either through the release or exposure of tissue factor into the blood, commonly termed the "extrinsic pathway," or through the activation of the contact factors of plasma, commonly termed the "intrinsic pathway." Both initiation pathways feed into a common pathway at a point where the prothrombinase complex catalyses the conversion of prothrombin to thrombin. Once formed, thrombin cleaves soluble fibrinogen to insoluble fibrin which crosslinks to form a blood clot. Many clinical disorders are characterized by the abnormal or undesirable activation of the clotting pathway. Such disorders include acute myocardial infarction (AMI), deep vein thrombosis (DVT), disseminated intravascular coagulation (DIC), pulmonary embolism (PE), coronary artery disease, non- hemorrhagic stroke and unstable angina, as well as rethrombosis after successful thrombolysis during AMI. Thus, there is a need for agents that can be used to interact with one or more of the components of the coagulation cascade and inhibit blood clotting. SUMMARY OF THE DISCLOSURE

It is surprisingly discovered that a polypeptide secreted from the salivary gland of the sand fly Lu. longipalpis has potent anticoagulation activity and acts by specifically binding the catalytic site of Factor Xa of the coagulation cascade. The sand fly anticoagulant polypeptide, referred to as LJL143, has an amino acid sequence set forth as residues 24-301 of SEQ ID NO: 15 and is encoded by nucleic acid residues 115-948 of SEQ ID NO: 16. Thus, methods are provided herein for using the disclosed sand fly salivary gland anticoagulation polypeptides, and polynucleotides encoding the anticoagulation polypeptides, to inhibit coagulation of blood in vitro or in vivo.

These methods include inhibiting coagulation of a blood sample or a plasma sample in vitro by contacting the blood sample or the plasma sample with an effective amount of a polypeptide, wherein the polypeptide has at least 90% (for example, at least 95% or 98%) sequence identity to the amino acid sequence set forth as residues 24-301 of SEQ ID NO: 15, thereby inhibiting coagulation of the blood sample or the plasma sample. Inhibiting coagulation of the blood sample or the plasma sample comprises at least 50%, for example at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% inhibition, compared to a sample that has not been contacted with the polypeptide.

In another embodiment of the method, a subject is treated for a disorder associated with increased blood coagulation by (a) selecting a subject with a pro- coagulant condition, and (b) administering to the subject a therapeutically effective amount of a polypeptide having at least 90% (for example, at least 95% or 98%) sequence identity to the amino acid sequence set forth as residues 24-301 of SEQ ID NO: 15, thereby treating the subject for the disorder associated with increased blood coagulation. In some embodiments, treating the subject comprises inhibiting coagulation of a blood sample or a plasma sample obtained from the subject by at least 50%, for example at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, compared to a subject who has not been administered a therapeutically effective amount of the polypeptide. In other methods, a subject is treated for a disorder associated with increased blood coagulation by (a) selecting a subject with a pro-coagulant condition, and (b) administering to the subject a therapeutically effective amount of a nucleic acid sequence encoding a polypeptide having at least 90% (for example, at least 95% or 98%) sequence identity to the amino acid sequence set forth as residues 24-301 of SEQ ID NO: 15, thereby treating the subject for the disorder associated with increased blood coagulation. In one embodiment, the nucleic acid sequence comprises the sequence set forth as residues 115-948 of SEQ ID NO: 16, or a degenerate variant thereof. The nucleic acid may be operably linked to an expression control sequence, wherein the expression control sequence is a promoter. The promoter can be an inducible or a constitutive promoter.

The subject can be treated to prevent new blood clots from forming or to inhibit existing clots from enlarging. In one embodiment the polypeptide is not administered to the subject with or for the purpose of treating a leishmania infection. In other embodiments, the subject being treated previously had, is currently suffering from, or is at risk for an acute myocardial infarction (AMI), deep vein thrombosis (DVT), disseminated intravascular coagulation (DIC), pulmonary embolism (PE), coronary artery disease, non-hemorrhagic stroke and unstable angina, as well as rethrombosis after successful thrombolysis during AMI. The anticoagulant polypeptides provided herein, or polynucleotides encoding the polypeptides, can be administered alone or in combination with a second anticoagulant that is different that the anti-coagulant peptide provided herein. The combination of two or more different anticoagulants can be administered to the subject simultaneously or sequentially.

The foregoing and other features and advantages of the invention will become more apparent from the following detailed description of a several embodiments which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a set of graphs and a table identifying a Lutzomyia longilpalpis salivary anticoagulant factor. Fig. IA is a bar graph demonstrating that saliva from half a pair, one pair, or two pair of Lutzomyia longipalpis salivary glands (left panel), and the recombinant protein LJL143 (right panel) increase the recalcification time of human plasma, indicating that a component of the saliva, and more specifically LJL143, has anticoagulant activity. Fig. IB is a graph illustrating that LJL143 inhibits thrombin formation in an in vitro prothrombinase assay. Fig. 1C is a graph showing direct binding of LJL143 to Factor Xa using surface plasmon resonance. Fig. ID is a graph demonstrating specific binding of LJL143 to the Factor Xa active site using surface plasmon resonance. Fig. IE is a table demonstrating that LJL143 inhibits specifically Factor Xa and not other serine proteases.

Figure 2 is a series of images demonstrating the characteristics of LJLl 43. Fig. 2A is an image of a Western blot analysis of recombinant LJL143 (rLJL143) and salivary gland homogenate (SGH) showing that LJL143 migrates at approximately 40 kDa. Fig. 2B is a representation of the nucleic acid sequence (SEQ ID NO: 16) encoding the unprocessed (mature form) of LJLl 43 (including 5' and 3' untranslated regions) and the amino acid sequence (SEQ ID NO: 15) of the unprocessed LJL143. Bolded amino acid residues (amino acids 1-23) correspond to the signal sequence of LJL143. Fig. 2C is an alignment of the amino acid sequence of unprocessed LJL143 (SEQ ID NO: 15) and salivary gland homologs from various sand flies (Phlebotomus ariasi: SEQ ID NO: 72; Phlebotomus perniciosus: SEQ ID NO: 73; Phlebotomus argentipes: SEQ ID NO: 74; Phlebotomus duboscqi: SEQ ID NO: 75). Underlined residues correspond to the signal sequence. Black shaded areas correspond to identical residues and grey shaded areas correspond to conservative residue variations between the aligned sequences. Fig. 2D is a schematic representation of the phylogenetic tree of LJL143 and sand fly salivary homologs. SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. §1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 is the amino acid sequence of LJL34.

SEQ ID NO: 2 is the nucleic acid sequence of LJL34. SEQ ID NO: 3 is the amino acid sequence of LJLl 8.

SEQ ID NO: 4 is the nucleic acid sequence of LJL18.

SEQ ID NO: 5 is the amino acid sequence of LJS 193.

SEQ ID NO: 6 is the nucleic acid sequence of LJS193.

SEQ ID NO: 7 is the amino acid sequence of LJS201. SEQ ID NO: 8 is the nucleic acid sequence of LJS201.

SEQ ID NO: 9 is the amino acid sequence of LJLl 3.

SEQ ID NO: 10 is the nucleic acid sequence of LJL13.

SEQ ID NO: 11 is the amino acid sequence of LJL23.

SEQ ID NO: 12 is the nucleic acid sequence of LJL23. SEQ ID NO: 13 is the amino acid sequence of LJMlO.

SEQ ID NO: 14 is the nucleic acid sequence of LJMlO.

SEQ ID NO: 15 is the amino acid sequence of LJL143.

SEQ ID NO: 16 is the nucleic acid sequence of LJL143.

SEQ ID NO: 17 is the amino acid sequence of LJS 142. SEQ ID NO: 18 is the nucleic acid sequence of LJS142.

SEQ ID NO: 19 is the amino acid sequence of LJL17.

SEQ ID NO: 20 is the nucleic acid sequence of LJL17.

SEQ ID NO: 21 is the amino acid sequence of LJM06.

SEQ ID NO: 22 is the nucleic acid sequence of LJM06. SEQ ID NO: 23 is the amino acid sequence of LJM17.

SEQ ID NO: 24 is the nucleic acid sequence of LJM17. SEQ ID NO: 25 is the amino acid sequence of LJL04.

SEQ ID NO: 26 is the nucleic acid sequence of LJL04.

SEQ ID NO: 27 is the amino acid sequence of LJMl 14.

SEQ ID NO: 28 is the nucleic acid sequence of LJMl 14. SEQ ID NO: 29 is the amino acid sequence of LJMl 11.

SEQ ID NO: 30 is the nucleic acid sequence of LJMl 11.

SEQ ID NO: 31 is the amino acid sequence of LJM78.

SEQ ID NO: 32 is the nucleic acid sequence of LJM78.

SEQ ID NO: 33 is the amino acid sequence of LJS238. SEQ ID NO: 34 is the nucleic acid sequence of LJS238.

SEQ ID NO: 35 is the amino acid sequence of LJS 169.

SEQ ID NO: 36 is the nucleic acid sequence of LJS169.

SEQ ID NO: 37 is the amino acid sequence of LJLIl.

SEQ ID NO: 38 is the nucleic acid sequence of LJLIl. SEQ ID NO: 39 is the amino acid sequence of LJL08.

SEQ ID NO: 40 is the nucleic acid sequence of LJL08.

SEQ ID NO: 41 is the amino acid sequence of LJS 105.

SEQ ID NO: 42 is the nucleic acid sequence of LJS105.

SEQ ID NO: 43 is the amino acid sequence of LJL09. SEQ ID NO: 44 is the nucleic acid sequence of LJL09.

SEQ ID NO: 45 is the amino acid sequence of LJL38.

SEQ ID NO: 46 is the nucleic acid sequence of LJL38.

SEQ ID NO: 47 is the amino acid sequence of LJM04.

SEQ ID NO: 48 is the nucleic acid sequence of LJM04. SEQ ID NO: 49 is the amino acid sequence of LJM26.

SEQ ID NO: 50 is the nucleic acid sequence of LJM26.

SEQ ID NO: 51 is the amino acid sequence of LJS03.

SEQ ID NO: 52 is the nucleic acid sequence of LJS03.

SEQ ID NO: 53 is the amino acid sequence of LJS 192. SEQ ID NO: 54 is the nucleic acid sequence of LJS192.

SEQ ID NO: 55 is the amino acid sequence of LJM19. SEQ ID NO: 56 is the nucleic acid sequence of LJMl 9.

SEQ ID NO: 57 is the amino acid sequence of LJL138.

SEQ ID NO: 58 is the nucleic acid sequence of LJL138.

SEQ ID NO: 59 is the amino acid sequence of LJL15. SEQ ID NO: 60 is the nucleic acid sequence of LJL15.

SEQ ID NO: 61 is the amino acid sequence of LJL91.

SEQ ID NO: 62 is the nucleic acid sequence of LJL91.

SEQ ID NO: 63 is the amino acid sequence of LJMl 1.

SEQ ID NO: 64 is the nucleic acid sequence of LJMl 1. SEQ ID NO: 65 is the amino acid sequence of LJS 138.

SEQ ID NO: 66 is the nucleic acid sequence of LJS138.

SEQ ID NO: 67 is the amino acid sequence of LJL124.

SEQ ID NO: 68 is the nucleic acid sequence of LJL124.

SEQ ID NO: 69 is the amino acid sequence of LJL35. SEQ ID NO: 70 is the nucleic acid sequence of LJL35.

SEQ ID NO: 71 is an oligonucleotide primer.

SEQ ID NOs: 72-75 is the amino acid sequence of various polypeptides from the sand fly salivary gland

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

/. Abbreviations

AMI acute myocardial infarction

DIC disseminated intravascular coagulation DVT deep vein thrombosis

FVa Factor Va

FXa Factor Xa

LuIoSP Lutzomyia longipalpis salivary proteins

PBS Phosphate buffered saline PE pulmonary embolism

PT prothrombin time aPTT activated partial thromboplastin time rLJL143 recombinant LJL143

SGH salivary gland homogenate TF tissue factor

TT thrombin time //. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19- 854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

Amplification of a nucleic acid molecule (for example, a DNA or RNA molecule): A technique that increases the number of copies of a nucleic acid molecule in a specimen. An example of amplification is the polymerase chain reaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to a nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. The product of amplification may be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing using standard techniques. Other examples of amplification include strand displacement amplification, as disclosed in U.S. Patent No. 5,744,311; transcription-free isothermal amplification, as disclosed in U.S. Patent No. 6,033,881; repair chain reaction amplification, as disclosed in WO 90/01069; ligase chain reaction amplification, as disclosed in EP 0320308; gap filling ligase chain reaction amplification, as disclosed in U.S. Patent No. 5,427,930; and NASBA™ RNA transcription-free amplification, as disclosed in U.S. Patent No. 6,025,134.

Antibody: immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for instance, molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. A naturally occurring antibody (for example, IgG, IgM, IgD) includes four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. However, it has been shown that the antigen-binding function of an antibody can be performed by fragments of a naturally occurring antibody. Thus, these antigen-binding fragments are also intended to be designated by the term "antibody." Specific, non-limiting examples of binding fragments encompassed within the term antibody include (i) an Fab fragment consisting of the V_L, V_H, CL, and CHl domains; (ii) an Fd fragment consisting of the V_H and CHl domains; (iii) an Fv fragment consisting of the V_L and V_H domains of a single arm of an antibody, (iv) a dAb fragment (Ward et al. , Nature 341 :544-546, 1989) which consists of a V_H domain; (v) an isolated complimentarity determining region (CDR); and (vi) an F(ab')₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region. Further, non-specific examples include single chain Fv proteins ("scFv") and disulfide stabilized Fv proteins ("dsFv"). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.

Immunoglobulins and certain variants thereof are known and many have been prepared in recombinant cell culture (for example, see U.S. Patent No. 4,745,055; U.S. Patent No. 4,444,487; WO 88/03565; EP 0256654; EP 0120694; EP 0125023; Faoulkner et al, Nature 298:286, 1982; Morrison, /. Immunol. 123:793, 1979; Morrison et al., Ann Rev. Immunol 2:239, 1984).

Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non- human mammals. Similarly, the term "subject" includes both human and veterinary subjects, such as dogs.

Anticoagulant: An agent that inhibits or reduces blood clot formation (coagulation) in vitro and/or in vivo. Such agents exhibit anticoagulant or anticoagulation activity. Anticoagulants can prevent new clots from forming or an existing clot from enlarging. Anticoagulants are given to subjects at risk for forming blood clots, such as those with artificial heart valves, or to subjects who have previously had, currently suffer from, or who are at risk of developing blood clots associated with a physiological, pro-coagulant condition such as atrial fibrillation, acute myocardial infarction (AMI), deep vein thrombosis, disseminated intravascular coagulation, pulmonary embolism, non-hemorrhagic stroke, unstable angina, as well as rethrombosis after successful thrombolysis during AMI. Anticoagulant drugs fall into three categories: inhibitors of clotting factor synthesis, inhibitors of thrombin, and antiplatelet drugs. A specific, non-limiting example of an anticoagulant that inhibits clotting factor synthesis includes warfarin (Coumadin®). Specific, non- limiting examples of anticoagulants that inhibit thrombin include heparin, low- molecular-weight heparin, argatroban, bivalirudin, and lepirudin. Specific, non- limiting examples of anticoagulants that inhibit platelets from forming clots include aspirin, ticlopidine, and clopidogrel (Plavix®).

Antithrombotic: Any compound (such as a pharmaceutical agent or molecule) that prevents or inhibits formation of a thrombus. Antithrombotic agents include anticoagulants (which limit the ability of platelets to clot), antiplatelet drugs (which limit the migration and aggregation of platelets) and thrombolytic drugs (which dissolve clots after they have formed).

Coagulation: A host defense mechanism also referred to as hemostasis (cessation of blood loss from an injured vessel). An enzymatic cleavage cascade leading to the catalysis of prothrombin to thrombin by Factor Xa. Thrombin then acts on fibrinogen to form fibrin monomers that serve as the structural basis of the fibrin clot, resulting in the transformation of blood or plasma from a liquid to a solid or gel phase. All coagulation factors exist as inactive zymogens in the circulating blood except tissue factor (TF), which is an integral membrane glycoprotein that only comes into contact with blood after vascular injury. Minor proteolysis of the soluble coagulation factors activates them into serine proteases (wherein the "a" in Factor Xa, designates it as the active form of Factor X), with the exception of Factors V and VIE, which become active cofactors. About 200 amino acid residues at the C-terminal end of each zymogen are homologous to trypsin and contain the active site Ser, Asp, His residues of the protease. Two coagulation pathways (intrinsic and extrinsic pathways) converge on a common coagulation pathway, which leads to the development of a fibrin clot (see Monroe et ah, Arterioscler Thromb. Vase. Biol., 26(l):41-48, 2006, incorporated by reference). The intrinsic and extrinsic pathways are each initiated by a distinct mechanism. The formation of a clot in response to an abnormal vessel wall in the absence of tissue injury is the result of the intrinsic pathway. Abnormal physiology, such as hyperlipidemic states (which can lead to atherosclerosis) or bacterial infiltration can result in the activation of thrombosis via the intrinsic coagulation pathway. The intrinsic pathway requires clotting Factors VIII, IX, X, XI, and XII, as well as prekallikrein, high-molecular weight kininogen, calcium ions, and phospholipids secreted from platelets. Factor Xa activation in the intrinsic pathway is catalyzed by cofactor Villa, calcium, and phospholipids. A subject with a prolonged activated partial thromboplastin time (aPTT) and a normal prothrombin time (PT) is considered to have a defect in the intrinsic coagulation pathway. Fibrin clot formation in response to tissue injury and exposure to TF is the result of the extrinsic pathway. TF, also known as Factor EI, is a cofactor in the Factor Vila-catalyzed activation of Factor X in the extrinsic pathway. TF is a non- enzymatic lipoprotein constitutively expressed on the surface of cells that are not normally in contact with plasma. A subject with a prolonged PT and a normal aPTT has a defect in the extrinsic coagulation pathway.

Factor Xa is the site where the intrinsic and extrinsic coagulation pathways converge. Prolongation of both the aPTT and PT suggests that a coagulation cascade defect lies in a component of the common coagulation pathway.

Conservative variants: Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). A non-conservative amino acid substitution can result from changes in: (a) the structure of the amino acid backbone in the area of the substitution; (b) the charge or hydrophobicity of the amino acid; or (c) the bulk of an amino acid side chain. Substitutions generally expected to produce the greatest changes in protein properties are those in which: (a) a hydrophilic residue is substituted for (or by) a hydrophobic residue; (b) a proline is substituted for (or by) any other residue; (c) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine; or (d) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl. Thus, in one embodiment, non-conservative substitutions are those that reduce an activity or antigenicity. cDNA (complementary DNA): A piece of DNA lacking internal, non- coding segments (introns) and expression control sequences. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells. Degenerate variant: A polynucleotide encoding a sand fly salivary gland polypeptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the disclosure as long as the amino acid sequence of the sand fly salivary gland polypeptide encoded by the nucleotide sequence is unchanged.

Effector molecule (EM): The portion of a chimeric molecule that is intended to have a desired effect on a cell or system or substance to which the chimeric molecule is targeted. The term effector molecule is interchangeable with effector moiety, therapeutic agent, diagnostic agent, and similar terms. Therapeutic agents include such compounds as nucleic acids, proteins

(including monoclonal antibodies and antigen-binding fragments of monoclonal antibodies), peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, recombinant viruses or toxins. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides. Diagnostic agents or moieties include radioisotopes and other detectable labels. Detectable labels useful for such purposes are also well known in the art, and include radioactive isotopes such as ³²P, ¹²⁵I, and ¹³¹I, fluorophores, chemiluminescent agents, and enzymes.

Expression Control Sequences: Nucleic acid sequences that control and regulate the expression of a nucleic acid sequence, such as a heterologous nucleic acid sequence, to which it is operably linked. Expression control sequences are operably linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, polyA signals, a start codon (for instance, ATG) in front of a protein-encoding polynucleotide sequence, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term "control sequences" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.

A promoter is a minimal sequence sufficient to direct transcription of a nucleic acid. Promoters may be cell-type specific or tissue specific. A promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. Both constitutive and inducible promoters are included (see for example, Bitter et al. , Methods in Enzymology 153:516-544, 1987).

For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac-hybrid promoter) and the like may be used. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (for example, metallothionein promoter) or from mammalian viruses (for example, the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences. A polynucleotide can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells. In one embodiment, the promoter is a cytomegalovirus promoter.

Factor Xa (prothrombinase or Stuart- Prower Factor): Factor Xa is a serine endoproteinase of the chymotrypsin family, and a member of the Sl peptidase family. It is the activated form of the zymogen Factor X. Factor X is activated in the intrinsic coagulation pathway by Factor IXa (in the presence of non-enzymatic cofactor Villa, calcium, and phospholipids) and in the extrinsic coagulation pathway by Factor Vila (in the presence of tissue factor and calcium). Thus, Factor Xa is the product of both the intrinsic and extrinsic pathways and is itself active in the common coagulation pathway. Factor Xa plays a critical role in the coagulation cascade by catalyzing the proteolytic conversion of prothrombin (Factor II) to active thrombin (Factor Ila). Factor Xa' s prothrombin conversion activity is greatly enhanced in vivo when complexed with non-enzymatice cofactor Va, calcium ions and phospholipids (to form the prothrombinase complex) on the activated platelet surface. Thrombin then converts fibrinogen to fibrin, which forms the primary hemostatic plug.

Factor Xa circulates in plasma as light and heavy chains connected by a single disulfide bond. The light chain N-terminal domain (residues 1-39) is the GIa domain, a protein domain rich in glutamate residues which are post-translationally modified by a vitamin K-dependent carboxylase to form γ-carboxy-glutamic acid, and which interacts with the phospholipid membrane. The GIa residues are responsible for the high-affinity binding of calcium ions. A conserved Gla-x(3)-Gla- x-Cys motif is found in the middle of the GIa domain, which seems to be important for substrate recognition by the carboxylase. Downstream from the GIa domain is a short region of hydrophobic residues (residues 40-45) and two epidermal growth factor-like repeats (EGF-like 1 domain, residues 46-84, and EGF-like 2 domain, residues 85-128). The heavy chain contains the catalytically active serine proteinase domain (254 amino acids; residues 16-269 in the chymotrypsinogen numbering system). Residues His57, AsplO2, and Serl95 (chymotrypsinogen numbering) form a catalytic triad at the active site cleft between the two subdomains of Factor Xa.

As used herein, coagulation Factor Xa, Factor Xa, FXa, prothrombinase, or Stuart-Prower Factor refer to any Factor Xa from any mammalian species that expresses the protein. For example, Factor Xa can be a human, non-human primate (such as baboon), mouse, dog, cat, cow, horse, pig, or rabbit polypeptide.

Hemostasis: Refers to the physiologic process whereby bleeding is halted. Hemostatic agents are those that prevent, treat or ameliorate abnormal bleeding, such as abnormal bleeding caused by a bleeding disorder or bleeding episode. Disorders of hemostasis include, for example, platelet disorders, such as idiopathic thrombocytopenic purpura, and disorders of coagulation, such as hemophilia. Hemostasis can also refer to the complex interaction between vessels, platelets, coagulation factors, coagulation inhibitors and fibrinolytic proteins to maintain the blood within the vascular compartment in a fluid state. The objective of the hemostatic system is to preserve intravascular integrity by achieving a balance between hemorrhage and thrombosis. An agent that promotes hemostasis can be an agent that reduces abnormal bleeding, such as by halting bleeding more rapidly, or by reducing the amount of blood loss.

Heterologous: A heterologous polypeptide or polynucleotide refers to a polypeptide or polynucleotide derived from a different source or species. For example, an immunoconjugate comprising a heterologous polypeptide refers to a fusion protein in which an antibody (or a portion of an antibody) is linked to a different polypeptide, such as a marker protein.

Host cells: Cells in which a vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used. Also includes the cells of the subject.

Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. An immune response can be a cellular response or a humoral response. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). The response can also be a nonspecific response (not targeted specifically to a polypeptide) such as production of lymphokines. In one embodiment, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a ThI or a Th2 (subsets of helper T cells) response. In yet another embodiment, the response is a B cell response, and results in the production of specific antibodies. Isolated: An "isolated" biological component (such as a nucleic acid or protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant technology as well as by chemical synthesis.

Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule. Specific, non- limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. In one example, a "labeled polypeptide" refers to incorporation of another molecule in the polypeptide. For example, the label is a detectable marker, such as the incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as ³⁵S or ¹³¹I), fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

Lutzomyia longipalpis (Lu. longipalpis): A species of sand fly endogenous to the New World (South and Central America). This sand fly is the principal vector of American visceral leishmaniasis, a potentially fatal disease that primarily affects children in several countries of South and Central America.

Lymphocytes: A type of white blood cell that is involved in the immune defenses of the body. There are two main types of lymphocytes: B cells and T cells. A lymphocyte can also be referred to as a leukocyte.

Mammal: This term includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects.

Oligonucleotide: A linear polynucleotide sequence of up to about 100 nucleotide bases in length. Open reading frame (ORF): A nucleic acid sequence having a series of nucleotide triplets (codons), starting with a start codon and ending with a stop codon, coding for amino acids without any internal termination codons. These sequences are usually translatable into a polypeptide.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame. Parenteral: Refers to administration other than through the alimentary canal (the digestive tract), such as by subcutaneous, intramuscular, intrasternal or intravenous administration.

Peptide tag: A peptide sequence that is attached (for instance through genetic engineering) to another peptide or a protein. In some embodiments the peptide tag provides a function to the resultant fusion. Peptide tags are usually relatively short in comparison to a protein to which they are fused; by way of example, peptide tags are four or more amino acids in length, such as 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more amino acids. Usually a peptide tag will be no more than about 100 amino acids in length, and may be no more than about 75, no more than about 50, no more than about 40, or no more than about 30.

Peptide tags can confer one or more different functions to a fusion protein (thereby "functionalizing" that protein), and such functions can include antibody binding (an epitope tag), purification, and differentiation (e.g., from a native protein). In addition, a recognition site for a protease, for which a binding antibody is known, can be used as a specifically cleavable epitope tag. The use of such a cleavable tag can provide selective cleavage and activation of a protein (e.g. , by replacing the cleavage site in TGF- βl with that for pro-caspase 3).

Detection of the tagged molecule can be achieved using a number of different techniques. These include: immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting ("western"), and affinity chromatography.

Epitope tags add a known epitope (antibody binding site) on the subject protein, providing binding of a known and often high-affinity antibody, and thereby allowing one to specifically identify and track the tagged protein that has been added to a living organism or to cultured cells. Examples of epitope tags include the myc, T7, GST, GFP, HA (hemagglutinin) and FLAG tags. The first four examples are epitopes derived from existing molecules. In contrast, FLAG is a synthetic epitope tag designed for high antigenicity (see, e.g., U.S. Patent Nos. 4,703,004 and 4,851,341). Purification tags are used to permit easy purification of the tagged protein, such as by affinity chromatography. A well-known purification tag is the hexa- histidine (6x His) tag, literally a sequence of six histidine residues. The 6x His protein purification system is available commercially from QIAGEN (Valencia, CA), under the name of QIAexpress®.

A single tag peptide can serve more than one purpose; any attached tag, for instance, will increase the molecular weight of the fusion protein and thereby permit differentiation between the tagged and native proteins. Antibodies specific for an "epitope tag" can be used to construct an immunoaffinity column, thus permitting an epitope tag to be used for purification of the tagged protein. Likewise, in some instances monoclonal antibodies specific for a purification tag are available (e.g. anti-6x His peptide monoclonal antibodies, which are available through QIAGEN or CLONTECH, Palo Alto, CA).

Pharmaceutical agent: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject or a cell.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 15th Edition, 1975, describes compositions and formulations suitable for pharmaceutical delivery of the anticoagulant polypeptides disclosed herein.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Phlebotomus ariasi (P. ariasi): A species of Phlebotomus (sand flies) genus endogenous to the Old World, in particular to southern Europe and Mediterranean countries, more particularly to Spain and France. This sand fly is a proven vector of visceral leishmaniasis. P. ariasi is a member of the subgenera of Phlebotomus Larroussius.

Phlebotomus papatasi (P. papatasi): A species of Phlebotomus (sand flies) genus endogenous to the Old World, in particular to southern Europe, and Mediterranean countries, more particularly to France, Italy, Greece, Morocco, and Spain. This sand fly is a proven vector of the visceral leishmaniasis.

Phlebotomus perniciosus (P. perniciosus): A species of Phlebotomus (sand flies) genus endogenous to the Old World, in particular to southern Europe, and Mediterranean countries, more particularly to France, Italy, Greece, Morocco, and Spain. This sand fly is a proven vector of the visceral leishmaniasis. P. perniciosus is a member of the subgenera of Phlebotomus Larroussius.

Polynucleotide: The term polynucleotide or nucleic acid sequence refers to a polymeric form of nucleotide at least 10 bases in length, thus including oligonucleotides and genes. A recombinant polynucleotide includes a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (for example, a cDNA) independent of other sequences. The polynucleotides can be ribonucleotides, deoxyribonucleo tides, or modified forms of either nucleotide. The term includes single -and double -stranded forms of DNA.

Polypeptide: Any chain of amino acids, regardless of length (thus encompassing oligopeptides, peptides, and proteins) or post-translational modification (for example, glycosylation, phosphorylation, or acylation). A polypeptide encompasses also the precursor, as well as the mature protein. In one embodiment, the polypeptide is a polypeptide isolated from Lu. longipalpis, or encoded by a nucleic acid isolated from Lu. longipalpis, such as the Lu. longipalpis polypeptides disclosed herein.

Polypeptide Modifications: Sand fly salivary gland polypeptides include synthetic embodiments of polypeptides described herein. In addition, analogues (non-peptide organic molecules), derivatives (chemically functionalized peptide molecules obtained starting with the disclosed polypeptide sequences) and variants (homologs) of these proteins can be utilized in the methods described herein. Each polypeptide of the disclosure is comprised of a sequence of amino acids, which may be either L- and/or D- amino acids, naturally occurring and otherwise.

Polypeptides may be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified polypeptides, and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxyl-terminal or side chain, may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ ester, or converted to an amide of formula NR₁R₂ wherein Ri and R₂ are each independently H or C₁-C₁₆ alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring. Amino groups of the peptide, whether amino-terminal or side chain, may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric, and other organic salts, or may be modified to Ci-Ci₆ alkyl or dialkyl amino or further converted to an amide.

Hydroxyl groups of the peptide side chains may be converted to Ci-Ci₆ alkoxy or to a Ci-Ci₆ ester using well-recognized techniques. Phenyl and phenolic rings of the peptide side chains may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine, or iodine, or with Ci-Ci₆ alkyl, Ci-Ci₆ alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the peptide side chains can be extended to homologous C₂-C₄ alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the peptides of this disclosure to select and provide conformational constraints to the structure that result in enhanced stability.

Peptidomimetic and organomimetic embodiments are envisioned, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid side chains, resulting in such peptido- and organomimetics of a polypeptide having measurable or enhanced anticoagulation activity. For computer modeling applications, a pharmacophore is an idealized, three-dimensional definition of the structural requirements for biological activity. Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drug design or CADD). See Walters, "Computer- Assisted Modeling of Drugs," Klegerman & Groves (eds.), 1993, Pharmaceutical Biotechnology, Interpharm Press: Buffalo Grove, IL, pp. 165-174 and Principles of Pharmacology Munson (ed.) 1995, Ch. 102, for descriptions of techniques used in CADD. Also included are mimetics prepared using such techniques.

Preventing, treating or ameliorating a disease: "Preventing" a disease refers to inhibiting the full development of a disease. "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. "Ameliorating" refers to the reduction in the number or severity of signs or symptoms of a disease, such as cancer.

Probes and primers: A probe comprises an isolated polynucleotide attached to a detectable label or reporter molecule. Primers are short polynucleotides. In one embodiment, polynucleotides are 15 nucleotides or more in length. Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, for example, by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art. One of skill in the art will appreciate that the specificity of a particular probe or primer increases with its length. Thus, for example, a primer comprising 20 consecutive nucleotides will anneal to a target with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, in order to obtain greater specificity, probes and primers may be selected that comprise at least 15, 20, 25, 30, 35, 40, 50 or more consecutive nucleotides.

Pro-coagulant: A substance that promotes the coagulation of blood. A pro- coagulant substance can be a protease of the coagulation cascade (i. e. , thrombin (Factor Ha), Factor Vila, Factor IXa, Factor Xa, Factor XIa, Factor XIIa, or kallikrein), or its precursor protein (i.e., prothrombin (Factor II), Factor VII, Factor IX, Factor X, Factor XI, Factor Xπ, or Prekallikrein). In some con

Pro-coagulant condition: A state or disorder that is characterized by the undesirable formation of blood clots. The pro-coagulant condition can result from normal coagulation in an abnormal location (for example, at the location of an atherosclerotic plaque or endothelial disruption) or from abnormal function of a component of the coagulation cascade. Disorders in which normal coagulation may occur at an abnormal location include acute myocardial infarction, deep vein thrombosis, disseminated intravascular coagulation, pulmonary embolism, coronary artery disease, non-hemorrhagic stroke and unstable angina, as well as rethrombosis after successful thrombolysis during AMI. Other subjects having a pro-coagulant condition include subjects receiving prostheses, such as heart valves, subjects undergoing open heart surgery or bypass surgery, or subjects who must stay in bed for a long time after certain types of surgery. These subjects can have normal coagulation of the blood, however the coagulation occurs in these subjects in an abnormal location. Pro-coagulant conditions can be the result of certain hereditary disorders where the body does not produce enough of a protein involved in controlling clotting, or one of these proteins is ineffective. Examples of such defective proteins include protein C, protein S, and antithrombin IE. In each of these disorders, the production of fibrin, which helps clots form, increases. In activated protein C (APC) resistance (Factor V Leiden mutation), APC does not respond when protein C signals to stop clotting. In another hereditary disorder (prothrombin G20210A polymorphism), production of prothrombin, another clotting factor, is increased, leading to increased clotting. Hereditary defects in one or more of the clotting factors can cause the formation of potentially dangerous blood clots (thrombosis); such clotting disorders are collectively called thrombophilia. Promoter: A promoter is an array of nucleic acid control sequences that directs transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription, for example, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. Both constitutive and inducible promoters are included (see for example, Bitter et ah, Methods in Enzymology 153:516-544, 1987).

Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified polypeptide preparation is one in which the polypeptide is more enriched than the polypeptide is in its natural environment. A polypeptide preparation is substantially purified such that the polypeptide represents several embodiments at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%, of the total polypeptide content of the preparation. The same applies for polynucleotides. The polypeptides disclosed herein can be purified by any of the means known in the art (see, for example, Guide to Protein Purification, Deutscher (ed.), Meth. Enzymol. 185, Academic Press, San Diego, 1990; and Scopes, Protein Purification: Principles and Practice, Springer Verlag, New York, 1982).

Recalcification time: Blood or plasma (blood depleted of platelets and other blood cells) is prevented from clotting in vitro by the addition of EDTA or citrate, which binds calcium in the sample. Recalcification time is a measure of the clotting time of an in vitro plasma sample (to which EDTA or citrate has been added) after the addition of excess calcium ions (recalcification) to the sample. Recalcified plasma will clot in 2-4 minutes. The recalcification (clotting) time can be shortened to 60-85 seconds by adding an emulsion of negatively-charged phospholipids (PL) to the sample. This time can be further shortened to 21-32 seconds by pre-incubation of the plasma with particulate substances such as kaolin (insoluble aluminum silicate). Measurement of the recalcification time of plasma in the presence of kaolin (or glass or another artificial surface), phospholipid, and calcium is termed the "activated partial thromoboplastin time" (aPTT) test. The clotting time of recalcified plasma can be shortened to 11-12 seconds by adding thromboplastin (a saline brain extract containing tissue factor). Thus, measurement of the recalcification time of plasma in the presence of thromboplastin and calcium is termed the "prothrombin time" (PT) test.

Recombinant: A recombinant polynucleotide is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. In one embodiment, a recombinant polynucleotide encodes a fusion protein.

Selectively hybridize: Hybridization under moderately or highly stringent conditions that excludes non-related nucleotide sequences.

In nucleic acid hybridization reactions, the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (for example, GC v. AT content), and nucleic acid type (for example, RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter. A specific, non- limiting example of progressively higher stringency conditions is as follows: 2 x SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2 x SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2 x SSC/0.1% SDS at about 42°C (moderate stringency conditions); and 0.1 x SSC at about 68°C (high stringency conditions). One of skill in the art can readily determine variations on these conditions (for example,

Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al, CoId Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). The hydridization conditions can be carried out over 2 to 16 hours. Washing can be carried out using only one of the above conditions, for example, high stringency conditions, or each of the conditions can be used, for example, for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.

Sequence identity: The similarity between amino acid sequences is expressed in terms of the percentage identity between the sequences. The higher the percentage, the more similar the two sequences are. Homologs or variants of a sand fly salivary gland polypeptide will possess a relatively significant high degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, /. MoI. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. ScL USA 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al, Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. ScL USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994 presents a detailed consideration of sequence alignment methods and identity calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, J. MoI. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.

Homologs and variants of a sand fly salivary gland polypeptide are typically characterized by possession of at least about 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full length alignment with the amino acid sequence of the polypeptide using the NCBI Blast 2.0, gapped blastp set to default parameters. The comparison between the sequences is made over the full length alignment with the amino acid sequence given in this present disclosure, employing the Blast 2 sequences function using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).

When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologues and, variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologues could be obtained that fall outside of the ranges provided.

Specific binding agent: An agent that binds substantially only to a defined target. Thus, for example, a Lu. longipalpis specific binding agent is an agent that binds substantially to a Lu. longipalpis polypeptide. In one embodiment, the specific binding agent is a monoclonal or polyclonal antibody that specifically binds the Lu. longipalpis polypeptide.

Subject: Living multi-cellular vertebrate organisms, a category that includes both human veterinary subjects, including human and non-human mammals. In one embodiment, the subject is a member of the canine family, such as a dog. In another embodiment, the subject is a human.

Therapeutically active polypeptide: An agent, such as a sand fly salivary gland polypeptide, that alters (inhibits or reduces) blood coagulation in a subject. Therapeutically active molecules can also be made from nucleic acids. An example of a nucleic acid based therapeutically active molecule is a nucleic acid sequence that encodes a sand fly salivary gland polypeptide, wherein the nucleic acid sequence is operably linked to a control element, such as a promoter. Therapeutically active agents can also include organic or other chemical compounds that mimic the effects of the sand fly salivary gland polypeptide. The term "therapeutically effective fragment of a sand fly salivary gland polypeptide" includes any fragment of the sand fly salivary gland polypeptide, or variant of the sand fly salivary gland polypeptide, or fusion protein including a sand fly salivary gland polypeptide, that retains a function of the sand fly salivary gland polypeptide (such as altering Factor Xa activity), or retains the ability to reduce the symptoms related to a disorder associated with abnormal blood clotting. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations that have been shown to achieve a desired effect in vitro.

Thrombosis: The formation or presence of a clot (also called a "thrombus") inside a blood vessel, obstructing the flow of blood through the circulatory system. Thrombosis is usually caused by abnormalities in the composition of the blood, quality of the vessel wall, and/or nature of the blood flow. The formation of a clot is often caused by an injury to the vessel wall (such as from trauma or infection) and by the slowing or stagnation of blood flow past the point of injury. Abnormalities in coagulation also cause thrombosis.

Transduced: A transduced cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. As used herein, the term transduction encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transduced host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Hence "comprising A or B" means including A, or B, or A and B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All sequence database references are incorporated by reference as of the date of the filing of this application. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Anticoagulant Compositions

The sand fly salivary gland polypeptides disclosed herein exhibit anticoagulant activity. A polypeptide with anticoagulant activity (referred to herein as an anticoagulant polypeptide) can be a polypeptide isolated from the salivary gland or saliva of any sand fly, for example (but not limited to) Phlebotomus papatasi, Phlebotomus ariasi, Phlebotomus perniciosus, Lutzomyia longipalpis, Phlebotomus argentipes, Phlebotomus orientalis, Phlebotomus duboscqi, Lutzomyia intermedia, Lutzomyia shanoni, Lutzomyia whitmani. Examples of these polypeptide sequences are disclosed in PCT/US2003/034453 filed October 29, 2003; PCT Application No. PCT/US2003/029833, filed September 18, 2003; and PCT/US02/19663, filed June 18, 2002, which are incorporated herein by reference. A particular Lutzomyia longipalpis (Lu. longipalpis) salivary polypeptide (residues 24-301 of SEQ ID NO: 15; also referred to as the mature form of LJL143) exhibited surprisingly potent anticoagulation activity, when compared to other Lu. longipalpis salivary gland polypeptides. For example, residues 24-301 of SEQ ID NO: 15 exhibited a complete inhibition of coagulation following recalcification of a human plasma sample, whereas other Lu. longipalpis salivary gland polypeptides had no effect on coagulation when tested under the same conditions. In one specific, non- limiting embodiment, residues 24-301 of SEQ ID NO: 15 exhibited a recalcification time at least twice as long as other Lu. longipalpis salivary gland polypeptides.

The sand fly salivary gland polypeptides disclosed herein have an inhibitory effect on coagulation of blood or plasma. An inhibition (or reduction) of blood or plasma clotting can be measured by any method known to one of skill in the art, for example, by an increased recalcification time, an increased activated partial thromboplastin time (aPTT), or an increased prothrombin time (PT) in vitro. In some embodiments, the reduction or inhibition of blood or plasma clotting is at least 50%, for example at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%. In other embodiments, an increase in recalcification time, aPTT, or PT is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 125%, at least 150%, at least 200%, or more. In yet other embodiments, blood or plasma clotting is measurable by a clinical response in vivo, such as a measurable reduction in symptoms related to a pro-coagulant condition, state, or disorder, for example acute myocardial infarction (AMI), deep vein thrombosis (DVT), disseminated intravascular coagulation (DIC), or pulmonary embolism (PE), non-hemorrhagic stroke, unstable angina, rethrombosis after successful thrombolysis during AMI, activated protein C resistance (Factor V Leiden mutation), prothrombin 20210 mutation; or a deficiency of protein C, protein S, or antithrombin.

In some embodiments, a sand fly salivary gland polypeptide disclosed herein has an inhibitory effect on a pro-coagulant protease of the coagulation cascade {i.e. , thrombin (Factor Ila), Factor Vila, Factor IXa, Factor Xa, Factor XIa, Factor XIIa, or kallikrein), or on its precursor protein {i.e. , prothrombin (Factor II), Factor VII, Factor IX, Factor X, Factor XI, Factor XII, or Prekallikrein), such that coagulation is inhibited or reduced. In one specific, non-limiting example, a sand fly salivary gland polypeptide (for example a Lu. longipalpis polypeptide, such as residues 24-301 of SEQ ID NO: 15 or variants or fragments thereof) inhibits or reduces coagulation by binding Factor Xa and altering (reducing or inhibiting) Factor Xa activity.

The first 23 residues of SEQ ID NO: 15 form a signal sequence. Thus, in another specific, non-limiting example, a sand fly salivary gland polypeptide (for example a Lu. longipalpis polypeptide, such as SEQ ID NO: 15 or variants or fragments thereof) inhibits or reduces coagulation by binding Factor Xa and altering (reducing or inhibiting) Factor Xa activity. Specific, non-limiting examples of sand fly salivary gland anti-complement polypeptides include the Lu. longipalpis polypeptide having an amino acid sequence as set forth as residues of 1-50, 51-75, 76-100, 101-125, 126-150, 151-175, 176-200, 201-225, 226-250, 251-275, 276-301, 24-50, 24-75, 24-100, 24-125, 24-150, 24-175, 24-200, 24-225, 24-250, 24-275, 24- 301, 50-301, 75-301, 100-301, 125-301, 150-301, 175-301, 200-301, 225-301, 250- 301, 275-301 of SEQ ID NO: 15, or variants or fragments thereof. Yet other specific non-limiting examples of anti-complement polypeptides include an amino acid sequence set forth as residues 25-301, 26-301, 27-301, 28-301, 29-301, 30-301, 31- 301, 32-301, 33-301, etc of SEQ ID NO: 15, or variants or fragments thereof. An altered Factor Xa activity in turn alters (reduces or inhibits) the conversion of prothrombin (Factor II) to thrombin (Factor Ila) and thereby alters (reduces or inhibits) blood clotting. In a particular embodiment, SEQ ID NO: 15, residues 24- 301 of SEQ ID NO: 15, or variants or fragments thereof, bind the active (catalytic) site of Factor Xa. Thus, the anticoagulant polypeptides disclosed herein can inhibit or reduce coagulation by binding Factor Xa and inhibiting or reducing Factor Xa activity.

In one embodiment, the anticoagulant polypeptides disclosed herein include homologous polypeptides having an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the anticoagulant polypeptides. One specific, non-limiting example of a longer homologous sequence that contains residues 24- 301 of SEQ ID NO: 15 and has anticoagulant activity is the full-length polypeptide set forth as SEQ ID NO: 15 (residues 1-301 of SEQ ID NO: 15). Other specific, non-limiting examples of a longer homologous sequence that contains residues 24- 301 of SEQ ID NO: 15 and has anticoagulant activity includes polypeptides having an amino acid sequence set forth as residues 2-301 of SEQ ID NO: 15, 3-301 of SEQ ID NO: 15, 4-301 of SEQ ID NO: 15, 5-301 of SEQ ID NO: 15, 6-301 of SEQ ID NO: 15, 7-301 of SEQ ID NO: 15, 8-301 of SEQ ID NO: 15, 9-301 of SEQ ID NO: 15, 10-301 of SEQ ID NO: 15, etc, or variants or fragments thereof.

Fusion proteins including an anticoagulant polypeptide can also be produced using methods known to one of skill in the art. In one embodiment, a fusion protein includes an amino acid sequence set forth as residues 24-301 of SEQ ID NO: 15, or conservative variants or fragments thereof, and a marker polypeptide. Fusion proteins, which include the anticoagulant polypeptide and retain the anticoagulant activity, are also disclosed herein. Such fusion proteins can include, in addition to the anticoagulant polypeptide, an effector molecule (such as a monoclonal antibody), a label (such as enzymatic labels, polypeptide epitopes, or fluorescent proteins), or a peptide tag (such as peptide tags that are four or more amino acids in length, such as 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more amino acids). Homologs, fragments, variants, and fusion proteins including the anticoagulant polypeptides exhibit substantially the same activity as the anticoagulant polypeptides. Thus, homologs, fragments, variants, and fusion proteins including the anticoagulant polypeptides inhibit or reduce coagulation. In some embodiments, homologs, fragments, variants, and fusion proteins including the anticoagulant polypeptides, inhibit or reduce coagulation by having an inhibitory effect on a protease of the coagulation cascade (i.e., thrombin (Factor Ila), Factor Vila, Factor IXa, Factor Xa, Factor XIa, Factor XIIa, or kallikrein), or on its precursor protein (i.e., prothrombin (Factor II), Factor VII, Factor IX, Factor X, Factor XI, Factor XII, or Prekallikrein). In other embodiments, homologs, fragments, variants, and fusion proteins including the anticoagulant polypeptides bind to Factor Xa, or inhibit or reduce Factor Xa activity. Fragments and variants of the sand fly salivary gland anticoagulant polypeptides disclosed herein can readily be prepared by one of skill in the art using molecular techniques. In one embodiment, a fragment of an anticoagulant polypeptide includes at least 8, 10, 15, 19, 20 23, 25, or 30 consecutive amino acids of an anticoagulant polypeptide. In some embodiments, fragments of the anticoagulant polypeptide include the amino acid sequence set forth as residues 24- 301 of SEQ ID NO: 15 having truncations or internal deletions. The truncations or internal deletions can include at least 1, 2, 3, 4, 5, 10, 15, 20, 30, or more amino acids. In other embodiments, a fragment of an anticoagulant polypeptide includes the N-terminal half or the C-terminal half of the polypeptide. In yet another embodiment, a fragment of an anticoagulant polypeptide includes a specific antigenic epitope found on a full-length anticoagulant polypeptide.

One skilled in the art, given the disclosure herein, can purify an anticoagulant polypeptide, for example residues 24-301 of SEQ ID NO: 15 or variants or fragments thereof, using standard techniques for protein purification and as described herein. The substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel. The purity of the anticoagulant polypeptide can also be determined by amino-terminal amino acid sequence analysis.

Minor modifications of the anticoagulant polypeptide primary amino acid sequences may result in peptides which have substantially equivalent activity as compared to the unmodified counterpart polypeptide described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein.

In another embodiment, the anticoagulant composition includes an isolated polynucleotide having a nucleic acid sequence encoding the sand fly salivary gland polypeptides disclosed herein. The polynucleotide can be from any sand fly, for example (but not limited to) Phlebotomus papatasi, Phlebotomus ariasi, Phlebotomus perniciosus, or Lutzomyia longipalpis. Examples of these polynucleotide sequences are disclosed in PCT/US2003/034453 filed October 29, 2003; PCT Application No. PCT/US2003/029833, filed September 18, 2003; and PCT/US02/19663, filed June 18, 2002, which are incorporated herein by reference.

Encompassed by this disclosure are polynucleotides encoding an anticoagulant salivary polypeptide such as residues 24-301 of SEQ ID NO: 15 or variants or fragments thereof. A specific, non-limiting example of an anticoagulant nucleic acid sequence includes residues 115-948 of SEQ ID NO: 16 and degenerate variants thereof. Another specific, non-limiting example of an anticoagulant nucleic acid sequence includes residues 46-948 of SEQ ID NO: 16 and degenerate variants thereof. These polynucleotides include DNA, cDNA, and RNA sequences that encode an anticoagulant polypeptide. It is understood that all polynucleotides encoding an anticoagulant polypeptide are also included herein, as long as they encode a polypeptide with the recognized activity, such as having an anticoagulant activity, binding to Factor Xa, or inhibiting or reducing Factor Xa activity, thereby inhibiting a component of the coagulation cascade. As used herein, the phrase "nucleic acid molecules encoding the anticoagulation polypeptide" includes both nucleic acid molecules encoding the anticoagulation polypeptide with the signal sequence and nucleic acid molecules encoding the anticoagulation polypeptide without the signal sequence.

The polynucleotides of the disclosure include sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the disclosure as long as the amino acid sequence of the anticoagulant polypeptide encoded by the nucleotide sequence is functionally unchanged.

Also included are fragments of the above-described nucleic acid sequences that are at least 33 bases, at least 36 bases, at least 42 bases or at least 48 bases in length, which is sufficient to permit the fragment to selectively hybridize to a polynucleotide that encodes a disclosed anticoagulant polypeptide under specified conditions. In some embodiments, fragments of the nucleic acid sequences include truncations or internal deletions. The truncations or internal deletions can include at least 1, 2, 3, 4, 5, 10, 15, 20, 30, or more nucleic acids. In other embodiments, a fragment of an anticoagulant polynucleotide includes the N-terminal half or the C- terminal half of the sequence encoding the polypeptide. The term "selectively hybridize" refers to hybridization under moderately or highly stringent conditions, which excludes non-related nucleotide sequences.

Another specific non-limiting example of a polynucleotide encoding an anticoagulant polypeptide is a polynucleotide having at least 75%, 85%, 90%, 95%, or 99% homology to a nucleotide sequence that encodes a polypeptide having an antigenic epitope or function of an anticoagulant polypeptide. Yet another specific non-limiting example of a polynucleotide encoding an anticoagulant polypeptide is a polynucleotide that encodes a polypeptide that is specifically bound by an antibody that specifically binds the anticoagulant polypeptide.

One specific, non-limiting example of a longer homologous sequence that contains residues 115-948 of SEQ ID NO: 16 and encodes a polypeptide that has anticoagulant activity is residues 46-948 of SEQ ID NO: 16, which codes for the full-length polypeptide sequence. Another specific, non-limiting example of a longer homologous sequence that contains residues 115-948 of SEQ ID NO: 16 and encodes a polypeptide that has anticoagulant activity is the full-length nucleic acid sequence set forth as SEQ ID NO: 16 (residues 1-948 of SEQ ID NO: 16). SEQ ID NO: 16 includes 5' and 3' expression control sequences, such as a start codon (ATG), a stop codon, and a poly A signal. Other specific, non-limiting examples of a longer homologous sequence that contains residues 115-948 of SEQ ID NO: 16 and encodes for a polypeptide having anticoagulant activity includes polynucleotides having an amino acid sequence set forth as residues 2-948 of SEQ ID NO: 16, 3-948 of SEQ ID NO: 16, 4-948 of SEQ ID NO: 16, 5-948 of SEQ ID NO: 16, 6-948 of SEQ ID NO: 16, 7-948 of SEQ ID NO: 16, 8-948 of SEQ ID NO: 16, 9-948 of SEQ ID NO: 16, 10-948 etc, of SEQ ID NO: 16, or degenerate variants thereof.

The anticoagulant polynucleotides include a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (for example, a cDNA) independent of other sequences. The nucleotides can be ribonucleotides, deoxyribonucleo tides, or modified forms of either nucleotide. The term includes single and double forms of either nucleotide. Recombinant vectors are also disclosed herein that include a polynucleotide encoding a polypeptide or a fragment thereof according to the disclosure. Recombinant vectors include plasmids and viral vectors and may be used for in vitro or in vivo expression.

A plasmid may include a DNA transcription unit, for instance a nucleic acid sequence that permits it to replicate in a host cell, such as an origin of replication (prokaryotic or eukaryotic). A plasmid may also include one or more selectable marker genes and other genetic elements known in the art. Circular and linear forms of plasmids are encompassed in the present disclosure.

For in vivo expression, the promoter is generally of viral or cellular origin. In one embodiment, the cytomegalovirus (CMV) early promoter (CMV-IE promoter), including the promoter and enhancer, is of use. The CMV-IE promoter can be of human or murine origin, or of other origin such as rat or guinea pig (see EP 0260148; EP 0323597; WO 89/01036; Pasleau et al, Gene 38:227-232, 1985; Boshart M. et al , Cell 41:521-530, 1985). Functional fragments of the CMV-IE promoter may also be used (WO 98/00166). The SV40 virus early or late promoter and the Rous Sarcoma virus LTR promoter are also of use. Other promoters include but are not limited to, a promoter of a cytoskeleton gene, such as (but not limited to) the desmin promoter (Kwissa M. et al, Vaccine 18(22):2337-2344, 2000), or the actin promoter (Miyazaki J. et al, Gene 79(2):269-277, 1989). Either constitutive or inducible promoters can be used. When several genes are present in the same plasmid, they may be provided in the same transcription unit or in different units.

The plasmids may also comprise other transcription regulating elements such as, for example, stabilizing sequences of the intron type. In several embodiments the plasmids include the first intron of CMV-IE (Published PCT Application No. WO 89/01036), the intron II of the rabbit β-globin gene (van Ooyen et al, Science 206:337-344, 1979), the signal sequence of the protein encoded by the tissue plasminogen activator (tPA; Montgomery et al, Cell MoI Biol 43:285-292, 1997), and/or a polyadenylation signal (poly A), in particular the polyA of the bovine growth hormone (bGH) gene (U.S. Patent No. 5,122,458) or the polyA of the rabbit β- globin gene or of SV40 virus. In a specific, non-limiting example, the pVR1020 plasmid (VICAL Inc.;

Luke C. et al, Journal of Infectious Diseases 175:91-97, 1997; Hartikka J. et al, Human Gene Therapy 7:1205-1217, 1996)) can be utilized as a vector for the insertion of such a polynucleotide sequence, generating recombinant plasmids. Various viral vectors are also of use with a polynucleotide encoding an anticoagulant polypeptide. A specific, non-limiting example includes recombinant poxvirus, including avipox viruses, such as the canarypox virus. Another specific, non-limiting example includes recombinant poxvirus, including vaccinia viruses (U.S. Patent No. 4,603,112), such as attenuated vaccinia virus such as NYVAC (see U.S. Patent No. 5,494,807) or Modified Vaccinia virus Ankara (MVA, Stickl H. and Hochstein-Mintzel V., Munch. Med. Wschr. 113:1149-1153, 1971; Sutter G. et al., Proc. Natl. Acad. ScL USA 89: 10847-10851, 1992; Carroll M. W. et al, Vaccine 15(4):387-394, 1997; Stittelaar K. J. et al, J. Virol. 74(9):4236-4243, 2000; Sutter G. et al, Vaccine 12(ll): 1032-1040, 1994). When avipox viruses are used, canarypox viruses (U.S. Patent No. 5,756,103) and fowlpox viruses (U.S. Patent No. 5,766,599) are of use, such as attenuated viruses. For recombinant canarypox virus vectors, the insertion sites may be in particular in the ORFs C3, C5 or C6. When the expression vector is a poxvirus, the heterologous polynucleotide can be inserted under the control of a poxvirus specific promoter, such as the vaccinia virus 7.5kDa promoter (Cochran et al, J. Virology 54:30-35, 1985), the vaccinia virus I3L promoter (Riviere et al, J. Virology 66:3424-3434, 1992), the vaccinia virus HA promoter (Shida, Virology 150:451-457, 1986), the cowpox virus ATI promoter (Funahashi et al, J. Gen. Virol. 69:35-47, 1988), other vaccinia virus H6 promoter (Taylor et al, Vaccine 6:504-508, 1988; Guo et al, J. Virol. 63:4189-4198, 1989; Perkus et al, J. Virol. 63:3829-3836, 1989).

Other viral vectors of use are herpes virus or adenovirus vectors. Specific, non-limiting examples include a canine herpes virus (CHV) or canine adenovirus (CAV) vector (for example, see U.S. Patent No. 5,529,780; U.S. Patent No. 5,688,920; Published PCT Application No. WO 95/14102). For CHV, the insertion sites may be in particular in the thymidine kinase gene, in the ORF3, or in the UL43 ORF (see U.S. Patent No. 6,159,477). For CAV, the insertion sites may be in particular in the E3 region or in the region located between the E4 region and the right ITR region (see U.S. Patent No. 6,090,393; U.S. Patent No. 6,156,567). In one embodiment in CHV or CAV vectors the insert is in general under the control of a promoter (as described above for the plasmids), such as CMV-IE promoter.

Multiple insertions can be done in the same vector using different insertion sites or using the same insertion site. When the same insertion site is used, each polynucleotide insert is inserted under the control of different promoters. The insertion can be done tail-to-tail, head-to-head, tail-to-head, or head-to-tail. IRES elements (Internal Ribosome Entry Site, see European Patent EP 0803573) can also be used to separate and to express multiple inserts operably linked to the same promoter. Bacterial vectors can also be used for in vivo expression.

Any polynucleotide according to the disclosure can be expressed in vitro by DNA transfer or expression vectors into a suitable host cell. The host cell may be prokaryotic or eukaryotic. The term "host cell" also includes any progeny of the subject host cell. Methods of stable transfer, meaning that the foreign polynucleotide is continuously maintained in the host cell, are known in the art. Host cells can include bacteria (for example, Escherichia coli), yeast, insect cells, and vertebrate cells. Methods of expressing DNA sequences in eukaryotic cells are well known in the art.

As a method for in vitro expression, recombinant Baculovirus vectors (for example, Autographa California Nuclear Polyhedrosis Virus (AcNPV)) can be used with the nucleic acids disclosed herein. For example, polyhedrin promoters can be utilized with insect cells (for example, Spodoptera frugiperda cells, like Sf9 cells available at the ATCC under the Accession number CRL-1711, or Sf21 cells) (see for example, Smith et al. , MoI. Cell Biol. 3:2156-2165, 1983; Pennock et al., MoI. Cell Biol. 4: 399-406, 1994; Vialard et al, J. Virol. 64:37-50, 1990; Verne A., Virology 167:56-71, 1988; O'Reilly et al. , "Baculovirus expression vectors, A laboratory manual," New York Oxford, Oxford University Press, 1994; Kidd I. M. & Emery V.C., "The use of baculoviruses as expression vectors," Applied Biochemistry and Biotechnology 42:37-159, 1993; European Patent No. EP 0370573; European Patent No. EP 0265785; U.S. Patent No. 4,745,051). For expression the BaculoGold ™ Starter Package (Cat # 21001K) from Pharmingen (Becton Dickinson) can be used.

As a method for in vitro expression, recombinant E. coli can be used with a vector. For example, when cloning in bacterial systems, inducible promoters such as arabinose promoter, pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter), and the like may be used. Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl₂ method using procedures well known in the art. Alternatively, MgCl₂ or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transduction of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransformed with anticoagulant polynucleotide sequences, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector (see above), such as a herpes virus or adenovirus (for example, canine adenovirus 2), to transiently transduce eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). In addition, a transfection agent can be utilized, such as dioleoyl-phosphatidyl-ethanolamine (DOPE).

Isolation and purification of recombinantly expressed polypeptide may be carried out by conventional means including preparative chromatography (for example, size exclusion, ion exchange, affinity), selective precipitation and ultrafiltration. Such a recombinantly expressed polypeptide is part of the present disclosure. The methods for production of such a polypeptide are also encompassed, in particular the use of a recombinant expression vector comprising a polynucleotide according to the disclosure and of a host cell.

Any of the sand fly polypeptides disclosed herein can have anticoagulation activity. Specific, non-limiting examples of sand fly salivary gland polypeptides, and the nucleic acid sequences encoding them, include those derived from Lu. Longipalpis, as follows: The LJL34 unprocessed protein (SEQ ID NO: 1) is encoded by nucleic acid residues 30-842 of SEQ ID NO: 2, and the mature protein (amino acid residues 20- 271 of SEQ ID NO: 1) is encoded by nucleic acid residues 87-842 of SEQ ID NO: 2. The LJL18 unprocessed protein (SEQ ID NO: 3) is encoded by nucleic acid residues 56-532 of SEQ ID NO: 4, and the mature protein (amino acid residues 20-

159 of SEQ ID NO: 3) is encoded by nucleic acid residues 113-532 of SEQ ID NO: 4.

The LJS 193 unprocessed protein (SEQ ID NO: 5) is encoded by nucleic acid residues 216-502 of SEQ ID NO: 6, and the mature protein (amino acid residues 21- 304 of SEQ ID NO: 5) is encoded by nucleic acid residues 276-502 of SEQ ID NO: 6. The LJS201 unprocessed protein (SEQ ID NO: 7) is encoded by nucleic acid residues 48-353 of SEQ ID NO: 8, and the mature protein (amino acid residues 24- 102 of SEQ ID NO: 7) is encoded by nucleic acid residues 117-352 of SEQ ID NO: 8.

The LJL13 unprocessed protein (SEQ ID NO: 9) is encoded by nucleic acid residues 26-766 of SEQ ID NO: 10, and the mature protein (amino acid residues 20- 247 of SEQ ID NO: 9) is encoded by nucleic acid residues 83-766 of SEQ ID NO: 10.

The LJL23 unprocessed protein (SEQ ID NO: 11) is encoded by nucleic acid residues 18-992 of SEQ ID NO: 12, and the mature protein (amino acid residues 22- 325 of SEQ ID NO: 11) is encoded by nucleic acid residues 81-992 of SEQ ID NO: 12.

The LJMlO unprocessed protein (SEQ ID NO: 13) is encoded by nucleic acid residues 92-571 of SEQ ID NO: 14, and the mature protein (amino acid residues 20-

160 of SEQ ID NO: 13) is encoded by nucleic acid residues 149-571 of SEQ ID NO: 14.

The LJL143 unprocessed protein (SEQ ID NO: 15) is encoded by nucleic acid residues 46-948 of SEQ ID NO: 16, and the mature protein (amino acid residues 24-301 of SEQ ID NO: 15) is encoded by nucleic acid residues 115-948 of SEQ ID NO: 16. The LJS 142 unprocessed protein (SEQ ID NO: 17) is encoded by nucleic acid residues 25-507 of SEQ ID NO: 18, and the mature protein (amino acid residues 21-161 of SEQ ID NO: 17) is encoded by nucleic acid residues 85-507 of SEQ ID NO: 18.

The LJL17 unprocessed protein (SEQ ID NO: 19) is encoded by nucleic acid residues 28-342 of SEQ ID NO: 20, and the mature protein (amino acid residues 21- 105 of SEQ ID NO: 19) is encoded by nucleic acid residues 88-342 of SEQ ID NO: 20.

The LJM06 unprocessed protein (SEQ ID NO: 21) is encoded by nucleic acid residues 50-523 of SEQ ID NO: 22, and the mature protein (amino acid residues 20- 157of SEQ ID NO: 21) is encoded by nucleic acid residues 107-523 of SEQ ID NO: 22.

The LJM17 unprocessed protein (SEQ ID NO: 23) is encoded by nucleic acid residues 24-1264 of SEQ ID NO: 24, and the mature protein (amino acid residues 19-412 of SEQ IDNO: 23) is encoded by nucleic acid residues 83-1264 of SEQ ID NO: 24. The LJL04 unprocessed protein (SEQ ID NO: 25) is encoded by nucleic acid residues 30-914 of SEQ ID NO: 26, and the mature protein (amino acid residues 18- 295 of SEQ ID NO: 25) is encoded by nucleic acid residues 81-914 of SEQ ID NO: 26.

The LJMl 14 unprocessed protein (SEQ ID NO: 27) is encoded by nucleic acid residues 29-475 of SEQ ID NO: 28, and the mature protein (amino acid residues 25-148 of SEQ ID NO: 27) is encoded by nucleic acid residues 101-475 of SEQ ID NO: 28.

The LJMlI l unprocessed protein (SEQ ID NO: 29) is encoded by nucleic acid residues 24-1214 of SEQ ID NO: 30, and the mature protein (amino acid residues 19-397 of SEQ ID NO: 29) is encoded by nucleic acid residues 78-1214 of SEQ ID NO: 30.

The LJM78 mature unprocessed protein (SEQ ID NO: 31) is encoded by nucleic acid residues 42-1091 of SEQ ID NO: 32, and the mature protein (amino acid residues 21-350 of SEQ ID NO: 31) is encoded by nucleic acid residues 102- 11091 of SEQ ID NO: 32. The LJS238 unprocessed protein (SEQ ID NO: 33) is encoded by nucleic acid residues 27-206 of SEQ ID NO: 34, and the mature protein (amino acid residues 21-60 of SEQ ID NO: 33) is encoded by nucleic acid residues 87-206 of SEQ ID NO: 34. The LJS169 unprocessed protein (SEQ ID NO: 35) is encoded by nucleic acid residues 11-370 of SEQ ID NO: 36, and the mature protein (amino acid residues 23-120 of SEQ ID NO: 35) is encoded by nucleic acid residues 77-370 of SEQ ID NO: 36.

The LJLIl unprocessed protein (SEQ ID NO: 37) is encoded by nucleic acid residues 30-1745 of SEQ ID NO: 38, and the mature protein (amino acid residues 26-572 of SEQ ID NO: 37) is encoded by nucleic acid residues 105-1745 of SEQ ID NO: 38.

The LJL08 unprocessed protein (SEQ ID NO: 39) is encoded by nucleic acid residues 26-238 of SEQ ID NO: 40, and the mature protein (amino acid residues 24- 86 of SEQ ID NO: 39) is encoded by nucleic acid residues 95-238 of SEQ ID NO: 40.

The LJS 105 unprocessed protein (SEQ ID NO: 41) is encoded by nucleic acid residues 24-275 of SEQ ID NO: 42, and the mature protein (amino acid residues 20-84 of SEQ ID NO: 41) is encoded by nucleic acid residues 81-275 of SEQ ID NO: 42.

The LJL09 unprocessed protein (SEQ ID NO: 43) is encoded by nucleic acid residues 74-1954 of SEQ ID NO: 44, and the mature protein (amino acid residues 19-626 of SEQ ID NO: 43) is encoded by nucleic acid residues 128-1954 of SEQ ID NO: 44. The LJL38 unprocessed protein (SEQ ID NO: 45) is encoded by nucleic acid residues 40-165 of SEQ ID NO: 46, and the mature protein (amino acid residues 21- 42 of SEQ ID NO: 45) is encoded by nucleic acid residues 100-165 of SEQ ID NO: 46.

The LJM04 unprocessed protein (SEQ ID NO: 47) is encoded by nucleic acid residues 40-456 of SEQ ID NO: 48, and the mature protein (amino acid residues 21- 139 of SEQ ID NO: 47) is encoded by nucleic acid residues 100-456 of SEQ ID NO: 48.

The LJM26 unprocessed protein (SEQ ID NO: 49) is encoded by nucleic acid residues 96-1616 of SEQ ID NO: 50, and the mature protein (amino acid residues 18-446 of SEQ ID NO: 49) is encoded by nucleic acid residues 147-1616 of SEQ ID NO: 50.

The LJS03 unprocessed protein (SEQ ID NO: 51) is encoded by nucleic acid residues 41-553 of SEQ ID NO: 52, and the mature protein (amino acid residues 20- 166 of SEQ ID NO: 51) is encoded by nucleic acid residues 98-553 of SEQ ID NO: 52.

The LJS192 unprocessed protein (SEQ ID NO: 53) is encoded by nucleic acid residues 18-344 of SEQ ID NO: 54, and the mature protein (amino acid residues 24-109 of SEQ ID NO: 53) is encoded by nucleic acid residues 87-344 of SEQ ID NO: 54. The LJM19 unprocessed protein (SEQ ID NO: 55) is encoded by nucleic acid residues 16-360 of SEQ ID NO: 56, and the mature protein (amino acid residues 23- 115 of SEQ ID NO: 55) is encoded by nucleic acid residues 82-360 of SEQ ID NO: 56.

The LJLl 38 unprocessed protein (SEQ ID NO: 57) is encoded by nucleic acid residues 12-1238 of SEQ ID NO: 58 and the mature protein (amino acid residues 21-409 of SEQ ID NO: 57) is encoded by nucleic acid residues 72-1238 of SEQ ID NO: 58.

The LJL15 unprocessed protein (SEQ ID NO: 59) is encoded by nucleic acid residues 63-542 of SEQ ID NO: 60, and the mature protein (amino acid residues 20- 160 of SEQ ID NO: 59) is encoded by nucleic acid residues 120-542 of SEQ ID NO: 60.

The LJL91 unprocessed protein (SEQ ID NO: 61) is encoded by nucleic acid residues 63-542 of SEQ ID NO: 62, and the mature protein (amino acid residues 20- 160 of SEQ ID NO: 61) is encoded by nucleic acid residues 120-542 of SEQ ID NO: 62). The LJMIl unprocessed protein (SEQ ID NO: 63) is encoded by nucleic acid residues 20-1216 of SEQ ID NO: 64, and the mature protein (amino acid residues 19-399 of SEQ ID NO: 63) is encoded by nucleic acid residues 74-1216 of SEQ ID NO: 64. The LJS138 unprocessed protein (SEQ ID NO: 65) is encoded by nucleic acid residues 12-1238 of SEQ ID NO: 66, and the mature protein (amino acid residues 21-170 of SEQ ID NO: 65) is encoded by nucleic acid residues 72-138 of SEQ ID NO: 66.

The LJL 124 unprocessed protein (SEQ ID NO: 67) is encoded by nucleic acid residues 23-241 of SEQ ID NO: 68, and the mature protein (amino acid residues 21-73 of SEQ ID NO: 67) is encoded by nucleic acid residues 83-241 of SEQ ID NO: 68.

The LJL35 unprocessed protein (SEQ ID NO: 69) is encoded by nucleic acid residues 12-1238 of SEQ ID NO: 70, and the mature protein (amino acid residues 24-76 of SEQ ID NO: 69) is encoded by nucleic acid residues 72-1238 of SEQ ID NO: 70.

The above identified Lu. longipalpis polypeptides are further characterized in Valenzuela et al, J. Exp. Bio., 207:3717-3729, 2004, which is incorporated herein by reference.

Methods of Use of Anticoagulant Compositions The anticoagulation polypeptides disclosed herein, and the nucleic acid molecules encoding the anticoagulant polypeptides, can be used to modulate the activity of one or more components of the coagulation cascade either in vitro or in vivo. For example, the activity of a blood coagulation factor, such as Factor Xa, can be reduced or inhibited as a result of the interaction or contact with an anticoagulant composition disclosed herein, in order to inhibit or reduce coagulation. Thus, the anticoagulation compositions disclosed herein have a wide range of medical applications, in the treatment, prevention and diagnosis of diseases and conditions, as well as being useful research tools in the study of anticoagulation and of the inhibition of both the intrinsic and extrinsic pathways of coagulation. A number of laboratory assays for measuring the coagulation or clotting time of a blood or plasma sample are available. When blood is removed from the body and placed in a glass test tube, it clots fairly quickly (4-8 minutes). As calcium ions are required for this process, the addition of EDTA or citrate to the blood prevents clotting (clotting time is indefinite). Clotting time of a blood sample (or a plasma sample, which is a sample of blood depleted of platelets and other blood cells) can be tested at a later time, under various conditions, after adding back an excess of calcium ions. Recalcified plasma (plasma and excess calcium) from a normal, control, subject (a subject who does not have a clotting disorder) will clot in 2-4 minutes. The recalcification (clotting or coagulation) time of this sample can be shortened to 60-85 seconds by adding an emulsion of negatively-charged phospholipids (PL) to the recalcified sample. This time can be further shortened to 21-32 seconds by pre-incubation of the normal (control) plasma with particulate substances such as kaolin (insoluble aluminum silicate), glass, or another artificial surface. Measurement of the recalcification time of plasma in the presence of kaolin (or glass or another artificial surface), phospholipid, and calcium is termed the "activated partial thromoboplastin time" (aPTT) test. The clotting time of recalcified plasma from a normal or control sample can be shortened to 11-12 seconds by adding thromboplastin (a saline brain extract containing tissue factor). Measurement of the recalcification time of plasma in the presence of thromboplastin and calcium is termed the "prothrombin time" (PT) test. Thrombin time (TT) the time required for plasma fibrinogen to form fibrin, is measured as the time for clot formation after exogenous thrombin is added to citrated plasma. A reference value for the thrombin time test is 10-15 seconds, or within 5 seconds of the control sample.

The effect on clotting time of any sand fly salivary polypeptide disclosed herein, for example residues 24-301 of SEQ ID NO: 15 or variants or fragments thereof, can be measured using one or more of the clotting time assays described above. After contacting a sample with a sand fly salivary polypeptide of the disclosure, prolongation of the clotting time in any one of these assays, compared to a sample which does not contain the sand fly salivary polypeptide, demonstrates that the molecule can inhibit coagulation in plasma and is an anticoagulant polypeptide. These assays can also distinguish between different mechanisms of coagulation inhibition. A subject with a prolonged activated partial thromboplastin time (aPTT) and a normal prothrombin time (PT) is considered to have a defect in the intrinsic coagulation pathway. A subject with a prolonged PT and a normal aPTT has a defect in the extrinsic coagulation pathway. Prolongation of both the aPTT and PT suggests that a coagulation cascade defect lies in a component of the common coagulation pathway. The difference in thrombin time between a test and a normal (control) sample indicates an abnormality in the conversion of fibrinogen to fibrin. The blood or plasma sample can be obtained from a normal (control) subject

(a subject who is not in a pro-coagulant state) or a subject who has previously had, is currently suffering from, or who is at risk for acute myocardial infarction, deep vein thrombosis, disseminated intravascular coagulation, pulmonary embolism, coronary artery disease, non-hemorrhagic stroke and unstable angina, rethrombosis after successful thrombolysis during AMI, activated protein C resistance (Factor V Leiden mutation), prothrombin 20210 mutation, or a deficiency of protein C, protein S, or antithrombin. In one embodiment, an anticoagulant polypeptide disclosed herein can be used in vitro to measure the ability of the polypeptide to inhibit clotting of a blood or a plasma sample from a subject in the presence or absence of other agents, such as other (different) anticoagulants. In some embodiments, clotting time is decreased when the sample is contacted with an effective amount of an anticoagulant polypeptide disclosed herein. In other embodiments, clotting time is decreased when the sample is contacted with an effective amount of an anticoagulant polypeptide derived from a sand fly salivary gland, and another anticoagulant. Anticoagulant drugs fall into three categories: inhibitors of clotting factor synthesis, inhibitors of thrombin, and antiplatelet drugs. A specific, non-limiting example of an anticoagulant that inhibits clotting factor synthesis includes warfarin (Coumadin®). Specific, non- limiting examples of anticoagulants that inhibit thrombin include heparin, low-molecular-weight heparin, argatroban, bivalirudin, and lepirudin. Specific, non- limiting examples of anticoagulants that inhibit platelets from forming clots include aspirin, ticlopidine, and clopidogrel (Plavix®). A reduction in clotting time can be at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100%, compared to a control sample. Similarly, inhibiting or reducing coagulation of a blood sample or the plasma sample comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% inhibition, compared to a sample that has not been contacted with the anticoagulant polypeptide.

Abnormalities in any one of the many factors that comprise the coagulation cascade is one mechanism that can result in a pro-coagulant state (undesired clot formation). However, pro-coagulant states can also result from other common causes, such as endothelial disruption or injury. When administered to a subject, an anticoagulant composition, as described above, inhibits or reduces blood coagulation in a subject. Thus, anticoagulant compositions can be used for the treatment of an abnormality in the body's normal clotting mechanism, or for preventing normal clots from occurring in abnormal locations, thereby preventing new blood clots from forming or inhibiting existing clots from enlarging. In one embodiment, the disclosed anticoagulant polypeptides and nucleic acid sequences encoding the polypeptides can be used for the treatment of pro-coagulant disorders characterized by the undesirable formation of clots. Such disorders include acute myocardial infarction, deep vein thrombosis, disseminated intravascular coagulation, pulmonary embolism, coronary artery disease, non-hemorrhagic stroke and unstable angina, rethrombosis after successful thrombolysis during AMI, activated protein C resistance (Factor V Leiden mutation), prothrombin 20210 mutation, or a deficiency of protein C, protein S, or antithrombin. The disclosed anticoagulant polypeptides and nucleic acid sequences encoding the polypeptides also can be used for the treatment of subjects receiving prostheses such as heart valves and to reduce the chance of the formation of blood clots during open heart surgery or bypass surgery, or in patients who must stay in bed for a long time after certain types of surgery. The administration to a subject of a disclosed anticoagulant composition inhibits or reduces the formation of new blood clots in a subject, or inhibits existing blood clots from enlarging. In some embodiments, a subject with a pro-coagulant condition is selected for treatment. In other embodiments, the sand fly salivary gland anticoagulant composition is not administered to a subject with or for the purpose of treating a leishmania infection in the subject.

The sand fly salivary gland polypeptides disclosed herein (for example a Lu. longipalpis polypeptide, such as residues 24-301 of SEQ ID NO: 15 or variants or fragments thereof) have an inhibitory effect on a component of the blood coagulation cascade. In one specific embodiment, residues 24-301 of SEQ ID NO: 15 bind to Factor Xa and inhibit Factor Xa activity. More specifically, residues 24-301 of SEQ ID NO: 15 inhibit Factor Xa by specifically binding the catalytic site of Factor Xa, thereby inhibiting the conversion of prothrombin (factor II) to thrombin (factor Ha) and inhibiting or reducing blood clotting. In other embodiments, a sand fly salivary gland polypeptide disclosed herein has an inhibitory effect on a pro-coagulant protease {i.e., thrombin (Factor Ila), Factor Vila, Factor IXa, Factor Xa, Factor XIa, Factor XIIa, or kallikrein), or on its precursor protein {i.e., prothrombin (Factor II), Factor VH, Factor IX, Factor X, Factor XI, Factor XII, or Prekallikrein), such that coagulation is inhibited or reduced.

The anticoagulant compositions disclosed herein can be formulated in pharmaceutical compositions. In one embodiment, the anticoagulant composition includes a therapeutically effective amount of at least one sand fly salivary gland polypeptide disclosed herein. In one specific embodiment, the anticoagulant composition includes a polypeptide having an amino acid sequence as set forth as residues 24-301 of SEQ ID NO: 15. In other embodiments, the anticoagulant composition includes a sand fly salivary gland polypeptide, such as residues 24-301 of SEQ ID NO: 15, having at least 75%, at least 80%, at least 90%, at least 95%, or at least 99% homology to the polypeptides disclosed herein, a conservative variant or a homolog thereof, or a fragment comprising at least eight, at least nine, at least ten, at least eleven, or at least twelve consecutive amino acids of one of these polypeptides, or a combination of these polypeptides.

In another embodiment, the anticoagulant composition includes an effective amount of one or more sand fly salivary gland polypeptides. Examples of these polypeptide sequences are described above and are disclosed in PCT/US2003/034453 filed October 29, 2003; PCT Application No. PCT/US2003/029833, filed September 18, 2003; and PCT/US02/19663, filed June 18, 2002, which are incorporated herein by reference.

The polynucleotides disclosed herein (for example, a polynucleotide encoding a Lu. longipalpis polypeptide, or a polynucleotide having residues 115-948 of SEQ ID NO: 16 which encode residues 24-301 of SEQ ID NO: 15) also can be formulated in pharmaceutical compositions. In other embodiments, the polynucleotide encoding an anticoagulant polypeptide is a polynucleotide having at least 75%, 85%, 90%, 95%, or 99% homology to a nucleotide sequence that encodes an anticoagulant polypeptide. In one embodiment, the anticoagulant composition comprises an effective amount of a recombinant vector expressing at least one sand fly salivary gland polypeptide disclosed herein. The anticoagulant composition can include a nucleic acid sequence encoding two or more sand fly salivary gland polypeptides. In one embodiment, the two or more sand fly salivary gland polypeptides are encoded by the same recombinant vector. In another embodiment, the two or more polypeptides are encoded by different recombinant vectors.

The pharmaceutical compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable vehicle, excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal.

The anticoagulant polypeptides, and polynucleotides encoding these anticoagulant polypeptides, can be administered by any means known to one of skill in the art (See Banga, A., "Parenteral Controlled Delivery of Therapeutic Peptides and Proteins," Therapeutic Peptides and Proteins, Technomic Publishing Co., Inc., Lancaster, PA, 1995) such as by intramuscular (IM), intradermal (ID), subcutaneous (SC), or intravenous injection, but even oral, nasal, or anal administration is contemplated. In one embodiment, administration is by subcutaneous, intradermal, or intramuscular injection using a needleless injector (Biojector™, Bioject, Oregon, USA).

To extend the time during which the anticoagulant composition is available to inhibit blood coagulation, the composition can be provided as an implant, an oily injection, or as a particulate system. The particulate system can be a microparticle, a microcapsule, a microsphere, a nanocapsule, or similar particle, (see, for example, Banja, supra). A particulate carrier based on a synthetic polymer has been shown provide - a controlled release.

An anticoagulation composition according to the disclosure can be prepared in accordance with standard techniques well known to those skilled in the pharmaceutical arts. Administration is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. A therapeutically effective amount of an anticoagulant composition is an amount used to inhibit or reduce Factor Xa activity. In another embodiment, a therapeutically effective amount of an anticoagulant composition is an amount used to inhibit or decrease conversion of prothrombin (Factor II) to thrombin (Factor Ha) and to inhibit or reduce blood clotting. In yet another embodiment, a therapeutically effective amount of an anticoagulant composition is an amount to prevent or treat a disorder associated with increased blood clotting in a subject. A therapeutically effective fragment of an anticoagulant polypeptide includes any fragment of the sand fly salivary gland polypeptide, or variant of the sand fly salivary gland polypeptide, or fusion protein including a sand fly salivary gland polypeptide, that retains a function of the sand fly salivary gland polypeptide (such as altering Factor Xa activity), or retains the ability to reduce the symptoms related to a disorder associated with increased blood clotting. Thus, in one embodiment, a therapeutically effective amount of an anticoagulant polypeptide or a fragment of an anticoagulant polypeptide is an amount used to bind to Factor Xa, or to inhibit or reduce Factor Xa activity. In another embodiment, a therapeutically effective amount of a fragment of sand fly salivary gland polypeptide is an amount of use to prevent or decrease conversion of prothrombin (Factor II) to thrombin (Factor Ha) and preventing or reduce blood clotting. In yet another embodiment, a therapeutically effective amount of a fragment of a sand fly salivary gland polypeptide is an amount to prevent or treat a disorder associated with abnormal blood clotting in a subject. Specific, non-limiting examples of a polypeptide fragment are the N-terminal half or the C-terminal half of one of the sand fly salivary gland polypeptide disclosed herein. It should be noted that fusion proteins are included, such as a fusion with six histidine residues, a c-myc tag, or any other polypeptide tag. Such fusions are known to one of skill in the art, and are often used in protein purification.

A blood or plasma sample obtained from a subject who is administered a therapeutically effective amount of an anticoagulant composition can be tested for change in clotting time, compared to a sample from a subject that has not been administered the anticoagulant composition. A reduction in clotting time can be at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100%, compared to a control sample. Similarly, inhibiting coagulation of a blood sample or a plasma sample obtained from a subject who is administered a therapeutically effective amount of an anticoagulant composition can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% inhibition, compared to a subject who has not been administered a therapeutically effective amount of the anticoagulant composition.

The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts, taking into consideration such factors as the age, sex, weight, species, and condition of the particular subject, and the route of administration. The route of administration can be via any route that delivers a safe and therapeutically effective dose of a composition of the present disclosure to the animal or human. Forms of administration, include, but are not limited to, topical, enteral, and parenteral routes of administration. Enteral routes include oral and gastrointestinal administration. Parenteral routes include intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, transdermal, and transmucosal administration. Other routes of administration include epidural or intrathecal administration. The effective dosage and route of administration are determined by the therapeutic range and nature of the compound, and by known factors, such as the age, weight, and condition of the patient, as well as LD50 and other screening procedures that are known and do not require undue experimentation. Examples of the techniques and protocols mentioned above can be found in Remington 's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

The term "dosage" as used herein refers to the amount of an anticoagulant composition administered to an animal or human. The therapeutic agent may be delivered to the recipient as a bolus or by a sustained (continuous or intermittent) delivery. When the delivery of a dosage is sustained over a period, which may be in the order of a few minutes to several days, weeks or months, or may be administered chronically for a period of years, the dosage may be expressed as weight of the therapeutic agent/kg body weight of the subject/unit time of delivery. In one embodiment of the present disclosure, an anticoagulant composition is administered as a bolus to a subject in need thereof, to inhibit or reduce coagulation, in a dose of about 0.1 ng to about 500 mg per kg of body weight, about 10 ng to about 300 mg per kg of body weight, from about 100 ng to about 200 mg per kg of body weight, from about 1 μg to about 100 mg per kg of body weight, from about 1 μg to about 50 mg per kg of body weight, or from about 1 μg to about 1 mg per kg of body weight. Alternatively, the amount of an anticoagulant composition administered to achieve a therapeutically effective dose is about 0.1 ng, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 30 mg, 40 mg, or 50 mg per kg of body weight or greater. In one embodiment, the anticoagulant polypeptide is residues 24-301 of SEQ ID NO: 15 or a variant thereof, or is residues 115-948 of SEQ ID NO: 16 or a degenerate variant thereof, and is administered parenterally, preferably intravenously.

In another embodiment, an anticoagulant composition is administered continuously to a subject in need thereof, to inhibit or reduce coagulation, in a dose of about 0.1 ng, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg, or 1 mg per kg of body weight per minute or greater. In one embodiment, the anticoagulant polypeptide is residues 24-301 of SEQ ID NO: 15 or a variant thereof, or is residues 115-948 of SEQ ID NO: 16 or a degenerate variant thereof, and is administered parenterally, preferably intravenously.

In yet another embodiment, an anticoagulant composition is administered to a patient in need thereof, to inhibit or reduce coagulation, in a dose sufficient to achieve a blood plasma concentration of 0.1 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 700 ng/ml, 800 ng/ml, 900 ng/ml, 1 μg/ml, 2 μg/ml, 3 μg/ml, 4 μg/ml, 5 μg/ml, 6 μg/ml, 7 μg/ml, 8 μg/ml, 9 μg/ml, 10 μg/ml, 11 μg/ml, 12 μg/ml, 13 μg/ml, 14 μg/ml, 15 μg/ml, 16 μg/ml, 17 μg/ml, 18 μg/ml, 19 μg/ml, 20 μg/ml, 30 μg/ml, 40 μg/ml, 50 μg/ml, 60 μg/ml, 70 μg/ml, 80 μg/ml, 90 μg/ml, or 100 μg/ml or greater. In one embodiment, the anticoagulant polypeptide is residues 24-301 of SEQ ID NO: 15 or a variant thereof, or is residues 115-948 of SEQ ID NO: 16 or a degenerate variant thereof, and is administered parenterally, preferably intravenously. A typical treatment course can comprise about six doses delivered over a 7 to

21 day period. Upon election by the clinician, the regimen can be continued six doses every three weeks or on a more frequent (daily, twice daily, four times a day, etc.) or less frequent (monthly, bimonthly, quarterly, etc.) basis. Of course, these are only exemplary times for treatment, and the skilled practitioner will readily recognize that many other time-courses are possible. The anti-coagulation polypeptides can be combined with any of a number of conventional treatment regimens. Regional delivery of sand fly salivary gland anticoagulation polypeptides is an efficient method for delivering a therapeutically effective dose to counteract the clinical disease.

Compositions comprising the disclosed sand fly salivary polypeptide or a polynucleotide encoding the sand fly salivary polypeptide (anticoagulant composition) may be administered alone or in combination with other anticoagulant treatments (such as an anticoagulant drug described above), either simultaneously or sequentially dependent upon the condition to be treated. Combination therapies are provided in which an anticoagulant composition disclosed herein is the primary active agent and is administered along with an additional active agent to a subject in order to inhibit or reduce coagulation in the subject. Such combination therapy may be carried out by administration of the different active agents in a single composition, by concurrent administration of the different active agents in different compositions, or by sequential administration of the different active agents. The additional active agent will generally, although not necessarily, be one that enhances or potentiates the effect of the salivary gland anticoagulant polypeptide.

Method of Screening for an Agent that Inhibits Factor Xa

Methods are provided for screening for agents that inhibit components of the coagulation cascade and therefore inhibit coagulation. Thus, methods are disclosed herein for identifying anticoagulant polypeptides that have an inhibitory effect on a protease of the coagulation cascade (i.e., thrombin (Factor Ha), Factor Vila, Factor IXa, Factor Xa, Factor XIa, Factor XIIa, or kallikrein), or on its precursor protein (i.e., prothrombin (Factor II), Factor VII, Factor IX, Factor X, Factor XI, Factor XII, or Prekallikrein).

In one specific embodiment, a sample (for example a plasma sample) is contacted with an agent of interest (for example a sand fly salivary gland polypeptide, such as residues 24-301 of SEQ ID NO: 15 or a variant thereof), and the effect of the agent on the coagulation of the sample (for example, clotting or recalcification time) is then assayed and compared to a control sample that has not been contacted with the agent. An increase in recalcification time indicates that the agent inhibits a component of the coagulation cascade, thereby inhibiting coagulation. Similarly, a decrease in the formation of thrombin from prothrombin in the presence of an agent of interest (for example a sand fly salivary gland polypeptide, such as residues 24-301 of SEQ ID NO: 15 or a variant thereof), indicates that the agent inhibits a component of the coagulation cascade, thereby inhibiting coagulation. In one specific embodiment, an increase in recalcification time indicates that the agent inhibits Factor Xa. In another specific embodiment, a decrease in the formation of thrombin from prothrombin in the presence of Factor Xa and the agent indicates that the agent inhibits Factor Xa.

Measuring a decrease in blood clotting time, and thus a change in activity of a component of the coagulation cascade, such as Factor Xa, can be assessed by any means known to one of skill in the art. The prothrombin time (PT) test and the activated partial thromboplastin time (aPTT) test are standard assays, well known to those skilled in the art, which enable the effects of factors on the extrinsic and intrinsic clotting pathways to be differentiated. In the prothrombin time test tissue factor is added to citrated plasma so that activation proceeds through the extrinsic pathway. Activated partial thromboplastin time (aPTT), can be used to measure the effectiveness of other clotting factors, for example clotting factors VIE, IX, XI, XII, or von Willebrand factor, in both the "intrinsic" and the common coagulation pathways.

Samples used in the above methods can be blood or plasma samples obtained from a subject, wherein the subject does not exhibit a pro-coagulation disorder or wherein the subject has previously had, currently suffers from, or is at risk for a pro- coagulant condition (undesirable clot formation), as described above. In addition, samples used in the above methods can be prepared in vitro, for example by combining in a buffer purified coagulation factors (such as prothrombin and Factor Xa), an appropriate substrate (if necessary) and an agent of interest. EXAMPLES

Example 1

Lutzomyia longipalpis salivary gland polypeptide has potent anti-Factor Xa activity

This example is provided to show that a salivary gland polypeptide of Lutzomyia longipalpis (a New World sand fly and the main vector of visceral leishmaniasis) is a surprisingly potent anticoagulant polypeptide. To find an anticoagulant factor in the saliva of Lu. longipalpis, eight different recombinant Lu. longipalpis proteins (His -tagged on their C terminus and purified through a mammalian expression system) were generated and tested in pharmacological assays. LJL143 (residues 24-301 of SEQ ID NO: 15), which has no homology reported in GenBank, was found to have anticoagulant activity. Moreover, LJL 143 has no structural prediction to be a serine protease inhibitor. This protein is found exclusively in sand flies and further biochemistry assays demonstrated that it acts by binding specifically the catalytic site of Factor Xa of the coagulation cascade. Thus, LJL143 is a useful molecule to inhibit Factor Xa in vivo or can be used to design specific inhibitors of Factor Xa. Because of its high specificity, LJL143 is envisioned as an anticoagulant in a number of pro-coagulant states, including deep venous thrombosis, coronary artery disease, non-hemorrhagic stroke and unstable angina.

The following protocols and data are provided to demonstrate the anticoagulant activity of LJL143.

Sandflies and preparation of salivary gland homogenate (SGH) - Lutzomyia longipalpis, Jacobina strain, were reared using as larval food a mixture of fermented rabbit feces and rabbit food. Adult sand flies were offered a cotton swab containing 20% sucrose and females were used for dissection of salivary glands at 4-7 days following emergence. Salivary glands were stored in groups of 10 pairs in 10 μl NaCl (150 mmol I^"1), Hepes buffer (10 mmol I^"1, pH 7.4) at -70⁰C until needed. Salivary glands were disrupted by ultrasonication within 1.5-mL conical tubes. The tubes were centrifuged at 10,000 g for 2 minutes and the resultant supernatant containing secreted salivary polypeptides (saliva) was diluted in phosphate buffered saline (PBS) and used for injections.

Cloning of Lu. longipalpis cDNA in His-tagged TOPO vector - VR2001-TOPO is a topoisomerase adaptation of VR1020 plasmid (Vical, Inc., San Diego, CA) described previously (Oliveira et al., Vaccine, 24:374-90, 2006). cDNA of each individual Lu. Longipalpis salivary protein (LuIoSP) was amplified by PCR using a specific forward primer deduced from the amino-terminus region and a specific reverse primer containing an ATGATGATGATGATGATG (SEQ ID NO: 71) motif between the stop codon and the carboxyterminus region. The expected amplified sequences were predicted to code for proteins starting after the natural cleavage site and containing a 6x His motif at the C-terminus region. PCR amplification conditions were: 1 hold of 94°C for 5 minutes, 2 cycles of 94°C for 30 seconds,

48°C for 1 minute, 72°C for 1 minute, 23 cycles of 94°C for 30 seconds, 58°C for 1 minute, 72°C for 1 minute, and 1 hold of 72°C for 7 minutes. Amplified products were extracted from a 1.0% agarose gel using Ultrafree-MC extraction kit (Millipore, Bedford, MA). 3 μL, of each PCR product was immediately incubated with 0.5 μL, of VR2001-TOPO, 1 μL, of salt solution (1.2 M NaCl, 0.06 M MgCl₂) and 1.5 μl H₂O for 5 minutes at room temperature. Transformation and selection of positive clones by sequencing were performed following standard procedures.

Expression and purification of Lu. longipalpis salivary proteins (LuIoSP) - For expression and purification of LuIoSP, an aliquot of a glycerol stock was added to 800 ml of LB/kanamycin (100 μg/ml) and incubated overnight on a shaker at 37°C. Plasmid purification was performed using the GenElute™ HP Endotoxin- Free Plasmid Megaprep Kit (Sigma- Aldrich, St. Louis, MO) following manufacturer's specifications. After elution, the plasmid was transferred to a Centricon plus-20 (Millipore, Bedford, MA) with a 100 kDa cutoff. The sample was washed three times with Ultra-pure Water (KD Medical, Columbia, MD) and once with PBS I x (9.0 g/1 NaCl, 232 mg/L KH₂PO₄, 703 mg/1 Na₂HPO₄, pH=7.2) (KD Medical, Columbia, MD) and purified through a 0.2 μM filter unit (Millipore, Bedford, MA). Recombinant proteins were produced by transfecting FreeStyle™ 293-F cells (Invitrogen, Carlsbad, CA) with 70 μg of purified plasmid following the manufacturer's recommendations (Invitrogen, Carlsbad, CA). After 72 hours, transfected cell cultures were harvested and the supernatant filtered through a 0.45 μM filter unit and concentrated to 15 ml in an Amicon^R concentrator device (Millipore Corp., Bedford, MA, USA) in the presence of Buffer A (20 mM NaH₂PO₄, 20 mM Na₂HPO₄. pH 7.4 and 500 mM NaCl). A HiTrap™ Chelating HP column (GE Healthcare) was charged with 5 ml of Ni₂SO₄0.1 M and washed with 10 ml of Milli-Q water and 30 ml of Buffer A using a vacuum manifold (Alltech associates, Inc., Deerfield, IL).

The concentrated recombinant protein was then added to the HiTrap Chelating HP column using a vacuum manifold (Alltech associates, Inc, Deerfield, IL). The column was then connected to a Summit station HPLC system (Dionex, Sunnyvale, CA) consisting of a P680 HPLC pump and a PDA-100 photodiode array detector in order to purify the recombinant protein. The column was equilibrated for 30 minutes with Buffer A at 1 ml/min and following equilibration of baseline the following gradient was used to elute the protein: minute 0-10, 100 % buffer A; minute 10-20, 0 % buffer A, 100% buffer B (20 mM NaH₂PO₄, 20 mM Na₂HPO₄, pH 7.4, 500 mM NaCI and 50 mM imidazole); minute 20-30 100% buffer B; minute 30-60 a gradient of 100% buffer B to 100% buffer C (20 mM NaH₂PO₄, 20 mM Na₂HPO₄, pH 7.4, 500 mM NaCl and 500 mM imidazole); minute 60-70 100% buffer C. Eluted proteins were detected at 280 nm and the eluted fractions were collected every minute on a 96 well microtiter plate using a Foxy 200 fraction collector.

An aliquot (5 μL) of each fraction was blotted on a nitrocellulose paper and the blot was blocked with 5 % milk for 1 hour and incubated for 1 hour with polyclonal anti-saliva antibodies against LJL143 and 1 hour with anti-mouse alkaline phosphatase-conjugated secondary antibody. Positive fractions were developed with WesternBlue® stabilized substrate for alkaline phosphatase (Promega, Madison, WI). An aliquot (5 μL) of each positive fraction was run on an SDS-PAGE gel and silver stained using SilverQuest™ (Invitrogen, Carlsbad, CA). Imidazole was removed from the positive fractions by dialyzing against PBS, pH 7.4.

Polyclonal antibodies against LJL143 - VR2001-TOPO plasmid containing coding sequences of LJL143 without His-tag extension were used to inject mice and generate polyclonal antibodies. Pre-immune samples were taken before the first injection of the DNA plasmid and immune serum samples were taken after three injections given in two week intervals. Each mouse serum sample was pooled for experimentation.

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and western blotting - The samples were treated with 4x NuPAGE LDS sample buffer (Invitrogen, Carlsbad, CA) and analyzed in NuPAGE 4-12 % Bis Tris gels (Invitrogen) with NuPAGE MES SDS running buffer (Invitrogen, Carlsbad, CA). To estimate the molecular weight of the samples, SeeBlue® Plus2 marker from Invitrogen (myosin, phosphorylase, BSA, glutamic dehydrogenase, alcohol dehydrogenase, carbonic anhydrase, myoglobin red, lysozyme, aprotinin and insulin B chain) was used. The proteins in the gel were transferred to nitrocellulose membrane using the iBlot™ device (Invitrogen, Carlsbad, CA). After blocking with 5 % milk in Tris buffered saline with 0.1 % Tween-20 (TBS-T), pH 8, the membrane was incubated with sera of mice immunized with LJL143 DNA plasmid (1/100 in TBS-T 5 % milk). After two washes with TBS-T, the membrane was incubated with alkaline phosphotase-conjugated goat anti-mouse IgG (ZyMax™, Invitrogen) at 1/10000 in TBS-T 5 % milk for 40 minutes at room temperature. After three washes with TBS-T, the blots were developed by addition of Western Blue® stabilized susbstrate for alkaline phosphatase (Promega, Madison, WI).

Serine protease inhibition assays - All assays were performed at 30⁰C in triplicates. 120 nM of LJL143 was pre-incubated with each enzyme to be tested for 5 minutes before the addition of the corresponding substrate. All enzymes used were of human origin, purified or recombinant. Thrombin and plasmin were purchased from Sigma (St. Louis, MO), Factor Xa (FXa) from EMD Biosciences (La Jolla, CA), kallikrein from Fitzgerald Industries International (Concord, MA), uPA from Molecular Innovations (Southfield, MI) and sequencing grade trypsin from Roche (Chicago, IL). The amount of enzyme used in each assay is shown in Table 1 (Figure IE). The assay buffers were: (i) for trypsin and thrombin, 50 mM Tris pH 8, 150 mM NaCl, 2OmM CaCl₂, 0.01 % Triton X-100, (ii) for kallikrein and plasmin, 20 mM Tris pH 8.5, 150 mM NaCl, 0.02% triton X-100, (iii) for FXa, 20 mM Tris pH 8, 200 mM NaCl, 5 mM CaCl₂, 0.1 %BSA, (iv) for uPA, 20 mM Tris pH 8.5, 0.05 % Triton X- 100. The substrates used were: Boc-Asp-Pro-Arg-AMC for thrombin and plasmin (Boc: butoxycarbonyl; AMC: 7-amino-4-methylcoumarin), Boc-Gln-Ala-Arg-AMC for trypsin and uPA (urokinase plasminogen activator) (Sigma, St. Louis, MO) and methylsulfonyl-D-cyclohexylalanyl-Gly-Arg-AMC acetate for FXa and kallikrein (American Diagnostica Inc., Stamford, CT). All substrates were used in 250 μM final concentration in all the assays. Substrate hydrolysis rate was followed in a Spectramax Gemini XPS 96 well plate fluorescence reader (Molecular Devices, Sunnyvale, CA) using 365 nm excitation and 450 nm emission wavelength with a cutoff at 435 nm.

Measurement of human plasma clotting activity - Clotting activity was measured by the recalcification time of human plasma using a Thermomax microplate reader (Molecular Devices) with a kinetic module. Briefly, 30 μL of citrated human platelet-poor plasma and 30 μL of sample (SGH or a recombinant LuIoSP diluted in PBS) were mixed in 96 well flat-bottom plates. LuIoSP were used at a final concentration of 10 nM. After incubation for 10 minutes at room temperature, 30 μL of 25 mM CaCl₂ were added using eight-channel multipipeter for delivering calcium aliquots. The plate was immediately mixed and maintained at 37 ⁰C during the kinetic experiment. Absorbance readings at 650 nm were taken at 20-sec intervals for 20 minutes.

Prothrombinase assembly - Activation of prothrombin by human FXa was performed in TBS-Ca²⁺ (20 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl₂, 0.3% I pH 7.5), using a discontinuous assay (Monteiro et al , Biochem J, 387:871-877, 2005). FXa (10 pM), was incubated with rLJL143 (0-10 nM) for 20 minutes at room temperature. Human Factor Va (1 nM) and Protein C/Protein S (10 μM) were added and incubated for 5 minutes. Reactions were initiated by addition of human prothrombin (1.4 μM). Aliquots of 25 μL were removed every minute into microplate wells containing 50 μL of TBS-EDTA (20 mM Tris-HCl, 150 mM NaCl, 20 mM EDTA, 0.1 % BSA, pH 7.5) to stop reactions. After addition of 25 μL of S- 2238 (312.5 μM; a chromogenic substrate for thrombin), absorbance at 405 nm was recorded at 37° C for 15 minutes at 11 -second intervals using a Thermomax microplate reader (Molecular Devices, Menlo Park, CA) (Francischetti et al., Biochemistry, 38: 16678-16685, 1999). Initial velocities (mOD/min) obtained were used to calculate the amount of thrombin formed, using a standard curve (Francischetti et al., Biochemistry, 38: 16678-16685, 1999). Absence of one of the components in the prothrombinase showed no thrombin formation.

Surface plasmon resonance - Binding of rLJLI43 to Factors Xa, X and Xa-DEGR

(active-site blocked factor Xa containing the fluorescent inhibitor Dansyl-EGRck) was analyzed at 25°C by surface plasmon resonance (SPR) spectrometry using a BIAcore TlOO instrument (BIAcore AB, Sweden). Factors X, Xa and Xa-DEGR were covalently immobilized on a CM5 sensor chip using the amine coupling kit supplied by Biacore, following the manufacturer's instructions. Blank flow cells were used to subtract the buffer effect on the sensograms. For kinetic experiments Factor Xa, in concentrations ranging from 3.25-500 nM was run for 120 seconds at 20 μl/min in HBS-P buffer (10 mM HEPES, 150 mM NaCl, 0.005 % surfactant P20, pH 7.4) over immobilized rLJL143. The complex dissociation was monitored for 600 seconds and the active cell was regenerated with a 3 second pulse (30 μL/min) of 10 mM HCl. The kinetic experiments were performed in triplicates on different days. The Biacore TlOO Evaluation software was utilized for kinetic evaluation and the sensograms fitted using the 1:1 binding model (Langmuir binding model).

Alternatively, an experiment was designed to test the binding specificity of rLJL143 towards Factors X, Xa and Xa-DEGR. The sensor chip was created in order to have the Factors X, Xa and Xa-DEGR in the same sensor chip. Different concentrations of rLJL143 (10-40 nM in HBS-P) were manually injected over the four flow cells in the sensor chip for 120 sec at a flow rate of 20 μl/min. The complex dissociation was monitored for 500 seconds and the sensor surface was regenerated by a pulse of 5 seconds of 10 mM HCl at 40μl/min. These experiments were carried out in duplicates.

Results - Salivary gland polypeptides of Lutzomyia longipalpis have an inhibitory effect in the coagulation cascade as it was observed that the recalcification time of human plasma was affected in the presence of 0.5 pair of salivary gland (Figure IA, left panel). To find the anti-coagulant factor in the saliva of Lu. longipaipis, eight recombinant salivary proteins were generated and expressed after transfection in HEK 293F cells and tagged with a 6x His tail in their C terminus. The eight proteins expressed recombinantly and tested in the system were LJMI7 (GenBank access number: AF132518), LJM04 (AF132517), LJL15 (DQ190946), UMI l (AY445935), LJL13 (AF420274), LJL143 (AY445936), LJMl 11 (DQ192488) and LJM26 (AY455913).

Of the eight Lu. longipaipis polypeptides tested, only one (LJL143) demonstrated a surprisingly complete (and potent) inhibition of recalcifiation of the sample (Figure IA, right panel). LJMI7, LJM04, LJL15, LJMIl, LJL13, LJMlI l, and LJM26 had recalcification times between 250 seconds and 300 seconds, which was similar to that of the control (phosphate buffered saline; PBS) sample. In contrast, LJL143 (1OnM) had a surprisingly long recalcification time (600 seconds), which was more than two times longer than the control sample or other Lu. longipaipis samples. LJL15 (C-type lectin protein) was previously suspected to have the anticoagulant activity of Lu. longipaipis saliva, but the corresponding recombinant protein showed no activity in this system (Figure IA, right panel). Thus, none of the seven other Lu. longipaipis proteins showed any anticoagulant effect.

The activation of prothrombin by Factor Xa (FXa) in the presence of rLJL143 was then assessed. As shown in Figure IB, rLJL143 inhibits the formation of thrombin by FXa. Thus, it was believed that rIJL143 interfered with Factor Xa binding to prothrombin. Surface plasmon resonance experiments were then performed and it was demonstrated that rLJLI43 binds FXa (Figure 1C), and more specifically to the active site of Factor Xa (Figure ID).

In order to check the substrate specificity of LJL143, several enzymatic tests were run in the presence of rLJL143. Several serine proteases were incubated in the presence of their respective substrates and rLJLI43 (12OnM). rLJLI43 specifically inhibited FXa activity. No inhibition of thrombin, kallikrein, trypsin, u-PA or plasmin by LJL 143 was reported (Figure IE), demonstrating the specificity of rLJL143 to FXa. In a similar experiment, LJM 19 had no inhibitory activity on Factor Xa or on thrombin, kallikrein, chymase, trypsin, α-chymotrypsin, β-tryptase, elastase, cathepsin, u-PA, plasmin matriptase, Factor Xia, Factor XIIa, or t-PA (Table 1), further demonstrating the specificity of the LJL143 salivary gland polypeptide in binding to Factor Xa.

Table 1

As discussed above, the recombinant LJL143 used in the above experiments is tagged with a 6x His tail in its C terminus. Native LJL 143 isolated from Lu. longipalpis salivary glands and LJL143 expressed in cells (without the His tag) following LJL143 cDNA vaccine administration are also able to inhibit Factor Xa, demonstrating that the LJL143 protein itself, and not the 6x His tail, is responsible for Factor Xa binding and that the addition of the six histidine residues does not affect the Factor Xa binding activity of LJL143. Moreover, other salivary proteins containing the 6x His tail did not bind Factor Xa, further demonstrating that the 6x His tail itself does not bind Factor Xa.

To demonstrate that LJL143 was secreted in Lu. longipalpis saliva, a Western blot of an SDS-PAGE analysis of a homogenate of 10 pairs of Lu. longipalpis salivary glands (SGH) was analyzed with sera of mice previously immunized with LJL143 cDNA. SGH reacted with anti-LJL143 antibodies (Figure 2A) and not with pre-immune serum. Moreover, the recombinant protein rIJL143 reacted with anti- LJL143 antibodies (Figure 2A) giving a band of comparable size to that seen in the SGH sample, but slightly higher, consistent with the addition of the 6x His tag motif at the C terminus of the recombinant protein. These results showed that LJL143 is actively secreted in Lu. longipalpis saliva. They also showed that rLJL143 electrophoresis mobility is about 40 kDa despite a predicted size of 32.4 kDa, suggesting post-translational modifications.

LJL143 sequence, alignment with homologs from Old World sand flies and phylogenetic tree are described respectively in Figures 2B, 2C, and 2D.

Example 2

Method of treating a subject with a sand fly salivary gland anticoagulation polypeptide

This example describes a protocol to prevent or treat a disorder in a subject characterized by abnormal or undesirable blood coagulation (clotting) using a sand fly salivary gland anticoagulation polypeptide described herein, or a polynucleotide encoding the polypeptide, for example a Lu. longipalpis salivary gland polypeptide, such as SEQ ID NO: 15 or residues 24-301 of SEQ ID NO: 15, or a variant thereof, or a polynucleotide such as SEQ ID NO: 16, residues 46-948 of SEQ ID NO: 16, or residues 115-948 of SEQ ID NO: 16, or a degenerate variant thereof. Such disorders include (but are not limited to) acute myocardial infarction, deep vein thrombosis, disseminated intravascular coagulation, pulmonary embolism, coronary artery disease, non-hemorrhagic stroke and unstable angina, rethrombosis after successful thrombolysis during AMI, activated protein C resistance (Factor V Leiden mutation), prothrombin 20210 mutation, or a deficiency of protein C, protein S, or antithrombin.. The disclosed methods also can be used for the treatment of subjects receiving prostheses such as heart valves and to reduce the chance of the formation of blood clots during open heart surgery or bypass surgery, or in patients who must stay in bed for a long time after certain types of surgery. This protocol is intended to serve as an example of such a treatment method, and is not meant to be limiting. Those of skill in the art will be able to modify the protocol to suit the needs of the subject, and to optimize for the particular compounds used. Subjects can, but need not, have received previous therapeutic treatments.

A sand fly salivary gland anticoagulation polypeptide or polynucleotide is administered orally or parenterally in dosage unit formulations containing standard, well known non-toxic physiologically acceptable carriers, adjuvants, and vehicles as desired. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intra- arterial injection, or infusion techniques. The sand fly salivary gland anticoagulation polypeptide or polynucleotide can be administered in various dosages, as described above, depending on the polypeptide or polynucleotide used. Therapeutically effective dosages of sand fly salivary gland anticoagulation polypeptides or polynucleotides can be determined by measuring clotting activity of the subject's plasma in the presence of various concentrations of the polypeptides. For example, clotting activity can be measured by titrating dosages of the anticoagulation polypeptides while measuring recalcification time or prothrombin time. A typical treatment course can comprise about six doses delivered over a 7 to

21 day period. Upon election by the clinician, the regimen can be continued six doses every three weeks or on a more frequent (daily, twice daily, four times a day, etc.) or less frequent (monthly, bimonthly, quarterly, etc.) basis. Of course, these are only exemplary times for treatment, and the skilled practitioner will readily recognize that many other time-courses are possible. The sand fly salivary gland anticoagulation polypeptides or polynucleotides can be combined with any of a number of conventional treatment regimens. Regional delivery of sand fly salivary gland anticoagulation polypeptides or polynucleotides is an efficient method for delivering a therapeutically effective dose to counteract the clinical disease. The sand fly salivary gland anticoagulation polypeptides or polynucleotides can be delivered to the subject before, after, or concurrently with the other anticoagulation agents.

Clinical responses can be defined by an acceptable measure. For example, a complete response can be defined by the disappearance of all measurable disease or symptoms of the disorder (for example, undesirable clotting) for at least a month. A partial response can be defined by a 50% or greater reduction of all measurable disease or symptoms of the disorder.

Of course, the above-described treatment regimes can be altered by those of skill in the art, who will be able to take the information disclosed in this specification and optimize treatment regimes.

In view of the many possible embodiments to which the principles of our invention may be applied, it should be recognized that illustrated embodiments are only examples of the invention and should not be considered a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A method of inhibiting coagulation of a blood sample or a plasma sample in vitro, comprising contacting the blood sample or the plasma sample with an effective amount of a polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as residues 24-301 of SEQ ID NO: 15, thereby inhibiting coagulation of the blood sample or the plasma sample.

2. The method of claim 1 , wherein inhibiting coagulation of the blood sample or the plasma sample comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% inhibition, compared to a sample that has not been contacted with the polypeptide.

3. The method of claim 1, wherein the polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as residues 24-301 of SEQ ID NO: 15.

4. The method of claim 3, wherein the polypeptide comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth as residues 24-301 of SEQ ID NO: 15.

5. The method of claim 4, wherein the polypeptide comprises an amino acid sequence set forth as residues 24-301 of SEQ ID NO: 15.

6. The method of claim 5, wherein the polypeptide comprises an amino acid sequence set forth as SEQ ID NO: 15.

7. The method of claim 1, wherein the polypeptide inhibits Factor Xa activity.

8. The method of claim 1 , further comprising measuring the clotting time of the blood sample or the plasma sample.

9. A method of treating a subject for a disorder associated with undesired blood coagulation, comprising: selecting a subject with a pro-coagulant condition; and administering to the subject a therapeutically effective amount of a polypeptide having at least 90% sequence identity to the amino acid sequence set forth as residues 24-301 of SEQ ID NO: 15, thereby treating the subject for the disorder associated with undesired blood coagulation.

10. The method of claim 9, wherein the polypeptide is not administered to treat a leishmania infection in the subject.

11. The method of claim 9, wherein the subject is treated to prevent new blood clots from forming or to inhibit existing blood clots from enlarging.

12. The method of claim 11 , wherein the subject previously had, is currently suffering from, or is at risk for an acute myocardial infarction, deep vein thrombosis, disseminated intravascular coagulation, pulmonary embolism, coronary artery disease, non-hemorrhagic stroke, or unstable angina.

13. The method of claim 12, wherein the subject is a human.

14. The method of claim 9, further comprising measuring the clotting time of a blood sample or a plasma sample obtained from the subject.

15. The method of claim 9, wherein the polypeptide is a first anticoagulant and the first anticoagulant is administered to the subject in combination with a second anticoagulant.

16. The method of claim 15, wherein the first and second anticoagulants are administered either simultaneously or sequentially.

17. The method of claim 9, wherein treating the subject comprises inhibiting coagulation of a blood sample or a plasma sample obtained from the subject by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, compared to a subject who has not been administered a therapeutically effective amount of the polypeptide.

18. The method of claim 9, wherein the polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as residues 24-301 of SEQ ID NO: 15.

19. The method of claim 18, wherein the polypeptide comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth as residues 24-301 of SEQ ID NO: 15.

20. The method of claim 19, wherein the polypeptide comprises an amino acid sequence set forth as residues 24-301 of SEQ ID NO: 15.

21. The method of claim 20, wherein the polypeptide comprises an amino acid sequence set forth as SEQ ID NO: 15.

22. The method of claim 9, wherein the polypeptide inhibits Factor Xa activity.

23. A method of treating a subject for a disorder associated with undesired blood coagulation, comprising: selecting a subject with a pro-coagulant condition; and administering to the subject a therapeutically effective amount of a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to the amino acid sequence set forth as residues 24-301 of SEQ ID NO: 15, thereby treating the subject for the disorder associated with undesired blood coagulation.

24. The method of claim 23, wherein the nucleic acid sequence comprises the sequence set forth as residues 115-948 of SEQ ID NO: 16, or a degenerate variant thereof.

25. The method of claim 23, wherein the nucleic acid is operably linked to an expression control sequence.

26. The method of claim 24, wherein the expression control sequence is a promoter.

27. The method of claim 25, wherein the promoter is an inducible or constitutive promoter.