TW200529870A - Therapeutic use of factor XI - Google Patents

Abstract

The present invention provides methods and compositions for treating bleeding episodes. The methods are carried out by administering to a patient in need thereof a preparation comprising a factor XI polypeptide, in an amount effective for such treatment. The methods of the invention result in one or more of: reduced clotting time; enhancement of hemostasis; increase in clot lysis time; increase in clot strength; and/or increase in overall clot quality (OCQ) in said patient.

Description

200529870 IX. Description of the invention: [Technical field to which the invention belongs] The present invention relates to the medical use of human factor XI for the prevention and / or treatment of bleeding incidents, a method for purifying factor XI and factor polypeptide from biological fluids, and medicine Preparations. [Prior Art] Background of the Invention Human factor XI is a serine protease containing two identical subunits, each of which has a molecular weight of approximately 80 kr) a. FXI circulates in the plasma with disulfide-linked elemental dimers with a molecular weight of ~ 160 kDa. The 50-kDa amine-terminated heavy chain and the 35-kDa carboxy-terminated light chain are formed by cutting M- and nem between each monomer, and the two are connected by disulfide bonds. The protein is encoded by a 23 kb gene located on chromosome 4 (4q35). It consists of 15 exons and 14 introns. It encodes an mRNA containing 2,097 nucleotides and thus contains 18 amine groups. The amino-terminal signal (leader) peptide of the acid and the 607 amino acids present on each monomer of the mature protein. The exon ηΐ-χ encodes four adapter repeats 歹 J (Apple domain) 'which are homologous to similar domains found in human plasma ρκ (the same as 58 / 〇fan Wang). Exons 乂 1-: ^ encode a typical trypsin-like catalytic domain, which proteolytically cleaves internal bonds of the zymogen to produce a heavy chain (369 amino acids) and a light chain containing four apple domains or a catalytic Domain (23 8 amino acids) to be activated. The initiation mechanism of the blood effect of the aunt is by converting the tissue factor (D) to 200529870 and then converting the plasma factor VII (FVII) into the activated factor TF-FVIIa complex and using protein X (FXa) (Hi) Exposing the coagulation ring to the injured site with Fxa, tightly binding to TF and proteolytic x (FXa); (ii) Converting factor X to proteolysis to turn into an activated factor tissue factor pathway inhibitor (Tfpi) zymogen is converted to thrombin; and (iv) a complex is formed between FXa and FXa, and TF-FVIIa can regulate the activation of thrombin by TFPI: FXa complex and TF-ganglion FXa and And restrict the flow of thrombin produced by the TF it pathway. A relatively small amount of thrombin produced during this phase will activate FXI to FXIa (which activates factor IX to FIXa) and activate factor v on the surface of platelets and further activate factor X. These events further promote the formation of sufficient thrombin (so-called "thrombin release") to convert fibrinogen to fibrin, thereby stabilizing the initial platelet embolism and causing proper hemostasis. FXI of the binomial circulates in the plasma with zymogen, non-covalently complexes with the high molecular weight kininogen (HK) cofactor, promotes the binding of FXI to the negatively charged surface, and promotes FXI by its homologous protease, Fxna , And thrombin activation. The binding site of HK to FXI involves multiple apple domains (A 丨, A2, A4), and the A2 domain is the most important domain. Formation of complexes with hk in the presence of Znh ions has been shown to promote FXI binding to activated platelets. The interaction of fxi with the surface of activated platelets has been shown to be regulated by Ser248-Val271 residues in the A3 domain in fxi. ; Residues Ser_, Arg ·,

LyS2 ”Phe26〇 and GIn263 have also been suggested to participate in this reaction. The A3 domain of fxi also contains a heparin binding site in residue Thr249_phe㈤, and residues Lysw and Lysw have been suggested to participate in platelet binding. Although fxi and 200529870 HK are non-co-combined Valence complexes circulate in the plasma. The interaction of FXI with the surface of platelets obviously does not require the binding of HK-FXI complexes. Instead, it is clear that FXI monomers bind directly to specific sites of high affinity for activated platelets ( Approximately 1500 sites / platelets; Kd binds to approximately the same sites of activated platelets and has the same affinity as FXI dimers at approximately isolated recombinant A3 domains. Activated enzyme FXIa has been shown to bind to activated platelets High affinity, sites that can be saturated (Kd approximately 800pM; 500 sites / per platelet), and FIX can be activated in a ratio similar to that observed in solution. Substance-fixing FIX binding sites in FIX It involves the secondary domain (Ala) 34-Leu172 in the ... domain and two secondary domains (ne ...- vaim and Serm-Sei ^ 5) in the A3 domain. The binding is regulated by a glycoprotein complex using an FX; [dimer polypeptide chain, so another monomer appears as the binding site for FIX. It is possible that the FDU catalyzed by the manufacturer of Fixa ^ FX activation is localized on the surface of platelets, and it also promotes the activation of prothrombin by FXa. In addition to the formation of a membrane-bound complex leading to the production of locally explosive thrombin on the surface of platelets, FXIa is also subject to various blood aggregation and platelet proteins. The regulation of the preparation, and the functional activity of the inhibitor shows whether it can be bound to the surface of platelets or whether it is free in solution. Therefore, there are several types of serine protease inhibitors including alpha protease inhibitors, anticoagulants M, cl • inhibitors… · antifibrinolytic enzymes, plasminogen activator inhibitors, and protein; protein C inhibitors have been shown to inactivate FXIa within the blood aggregation interval. In the environment of activated platelets, 200529870 seems to be the most physiologically relevant inhibitor of FXIa is protease connexin II (PN II), which was found to have a non-plasma concentration It is usually low, but it is secreted from platelet alpha particles (1-1 · 5ηΜ PNΙΙ is released per 108 platelets), which shows that its cytoplasm concentration is 3-5ηΜ under normal physiological conditions. PNΙΙ is an effective FXIa inhibitor with a Ki value of 300-5 00 pM, which is significantly promoted in the presence of heparin.

In the presence of Zn2 + ions or in the presence of prothrombin and Ca2 +, the binding of FXia to the surface of platelets is protected by PNII and α-; [_ protease inhibitors without loss of activity. FXIa activity generated on the surface of platelets is located in the hemostatic thrombus. Whereas FXIa is regulated by PNII and other protease inhibitors in solution. It is also possible that epidermal cells contain heparan sulfate, glucose, and luciferin, which promotes the combination of FXIa / PNn complexes, thereby increasing the potential for FXIa to be inhibited on the endothelium. "FXI is involved in thrombin production on the surface of the blood clot and is also thought to play a role in inhibiting fibrinolysis by a thrombin-activatable fibrinolytic inhibitor (tafi). Residue removal and insertion, where the _ & shows that the lysine plays a role in the binding and activity of plasminogen. Let ’s look at each other — only a whole FXI feedback loop It is necessary to produce a sufficient amount of thrombin due to the activation of TAFI activation. It is worth noting that i α platelets and megakaryocytes apparently synthesize DXI-form FXI. In a, he gained the favor of a platelet-derived FXI (pd-FXI), which differs from the circulating form on exon V, i.e., in two exons lacking the exodomain, K 疋, Shows better platelets for Shima's second apple; exon 111 'and in vitro studies have shown that the factor Xla substrate may be blood "magic" rather than 200529870 Πχ. Platelet FXI (molecular weight 220KDa) has been found to bind to the plasma membrane of platelets. Platelets contain approximately 300 pd_Fx " per cell. Deficiency is a somatic recessive syndrome characterized by a erratic bleeding tendency. Even more severe, the deficiency may be asymptomatic on the L bed until the patient is challenged by surgical trauma; however, in some cases, the severity of bleeding may occur regardless of the deficiency. The optimal management of patients with FXI requires attention to several specialties in addition to FXI concentration. First, it is important to assess the bleeding tendency of individuals with partial deficiencies and whether other factors contribute significantly. Such studies should include measurements of FVIIIC and von Willebrand factor concentrations, bleeding time, and platelet coagulation. Fresh frozen plasma has been used to treat the first known cases of fxi deficiency and was the main treatment until FXI concentrates were developed. The main disadvantages of plasma are the large volume required, the possibility of allergic reactions and the penetration of infectious agents. In addition, there have been reports of a more variable FXJ inclusion in this product. Two FXI concentrates are currently available. The fxi concentrate from Bio Products Laboratory (BPL) (UK) was formulated with high concentrations of antithrombin (average 102iu / ml) and heparin (10u / ml) which is considered to be resistant to any residual Fxja. The second ρχζ Chinese condensate was produced by Hemoleven (France), and the product was formulated with 3-5 u / ml heparin, 2-3 iu / ml antithrombin and C1 inhibitor. Furthermore, pasteurization of fresh pooled frozen plasma has been reported to retain 75-95% of FXI activity. Although patients with severe FXI deficiency can be treated with FXI concentrates, patients with mild FXI deficiency are generally treated with fresh 200529870 frozen plasma. Patients with excessive bleeding or severe trauma associated with surgery and in need of blood transfusion (including those without a congenital deficiency) will have more complications than patients who have not experienced any bleeding. Even the need to administer human blood products (such as platelets, white blood cells, concentrated blood cells ...: Therapy: Immobilization = persons) can alleviate bleeding which can lead to human viruses (hepatitis, chat, virus, and other currently known viruses) Complications associated with the risk of metastasis. Extensive bleeding that requires large blood transfusions can lead to multiple organ failures, including impaired lung and kidney function. -Once a subject has these severe eruptions, a cascade of events involving several cytokines and inflammatory responses will begin, making any treatment extremely difficult or often unsuccessful. Therefore, one of the main goals of T and the treatment of major tissue damage is to avoid or minimize bleeding. "In order to avoid or minimize such bleeding, it is important to confirm that the formation is stable and not to be dissolved by the fibroin enzymes = embolism. And it ’s important to ensure that such a king or blood clot is formed quickly and effectively. W00200307983 reveals the use of factor VHa and now both for the treatment of bleeding incidents. In other words, an improved hemostatic treatment mode is needed in the art to make stable, rapid and regulated formation of fibrin clots. [Summary of the Invention] Summary of the Invention The present invention provides a method and a composition for treating a bloody incident. The 10 200529870 method is performed by administering a therapeutically effective amount of a preparation containing Factor XI (FXI) to a patient who is in need of I + Shi + Ding. The method of the present invention may cause one or more of the following results:> reducing the time of coagulation, increasing: hemostasis, increasing blood clot dissolution time, increasing the strength of the blood clot, and / or increasing the overall blood clot product temporarily ^, ^

Xi, Q). In certain specific examples, the patient ' s exhibit an effective FXI plasma concentration of at least about 5 nM, 10 nM, or 30 n] vi after administration of FXI peptide. In certain specific examples, the FXI polypeptide comprises the sequence of SEQ ID NO: 1 or a fragment that retains at least one biological activity associated with ρχι. In a specific embodiment, the FXI polypeptide comprises the sequence of SEQ ID NO: 2 or a fragment thereof that is retained to) a biologically active fragment associated with FXI. In some specific examples: the FXI polypeptide comprises a pre-modified derivative of SEQ ID NO: 1 or SEQ ID NO: 2, or SEQ ID N with one or more than one amino acid sequence change O: 1 or a variant of SEq ID NO: 2. In some specific examples, the patient does not suffer from congenital FXI deficiency. In some specific cases, the incidental bleeding event is subordinate to surgery, medical procedures, trauma, or hemodilution. : The invention also provides methods and compositions for preventing incidents of bleeding. It is used to treat patients in need of this treatment, which is effective for preventing bleeding. In some specific examples, the method of the present invention further includes administering the following rules: 获取 obtaining a blood sample from the patient; ⑻ measuring at least the following FXI 'degree, Fxia: fxi ratio, required to restore coagulation The amount of exogenous yang; and ⑷ based on the result of step (b), determine the amount of FXI treated with 200529870. In a specific embodiment, the method of the invention does not include administering a factor VII / factor Vila coagulant. As used in this specification, Factor VII / Factor Vila Coagulant is a Factor VH polypeptide or a Factor V related polypeptide as described in 2000007983. The invention also provides for the treatment of incidental bleeding events. Method and composition, the patient is administered the first amount of the package ★ FXI polypeptide preparation and ⑻ = the preparation containing non-Factor VII / Factor clotting agent, wherein the first amount and the second amount are combined. The treatment is effective. Non-limiting examples of non-time VII / factor coagulants include: factor xm, · tissue factor pathway = preparation ™) inhibitors; factor ιχ, thrombin can activate fibrin: v (: AFI); fibrinolysis Zymogen activator inhibitor-i (PAC-!); Negative c inhibitor due to I 'protein; protein 3 inhibitor of activator of activator (tPA) and factor X. _, factor VIII, fibrinogen are also provided by the present invention Medical composition

Polymorphism and (ii) medical treatment 4. Isolated recombinant FXI (acceptable carrier or excipient). The present invention also provides a method for factor X, which method comprises Purified from the biological material, the sub-exchange chromatographic analysis material, and the hydrophobic continuous two-layer chromatographic analysis method are performed on the yanglin limestone chromatographic analysis material. Wang Li @ 色 层 分析 材料, and Jingji 12 200529870 DETAILED DESCRIPTION OF THE INVENTION The present invention is based on a surprising finding: exogenously administered factor XI (FXI) is effective as a general hemostatic agent in human blood without the application of factor vla / factor VIIa coagulants. The medical use of fxi according to the present invention provides one or more than one: shortened coagulation time, tighter clumps, and formed blood clots with improved resistance to fibrinolysis.

The invention provides a method and a preparation for medical use of FXI in a human-induced diseased meat field recognizing g # eight moth / Bingsi basket, in order to treat or prevent occasional bleeding events, improve hemostasis, increase clot dissolution time, And / or increase the strength of the blood clot. This method achieves one or more of these desired medical goals by administering an effective amount of Factor XI to a patient. The composition includes a pharmaceutical formulation for medical use in FXI ' which comprises an isolated recombination.

In specific examples of the series, the present invention relates to the administration of fxi to normal human patients. As used in this manual, "normal" humans are those who do not suffer from congenital FXI deficiency (ie, hemophilia C, see Selig Sohn (1993), Thrombin and Hemostasis, Section 7) Stage 〇: Stages, ~ Patients (reduced platelet count or activity), patients who are expected or undergoing surgical procedures, patients who have suffered trauma or organ damage, to show lower platelet numbers and And / or reduced fibrin FVIII, and / or other blood coagulation protein concentrations. For example, normal human patients cover experiencing due to bleeding, trauma, chemotherapy, liver disease, blood thinning ^ 13 200529870 if possible due to infusion of plasma swelling agents or saline In order to maintain blood volume or prevent shock), or other conditions not directly related to the congenital defect of the FXI gene, the plasma concentration of FXI (or other coagulation-related proteins or factors) causes a transient decrease in lipid levels. In other series of specific examples, the invention relates to administering isolated and / or recombinant FXI to patients suffering from congenital FXI deficiency.

In the practice of the present invention, any FXI that can effectively prevent or treat bleeding

Even peptides can be used. This includes blood or plasma or platelets or

FXI polypeptides produced by recombinant methods in any suitable host organism or cell. The invention also encompasses FXI polypeptides in their unfragmented (zymogen) form, as well as those that have been proteolytically processed to produce their corresponding biologically active forms (designated FXIa).

As used in this specification, FXI polypeptides encompass, but are not limited to, and FXI related polypeptides. The term FXI is intended to cover, but is not limited to, polypeptides having the amino acid sequence of FXI in human plasma of Richter type, for example, in F ... Teren, "Biochemistry", No. 25: p. 2417 (1986). And wild-type FXI from other species, such as cattle, pig, dog, rat, rabbit, and Salmon ™. -In general, it is better to use a syngeneic: FXI protein to reduce the risk of inducing an immune response. The preparation and characterization of non-human_m have been described, for example, by Gailani (1997 ^, "Blood", Issue% ', p. 1055. The present invention also covers veterinary procedures ^ the use of factor XI proteins. In some specific examples, the FXI polypeptide is wild-type human blood pack ™ (SEqidn0: 1). In other specific examples, the Fxi-type platelet derivative 14 200529870 FXI (pd-FXI) (SEQ ID No) ), As described by Hsu et al. (I998), Journal of Biochemistry, No. 273: 13787_93. FXI peptides also include FXI's possible variants of the different ligatures dual genes. At the same time, saccharification The extent and location of effects or other post-translational repairs may change in some cases, depending on the source of fxi-encoding nucleic acid, the host cell produced by FXI, and the conditions under which the production cell is maintained. Polymorphisms related to FXI include but Not limited to FXI polypeptides that have been chemically modified relative to human fx (ie, FXI street organisms) and / or ρχι polymorphic (ie, ™ mutations) that contain one or more than one amino acid sequence change associated with human FXI Body). This is related to FXI Related polypeptides can exhibit one or more changes in biological activity relative to human FXI, including but not limited to altered stability, altered phospholipid binding, altered I enzyme activity, altered immunogenicity, alteration Bioavailability, altered binding to one or more FXI binding partners, altered binding to caffeine inhibitor M and the like. ™ related polymorphisms encompass polypeptides in their uncut form «proto} ", And the relative biological activity of the proteolytic peptides that have been produced by the proteolytic enzymes of the" V-type peptide "can be referred to as" multiple FXI-related polypeptides associated with FXI. " Non-limiting examples of inferior FXI organisms include: wild-type has been acidified, sulfated, PEGylated, in vivo or in vitro, or by a species of transglucosidase and / or enzyme Modified by enzyme action

Hi Yijing, for example, et al., "Thrombin 82, 1283-8"). Non-limiting examples of FXI variants include:-one or more N_15 200529870 linkages or 0-linkages where the glycosylation is consistent has been modified, single-chain FXI (i.e., its monomeric polypeptide is not like wild Type is generally subject to in-chain proteolytic cleavage (FXI), and cysteine variants in which one or more than one cysteine residue is condensed or reset, including but not limited to altering the monomer or dimer Changes in the form of disulfide bonding. ’In a specific example, Cys! I (not considered to be involved in intermolecular or intramolecular disulfide bonding) is condensed or replaced. In a series of specific examples, FXI variants reduce half-life in plasma relative to wild-type human FXI. In a specific example, the FXI variant has a half-life of less than 50 hours. In a specific example, the FXI variant has a half-life of less than 24 hours. In a specific example, the FXI variant has a half-life of less than 12 hours. In a specific example, the FXI variant has a half-life of less than 6 hours. In a specific example, the FXI variant has a half-life of less than 3 hours. In a series of specific examples, FXI variants are polypeptides that have been destroyed by N-linked saccharification at one or more positions that have been modified by homologous sites that are consistent with N-linked saccharification, such as: Any of the following amino acids are independently substituted: N90, N126, N353, N450, N491, or a combination of any substitutions previously mentioned. Non-limiting examples of such variants include FXI-N72Q; FXI-N108Q; FXI-N335Q; FXI-N432Q; FXI-N473Q; FXI-N72Q / N108Q; FXI-N72Q / N1 08Q / N335Q; FXI-N72Q / N108Q / N3 3 5Q / N43 2Q; FXI-

N72Q / N108Q / N335Q / N432Q / N473Q; FXI-N72Q / N432Q; FXI-N72Q / N473Q; FXI-N1 08Q / N432Q; FXI-N1 08Q / N473Q 16 200529870 and FXI-N432Q / N473Q. Glycation that disrupts n-linking at one or more sites can also be achieved, for example by independently deleting any residues 72-74, 108-110, 335-337, 432-434, and 473_475 (i.e. One or more residues at one site may be deleted and not replaced by any other laurinic acid) '(ii) independently substituted N + 2 residues (such as T74 to any residue other than S, Replacing S110 with any residue other than τ,

Replace S337 with any residue that is not S, S434 with any residue that is not T, and S475 with any residue that is not butyl), · (丨 ⑴ Replace N + 1 with an amino acid that destroys saccharification Residues (for example but not limited to Puraminol (P)). We should understand that any combination of the above methods can be used to independently destroy the glycation of different parts of the FXI polypeptide. The present invention also covers the whole or part of the FXI polypeptide and other Chimeric or fusion polypeptides formed between heterologous peptide sequences. For example, one or more of the four apple domains are replaced by similar apple domains obtained from other polypeptides (see J Female Gailam et al. (1999 ), "Blood", No. 94 · · 621

B) Either one or more Apple domains are deleted in their entirety. In another selected male case, a LDL receptor-related protein (LRP) that does not have a binding moiety (such as a polypeptide containing Phe34r Asn346 residues due to + T Υα β m) has been shown to be a parent of LRP. Contributions to interactions, Rohlena et al. (2003), "Study of Life ^ 2" "Issue 278 · · page 9394) connected to FXI polymorphism has been>> pharmacokinetic properties. The nature of the dimers of FXI in the form of Tauto (as well as the asymmetric function of two monomers in, for example, the activation of FXI of the blood preparation lance) also makes the use of the present invention-containing heterodimer ( That is, it is feasible to combine two incomplete FXI (or FXI-related) monomeric polypeptides of FXI (2005 20057070). All that is required is that the heterodimer exhibit one or more beneficial aspects of biological activity. The FXI polypeptide used in the present invention includes, but is not limited to, a polypeptide exhibiting biological properties that are substantially the same as or enhanced in nature with wild-type human-FXI, and the biological activity of FXI relative to the wild-type human FXI activity has been essentially Repair or reduce peptides.

In the present invention, beneficial compositions containing FXIa or FXIa-related polypeptides, including k allogenes, are included when wild-type FXI equivalents have been obtained from the same source or produced in the same cell type, and the activity comparison has been By performing in the same FXI activity test of item 70, it can show at least about 10%, at least about 20%, at least about 30%, at least about 40% 'at least about 50%, at least about, at least about / 〇, at least about 80%, at least about 90%, at least about 100%, at least about no%, at least about 120%, at least about including a specific activity of only wild-type FXI composition. As used in this specification, the terms "activity" and "specific activity" apply individually or collectively to any one or more aspects of FXI biological activity. In some specific examples, the ratio of the specific hydrolytic activity of XI-related peptides to the egg-type hydrolytic activity of wild-type humans when tested in the FXI amidine hydrolysis test is $ / 1, and the hand to ^ is about 1.25; In another specific example, the ratio is at least about 2.0 square U. In a further specific example, the ratio is less than about 4.0. FXI biological activity 18 200529870 In the practice of the present invention, one or more different aspects of FXI biological activity can be quantified or used in the following aspects: ⑴ Select the appropriate FXI composition and formulation for medical yoke , Methods of FXI production or purification, and the like; (ii) assess the effectiveness of different medical methods. I should understand that Fxia's "specific biological activity" or any aspect of these biological activities is expressed in units of active units per unit mass of FXI polypeptide. These aspects include the following: I. Proteolytic activity: (a) The amidolytic activity can be quantified in vitro using an appropriate chromophore and substrate, such as S2355 (produced by Chromogenix), as described by Ekdahl et al. U999 ), "Platelet and Hemostasis", No. 82: p. 8 The measured activity is compared with a standard FχIa preparation with a defined specific activity (enzyme research laboratory) and its value is expressed as au of Fxia activity. (b) The activity of FXI activation can be quantified in vitro directly by measuring the proteolytic conversion of the factor ιχ to IXa, as described in, for example, Yi et al. (2001), "Blood", No. 97 · · 3 ΐ 7-3 ΐ 22 pages. 11 · Binding Activity and Inhibition IX (FIX) 'and platelet-related FXI receptors In the practice of the present invention, any conventional test can be used for the affinity of any of these (or other) binding partners. Wild-type FXI has several binding partners, including kininogen (PK), high molecular weight kininogen (HK), clotting / thrombin, and factor 'is called GPlb-IX. Quantitative FXI Peptide Pairs in this binding test kit 19 200529870-.... Partner-labeled Competitive Knot ™ Peptide Binding Partner also includes :: test. But it is not limited to antithrombin m, c] 3 x ι active site inhibitors, including 此, 于, 月, 月, and protease connexin II (PN II), dioxin 5 polymorphism for FXI). Quantitative test method; or this record "Effective to use the traditional proteolytic activity of the proteolytic activity of the inhibitory activity can be measured using hydrazine hydration test. / Ί 活 III. Coagulation weight blood-time, blood clot dissolution time and The strength of the block is a parameter of the hemostatic system bed. The patient's + time gate np—blood kappa-like 施用 was obtained by Shita's occupational patient after the application of TM ™ and tested for these parameters. Buben ϋντ ^ tir Xi Wang Lu… Second time 'FXI peptide preparations can be used for in vitro / in vitro treatment of blood collected from human subjects. Edges τ can be tested using standard ρτ or apTT tests. Dissolution time and clot strength can be measured by thromboelastography, using, for example, Vig et al. (2001) "Blood coagulation and fibrinolysis", Issue 12: pp. 555-561 and Sorensen ( (2003) Thrombin and Hemostasis, H. p.551 · 558. Or 'clot strength can be borrowed from Carr et al. (1991),' American Journal of Medical Science, ' 3G2: Issue 丨 3_8 f The method described is examined. TEG reaction method to measure clotting activity of FXI one shell ί Cloth overall clot quality number (overall clot quality) "(qcq). 20 200529870-Once blood clot formation starts ㈣), the measurement of blood clot strength as a function of time is the maximum velocity (max vel) of blood clot formation and the daytime interval U required to reach the maximum velocity. Further, the addition of tissue plasminogen activator (tpA) can measure fibrin, and cross S2 -rf Θ ίΐΐ 2: Ϊ / 1 reed white A solution and pay the required speed to reach the maximum rate of fibrin dissolution Time (tminre,). The OCQ is calculated as: (MU Vel / tmax rel) X din rel _tniax "丨" IY · pharmacokinetic parameters wild-type human FX] [believed to have a half-life in plasma of about 50 hours, and a half-life of at least 50 Hz Partially regulated by its effects. In the practice of the present invention, FXI polymorphisms can be used, which exhibit pharmacokinetic parameters different from natural FXI. Non-limiting examples include those that have been treated with sialidase to self-extract from FXI FXI polypeptides from which one or more incomplete sialic acid residues have been removed from the associated sugar collection, Fxl polypeptides that have been modified by the pEG reaction, and ρχι polypeptides that exhibit altered interactions with HK. When invented, the pharmacokinetic properties can be calculated using, for example, WinNonlin Professional Edition 3.1 (produced by PharSight, located in Mountain View, California, USA). If more than one value exists, the calculation can be used every time The average concentration value of the point is calculated. The following pharmacokinetic parameters can be calculated using the following formula: AUC, AUC% Extrap, Cmax, tmax,, ti / 2, CL and Vz: 21 200529870

The area of AUC from 0 to infinity under the plasma concentration · time curve. It is calculated by extrapolating the linear AoG trapezoid method to infinity. The linear trapezoidal method is used from time 0 to tmax: just (. ~) = ㈤ ^ 兔 ⑹) The 10g trapezoidal method is used from time tmax to the final time point t: (W-especially) £ (j) ΑίΤΐ) Using the following formula Extrapolate to infinity: AUC (i ^ GD) = ^ l Λ

AUC〇 / oExtrap r ^ max CL Obtained from the final concentration limit _ AUC percentage: AUCy body plate =-^ 7 ^ .100% AUC maximum extrapolated plasma concentration to time 〇 Total clearance

CL

Dose

AUC h / i λζ vz Maximum plasma concentration observed time _ In 2 iy = half-life: ′ 1 terminal rate constant. Calculated based on the 1-line reduction (average) concentration time. The distribution v__ Dose-product 1-AUC · obtained according to the final phase] Production and purification of FXI FXI polypeptides used in the present invention can be made using any of the appropriate methods of this technology by using the method 22 200529870 Prepared from blood or blood sources. As used in this specification, the term "isolated" refers to a ρχι polypeptide that has been isolated from the cells from which it is synthesized or from the substrates (such as plasma or blood) from which it is expressed. Isolation of the polypeptide from cells of its origin can be achieved by any method known in the art, including but not limited to removing the cell culture medium containing the desired product from the attached cell culture; centrifuging, or passing through; To remove non-adherent cells; and the like. If necessary, the ρχι polypeptide can be further purified. Purification can be achieved by any method known in the art, including, but not limited to, affinity chromatography, such as in an anti-fxi antibody column or peptide affinity column (non-limiting examples include heparin, This, Han Yi, arginine, nodules, peptides, other dyes, or Rp_chromatography); hydrophobic interaction chromatography; ion exchange chromatography analysis; particle size exclusion chromatography Analysis; electrophoresis procedures such as isoelectric focusing (IEF) for preparation, differential solubility (eg, any precipitation or crystallization method, using salt, pH, ammonium sulfate, or other additives), or extraction and the like, Proceed as described in more detail above. After purification, the formulation preferably contains less than about 2% to about 10% by weight ' more preferably less than about 5%, and most preferably less than about a non-FXI polypeptide derived from the host cell. Purification of FXI from serum can also be achieved by known methods, including, but not limited to, by Koide et al. (1977), Biochemistry, No. 2 p. 2279, and Bouma et al. ( 1977), Journal of Biochemistry, No. 252, p. 6432, which is incorporated by reference in this specification. Methods to prepare for restructuring FXI are known in the art. Please refer to the example ^ 23 200529870

Kemball_cook et al. (1994), Genes, No. 139: p. 275, Fujikawa et al. (1986), Biochemistry, No. 25: p. 2417, and Meijers et al. (1992), "Blood", No. 2 [5] [Complete references in this specification and humans may also be obtained from Enzyme Research Laboratoris, located in South Bend, Indiana. The present invention also relates to a method for purifying FXUJ Tairu recombinant FXI polymorph from pelvic biomaterials. The method comprises subjecting the substance f to chromatographic analysis on a chromatographic material having cation exchange action. The present invention also relates to a method for purifying an FXI polypeptide, such as a recombinant FXI polypeptide, from other biological materials. The method includes performing color layer analysis on a hydrophobically interacting color separation material. The present invention also relates to a method for purifying an FXI polypeptide, such as a recombinant FXI polypeptide, from other biological substances. The method includes performing a color layer analysis on a color-dialyzed material based on base wollastonite. The invention also relates to a method for purifying an FXI polypeptide, such as a recombinant FXI polypeptide, from other biological materials. The method includes separating the material in the positive ion; a coloring material for exchange, a coloring material for anaerobic interaction, and hydroxyapatite. Color layer analysis on the color separation material. I must understand that a series of color analysis numbers are performed in the order indicated. The "coloring material of hydroxyapatite" used in the present specification also refers to any known coloring material of base disc limestone that can be combined with PM in this specification, such as a hydroxyapatite matrix. The “cation exchange chromatography analysis material” used in this manual means 24 200529870 any analysis material of this technology. The cation-exchange chromatographic layer that is known to combine polymorphism with F XI in this paper. The "hydrophobic interaction chromatographic analysis material" used in this specification. "Any chromatographic chromatographic layer known to be capable of binding to FXI polypeptide in this technology. Analyze materials. · In a specific example, the present invention relates to a method for purifying FXI polypeptide from biological material, which method comprises the following steps:

⑴ Apply the biological substance to the first cation exchange color layer material; (10 Unbound material is eluted from the first cation exchange color layer analysis material with buffer A, and buffer A is suitable for applying The material that fails to bind to the first 'cationic parent color change layer analysis material is eluted; and (ill) the buffer A' is used to elute the unbound material from the first cation exchange color layer analysis material, Buffer A, suitable for elution and dissolution of substances that fail to bind to the first cation exchange chromatography material; and

〇) Use buffer A ”to elute the FXI peptide from the first cation exchange chromatography analysis material 'Buffer A, suitable for eluting FXI peptide from the first cation exchange chromatography analysis material In a specific embodiment, the present invention relates to a method for purifying FXI from biological material, which method comprises the following steps: using a hydrophobic interaction color layer analysis material to eluate obtained from step (iv) Or use the liquid prepared in step (iv) for chromatographic analysis, which includes: 25 200529870 (V) applying the eluate obtained from step (iv) or the liquid prepared in step (iv) to the hydrophobic The material of the color layer analysis of the sexual interaction; (), the unbound material is eluted from the material of the color layer analysis of the hydrophobic interaction with the, and the liquid B. The buffer A is suitable for the material that fails to interact with the hydrophobicity. (Vn) using buffer B to elute the ρχι peptide from the hydrophobic interaction color analysis material, buffer B, which is suitable for FXI peptide from this The water-based interaction color layer analysis material elutes. In a specific example, the present invention relates to a method for purifying FXI from biological material, which method comprises the following steps: using a base apatite color layer analysis material to obtain Chromatographic analysis is performed from the eluate of step (vii) or using the liquid prepared in step (vii), which includes = (vm) the eluate from step (vii) or the use of step (ν 利用) The prepared liquid is applied to the hydroxyapatite color layer analysis material; (lx) unbound material is eluted from the hydroxyapatite gray layer analysis material with buffer solution C, and the buffer solution C is suitable for The substance bound by the hydroxyphosphorus gray layer analysis material elutes; and (X) using buffer C, the FXI polypeptide is eluted from the hydroxyphosphorus gray layer analysis material, buffer C, which is suitable for eluting the FXI polypeptide from the The hydroxyphosphorus gray layer was eluted from the analysis material. In a specific example, the present invention relates to a method for purifying FXI from biological material, which method comprises the following steps: 26 200529870 (a) exchange the first cation color layer analysis Chromatographic analysis of biological substances containing peptides, which includes: 层 applying the biological substance to the first cation exchange color material; 缓冲 using buffer A to remove unbound substances from the first- Buffer A is suitable for eluting substances that fail to bind to the first cation exchange chromatography material; and Buffer A 'is used to elute unbound substances from the first _A kind of cation exchange chromatographic analysis material elutes' buffer ， A, which is suitable for eluting substances that cannot be combined with the first type of cation exchange chromatographic analysis material; and using buffer A, FXI peptides are eluted from the first cation exchange chromatography analysis material, and buffer ⑨A is suitable for eluting ρχι polymorphism from the first cation exchange chromatography analysis material. (b) Colorimetric analysis of the eluate obtained in step ㈣ or the liquid prepared in step (iv) with a hydrophobic interaction color layer analysis material, which includes: ⑺ will be obtained from step (iv ) Eluate or liquid prepared in step ㈣ · Applied to the color analysis material of the hydrophobic interaction; (Vi) Using buffer B to remove unbound material from the color analysis material of the hydrophobic interaction Down 'Buffer A is suitable for eluting substances that fail to bind to the chromatographic analysis material of the hydrophobic parent; and ii) using buffer B to remove the FXI polypeptide from the chromatographic layer of the hydrophobic interaction 'Buffer B' eluted from the analysis material is suitable for eluting ρχι poly from the hydrophobic interaction color layer analysis material. 27 200529870 (c) Chromatographic analysis of the eluate obtained in step (vii) or the liquid prepared in step ii) using hydroxyapatite chromatographic analysis material, the chromatographic analysis method comprises: (viii) The eluate obtained in step (vii) or the liquid prepared in step (vii) is applied to the hydroxyapatite color layer to analyze the material; (IX) Unbound material is analyzed from the hydroxy-filled gray layer with buffer C to analyze Eluted from the material, buffer C is suitable for eluting substances that fail to bind to the hydroxy plate gray layer analysis material; and (X) Buffer C 'is used to analyze the material from the hydroxy phosphorus gray layer The buffer solution C is suitable for eluting the fxi polypeptide from the hydroxyphosphorus gray layer analysis material. Purification of FXI polypeptide is a process that increases the concentration of FXI polypeptide in a sample compared to other components in the sample, resulting in increased purity of the FXI polypeptide. We should understand that the concentration of FXI polypeptide in the sample compared to other components in the sample is not equal to the concentration of FXI polypeptide in the sample. The increase in the purity of the FXI polypeptide can then be measured using methods known in the art, such as by SDS-PAGE (sodium lauryl sulfate polyacrylamide gel electrophoresis), HPLC (high-performance liquid chromatography) or Bericome test (Dade Behring) Diagnostics) or blood clot activity test. Biological material can be any material obtained from or containing cells, cellular components, or cell products. A biological substance may be a biological liquid. A biological fluid may be any liquid obtained from or containing cells, cellular components or cell products. Biological fluids include, but are not limited to, cell cultures, cell culture supernatants, cell lysates, clarified cell lysates, cell single 200529870 extracts, tissue extracts, plasma, serum, all of which can also be homogenous or The filter and a part of the collected matter, for example, the non-partially collected biological fluid is collected by chromatography. FXI and more | too can be purified from a wide variety of biological materials, the second has included cell culture supernatants that can naturally produce FXI polypeptide, but can also be purified from cells genetically modified to produce FXI polypeptide, such as Mammalian cells (eg cho cells) transformed via DNA encoding a pxi polypeptide. There are several ways to treat biological material before applying it to the first cation exchange chromatography material. Such methods include, but are not limited to, separation and filtration. In a specific embodiment of the present invention, the biological liquid is a supernatant of a cell lysate. In a specific embodiment of the present invention, the biological fluid is a supernatant of a yeast cell lysate. In a specific embodiment of the invention, the FXI polypeptide is purified from a cell culture, such as a mammalian cell culture described above. Prior to the chromatographic analysis in step 哺乳动物, mammalian cells can be separated from the cell culture supernatant by centrifugation and / or transition. Inhibitors such as EDTA (ethylene diamine tetraacetate) and phenylhydrazone HC1 can be included before step (a) of the chromatographic analysis. A buffer solution is a solution containing a substance that prevents the solution from significantly changing in pH when a small amount of acid or alkali is added, thereby largely maintaining the original acidity or alkalinity of the solution. Buffers usually contain weak acids or weak salts and their salts. Prior to the chromatographic analysis of step (a), the pH of the biological fluid can be adjusted to the pH of buffer A, for example with 1M hci or iM NaOH or using other methods known in the art. The first type of cation exchange chromatography, He Bei can be any known in the art = Ion exchange chromatography analysis material 'can be under a series of conditions: the peptide binding subgroup and under different-a series of conditions τ will It is released by, for example, a cation exchange chromatography material containing a propyl propyl group. Examples of more non-limiting cation exchange chromatography materials f include dextran, agar, cellulose, polyacrylamide, and specialized silicone members, such as carboxymethyl. Identification of the appropriate cation exchange chromatography material f can be performed by applying a biological fluid containing f χ engineering polypeptide on the selected cation exchange chromatography material for cation exchange chromatography analysis, collecting part of the collection and determining the purity and Part of the contents of the collection, such as using SDS_PAGE (polysodium lauryl sulfate polyacrylamide gel electrophoresis), HPLC (high performance liquid chromatography) or

The Bericome test (produced by Dade Behring Diagnostics), monitors the absorbance of the eluate at a wavelength of 280 nm, and uses other methods of this technique. Examples of suitable cation exchange chromatography materials include, but are not limited to, Streamline SP XL (produced by Amersham Biosciences, catalog number 17-5073), Obelix ST CIEX (produced by Amersham Biosciences, catalog number 11-0010), Streamline Direct CST ( Produced by Amersham Biosciences, catalog number 17-5266), S-

Support Unosphere, BioRad catalog number 156-0113 or Toyopearl SP 5 5 0C Toso Haas catalog number 14028. In a specific example, Obelix ST CIEX is used. The first cation exchange chromatographic analysis material can be equilibrated with buffer A before applying the biological substance. 30 200529870 Buffer A may contain protease inhibitors such as ed and fat, but other commercially available protease inhibitors may also be used:: private) In a specific example of the present invention, the buffer 7 and 7 Between 9. In a specific embodiment, the pH of the buffer solution A is approximately 8800. In one embodiment of the present invention, the buffer solution α is approximately 40 mS / cm. Electricity and J%… Buffer A ”is used for Elution ™. Typically, the buffer used for the washing of step （(in this case, 1 缥 茶茶 疋, readings A and A,) -One or more, the degree will increase or decrease during the elution process, or-a new buffer is added, and the concentration of this component will decrease during the elution process. Such increase or decrease is known in the art It will occur continuously or in separate steps. For S, the material bound to the cation-exchange color-acoustic analysis material will be eluted, and the salt to be added, such as Na ^, will be added to the flushing liquid A. This This particular kind of yang, ^ ^ ^ Do not carve and change the resin can also be used as a sparse tree moon purpose, for this resin, I usually use

Propylene glycol / glycerin was added to Buffer A. The growth was better than that of Buffer A. 〇If NaCI === was added to the buffer, then ™ will be imported into the "collection"-a partial collection containing FXI peptide In order to further process this [into the lamp, for example, to exclude the elution at the beginning or end of the FXI continuation wash. Therefore, μ, subject to π 雑 疋 heat, which is known to this artist. The general techniques for performing cation exchange chromatography are as follows: pre-equilibrium, elution time, ^ M, Ca, M, and P4.% Ion exchange chromatography analysis materials are well known. 31 200529870 Elution of the FXI polypeptide in step (iv) M t) with fxi peptide protein inhibitors such as edta, ...] plus 3 EDTA (tetraacetic acid with diamine) and benzene fat washing T Step (V). The eluate is also maintained at, for example, 40 ° C for 24 hours or more, or at, for example, -8 °. ^. The hydrophobic interaction color layer analysis material used in step (b) can be any hydrophobic interaction color layer analysis material known in the art, which can bind FXI polypeptide under a series of conditions and under different series of conditions. Let's release them, such as a phenyl, butyl, or octyl or polyacrylic resin derivative I analysis of the hydrophobic interaction color layer. Non-limiting examples of suitable hydrophobic parent interaction color analysis materials are AmberchromTM CG71 (produced by Tosoh BioScience), Phenyl Sephar〇seTM High

Performance (Phenyl Sepharose High Performance Chromatography) (Amersham Company's Catalog No. 1 7-1082), Phenyl SepharoseTM 6 Fast Flow High Substitution (Phenyl Sepharose 6 Fast Flow High Substitution Chromatography) (Amersham Company, Catalog No. 1 7-0973), Toyopearl® butyl 650 (produced by Tosoh Bioscience), Toyopearl® phenyl (produced by Tosoh Bioscience), Source tm 15 Phe (produced by Amersham Biosciences, catalog number 17-0147), Butyl

Sepharose ™ High Performance High Substitution (Amersham Biosciences, Catalog No. 17-3100), Octyl-SepharoseTM (Amersham Biosciences, 'Cat. No. 17-0946), and Benpha SepharoseTM High Performance High Substitution (Amersham Biosciences) 32 200529870 and the like. . In a specific example of the present invention, the anaerobic interaction color layer analysis material uses butyl as a ligand. Buffer B and NaCl can be added to the eluate obtained from stage (iv) or a liquid prepared using the eluate obtained from stage (iv) before the chromatography in step (b) in an amount of approximately One to two volumes or more, or a concentrated form of buffer B—contains the same components as buffer b, but with, for example, twice the concentration, added to the eluate from stage (iv) or utilized Liquid prepared from eluate from stage (iv) in an amount approximately equal to the concentration buffer

Solution strength (add 1.5 times the volume of twice the volume of concentrated buffer). Buffer B has a pH of about 5 to about 9, such as about 8. In a specific example of the present invention, the buffer solution B has a package density greater than 25 ms / cm, such as greater than 70 mS / cm. This can be achieved, for example, using phosphate buffered saline or other methods known in the art such as NaC1. In a specific embodiment of the present invention, the conductivity of the eluate obtained from step (iv) or the liquid prepared using the eluate obtained from step (^) is adjusted so that its conductivity is at least about 60 rnS / crn.

Buffer B was used to elute the FXI polypeptide by a gradient elution method. In the gradient eluent, the composition of buffer B was changed during the elution. Typically, the concentration of one or more components of the washing buffer in step (vi) will increase or decrease during the elution in the case of buffer B, or a new component is added to the buffer. The concentration of the components increases during the elution. This increase or decrease may occur in successive or =: steps, as is well known in the art. In order to elute the substance bound to: water and interaction chromatography analysis material, we usually used 33 200529870 to dilute the washing buffer with water until to, 1 4 to 8 until V. The main part containing the bound FXI polypeptide was eluted. . Determine which eight Ι / Γ # 1 fraction collections contain FXI polypeptide to

It will be collected for further virtual search A 乂 treatment, for example, to exclude unnecessary impurities eluted at the beginning or the end of the FXI peptide elution process is known to those skilled in the art. Similarly, the general techniques of performing hydrophobic interaction chromatography are well known, such as pre-equilibrium, dissolution time, washing, and reorganization of hydrophobic interaction chromatography analysis materials. In one embodiment of the present invention, the eluate obtained from stage ii)

Or the liquid prepared by using the eluate of the vii segment (vii) is processed by a method comprising the following steps: ⑴ Adding-or more than-a stabilizer that can increase the degree of FXI polypeptide, the amount is effective Increasing near-term stability, and / or

This step, as well as other processed steps known in the art, may be performed separately or in combination if necessary, and the order of the performed steps is not important. Those skilled in the art can decide how and when to perform these steps. In a specific example of the present invention, a stabilizer capable of increasing the physical and / or chemical stability of the ρχι polypeptide is added to a part of the collection containing fxi. The "physical stability" of the FXI polypeptide used in the present specification The term means that the protein forms biologically inactive and / or insoluble aggregates or polymers due to exposure to temperature_mechanical pressure and / or interaction with destabilizing interfaces or surfaces such as hydrophobic surfaces and interfaces. The possible tendency. The physical stability of the FXI polypeptide present in buffer A can be utilized after exposing the formulations filled in the appropriate valleys | § to different temperatures at different times relative to each period of time. Visual inspection and / or turbidity measurement. Visual inspection of the FXI peptide in the buffer can be performed under a sharply focused light with a dark background. The degree of turbidity of the composition can be listed by visual score, such as a scale of 0 to 3 (a composition showing no turbidity is equivalent to a visual score of 0, and a composition under light Obvious visual turbidity is equivalent to visual scoring 3) When the composition is visible under light, it is classified as physically unstable due to protein aggregation. Alternatively, the turbidity of the composition can be measured by a simple method of turbidity known to those skilled in the art, for example, by measuring the optical density of the solution at a wavelength of 405 nm (0045). The physical stability of the aqueous protein composition can be evaluated using the spectroscopic analysis agent or probe of the eggshell structure. The probe is preferably a small molecule that preferentially binds to a non-natural conformation of the protein. An example of a small molecule spectroscopic probe for eggshell and sigma structure analysis is thioflavin T (Thiolavin). Thioflavin is a fluorescent dye that has been widely used to detect small fibers. When there are small fibers and perhaps other protein shell configurations, the combination of thioflavin and a small fibrin form results in a new maximum excited state at about 405 nm and an enhanced emission at about 482 nm. Unbound thioflavin is essentially non-fluorescent at this wavelength. , Bata Xiaoweizi can be used as a protein structure from natural to non-sky = ~ titanium needle. For example, the "hydrophobic block" probe preferentially breaks away from the hydrophobic block center of the protein. Hydrophobic blocks are generally buried in the tertiary structure of the natural state of protein 35 200529870, but when the protein begins to unravel or inactivate the code ‘Ai is exposed. The spectroscopic analysis probes for these small molecules are aromatic hydrophobic dyes such as anthracene, acridine, phenanthroline, or the like. Other spectroscopic analysis probes are metal-amino acid complexes, such as cobalt metal adducts of hydrophobic amino acids, such as benzyl alanine, leucine, isoleucine, sulfanilate, valine, Or similar. The term "chemical stability" of the FXI polypeptide used in this specification means chemical covalents in the structure of the protein that result in the formation of chemical decomposition products.

This degradation product may have lower bioavailability and / or possibly increased immunogenic properties compared to the natural protein. A variety of chemical decomposition products see the sky ..., the type and nature of the protein shell, and the formation of the environment to which the protein is exposed: reducing chemical decomposition is unlikely to be completely avoided, but in chemical products; During this period, two or two people who are familiar with the art often know well. Most proteins are prone to be hydrolyzed to form free aminoamidine or asparagine residues.

物 η 时间。 Object n process. Other decomposition pathways involve the formation of high molecular weight transitions :::::: or more than two protein molecules through transammonification, the parent interaction of Α—shigua bonds, dimerization f ^ /, buckle ,, Ό 5, leading to the formation of a covalent bond # Qualitative ", Ahern D. J. M. " Probe of Besilox σσ

Ma: mng Presses

Another chemical reaction of chemical decomposition (such as methionine) can be mentioned as J ～ another kind of chemical stability for 刀 野 1 卞: // The solution is in buffer B, when it is in the middle, ~生 f can be evaluated by the hunter in each case; for example, the formation of the chemical decomposition of the sub-test 1 often accelerates with the temperature of 200536 200529870 plus. The content of each individual decomposition product is often determined by separating the decomposition products using various chromatographic techniques (such as sec_hplc and / or RP-HPLC) depending on the molecular size and / or charge. Any reagent present in buffer B that significantly improves the physical and / or chemical stability of the Fxi polypeptide (e.g., turbidity measured over a period of time of 0%) can be used as a stabilizer. Suitable reagents as stabilizers, such as a salt (such as sodium chloride), sugar, alcohol (C ^ C: 8 alcohol), aldol, amino acid (such as glycine, histamine, arginine, Lysine, isoleucine, aspartic acid, tryptophan or threonine), polyethylene glycol (eg WEG400), or one or more than one of them. Any sugar such as a monosaccharide, a disaccharide, or a polysaccharide, or a water-soluble polysaccharide can be used. Aldol is a polyol having a structure of HOCH2- [CH (OH)] n_CH2OH, where η is 1, 2, 3, etc. Non-limiting examples of sugar, alcohol or aldol substances are fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sea sugar, dextran, amylopectin, dextrin, cyclodextrin, soluble Starch, transethyl starch, sodium mannitol_Na, mannitol, sorbitol, inositol, galactitol, eugenyl alcohol, xylitol, arabinitol, glycerol, propanol, alcohol (propyl: Alcohol), propylene glycol and butane ", 3-diol. The above-mentioned sugars, mandarin, and acid alcohols can be used individually or in combination. There is no fixed limit on the amount used, as long as the substance can be dissolved in the liquid The preparation also enhances the physical stability of FXI peptides in solution. In this regard, Yan Qing refers to "Remington's Western Medicine Science and Practice", 19th edition, 1995. In a specific example of the present invention -37 200529870 In a specific example of the present invention, the additive is selected from the group consisting of glycerin (propylene-1,2,3-triol), propylene glycol (propylene- 丨, 2 · diol), propane-, 3-diol, propanol (propanol) and isopropanol (2- One or more stabilizers in the group of propanol). In a specific example of the present invention, one or more selected from the group consisting of glycerin, propylene glycol, and propylene 1,3-diol In a stabilizer.

In a further specific embodiment of the present invention, when the stabilizer is a liquid alcohol or a liquid polyol [for example, glycerin, propylene glycol, propylene-, diol, propanol, or isopropanol], the stabilizer is It is present at a concentration of approximately volume ratio (v / v) to approximately 50% (ν / ν). In another specific example, the liquid alcohol or liquid polyol type stabilizer is present at a degree of about 10% (v / v) to about 50 / 〇 (v / v). In another specific example, the liquid alcohol or liquid poly70 alcohol type stabilizer is present at a concentration of about 10% (v / v) to about 20 / 〇 (ν / ν). In another specific example, the liquid alcohol or liquid polyol type stabilizer is present at a concentration of about 10% (v / v).

In still another specific example, the stabilizer of the liquid alcohol or liquid polyol type is present at a concentration of about 20% (v / v). The stability mentioned above should increase the physical and / or chemical stability of the FXI polypeptide. Any reagent that can significantly increase the physical and / or chemical stability of the polypeptide (e.g., by measuring the turbidity of OD over a period of time) can be used as a stabilizer. The eluate from step (V11) can be used for the preparation of a pharmaceutical composition. This can involve altering the buffer, and / or adjusting the conductivity and / or pH to the pH value and / or other effects to make the eluate acceptable for use in mammals such as humans; making such elution The use of objects in this way 38 200529870 The accepted methods are well known in the art. The eluate may also be stored at, for example, 4QC over a period of 24 hours or more, or at, for example, -800C. In a specific example of the present invention, the method further comprises making the eluate from the stage (vii) or the liquid prepared by using the eluate obtained from the stage (vii) a hydroxyapatite color layer analysis material The step of color layer analysis processing, the color layer analysis comprises: (vii) applying an eluate obtained from the phase (vii) or a liquid prepared using the eluate obtained from the phase (¥ 丨 丨) to the base Apatite chromatographic analysis material; (ix) Unbound substances are washed from the hydroxyapatite chromatographic analysis material with buffer C. This buffer C is suitable for the analysis of non-associated chroma The material bound to the material elutes; and (x) the FXI polypeptide is eluted from the hydroxyapatite chromatographic analysis material with a buffer solution C ', which is suitable for use in step (ix) The material bound to the chromatographic analysis material of the base lin limestone is eluted. The liquid prepared using the eluate obtained from step (vii) can be prepared, for example, before application. In a specific example of the present invention, the Elution from step (vii) or use of elution from phase (vi) The conductivity of the prepared liquid is adjusted to less than about 20 mS / cm by adding water. The pH is adjusted to 5.8 to 9. In a specific example, the pH is adjusted to 6.0. Buffer C and Buffer C can be changed from The desired final FXI polypeptide pharmaceutical composition is chosen based on the viewpoint. Such considerations are well known to those skilled in the art. In a specific example of the present invention, buffer C contains one or more than 39 200529870 In a stabilizing agent, the stability #j ^ This m above increases the physical and / or chemical stability of fxi polymorphism. Presence in the liquid C can significantly improve the physical and / or chemical stability of Fxi peptide (for example, Any reagent used to measure the turbidity of d4G5 over a period of time is used as a stabilizer in buffer solution C and buffer solution C. Youheng Shishi is the stable reliance of a gentle C towel, such as A salt (e.g. sodium carbonate), a sugar, an alcohol (c ρ contains ^ _48%), an aldol, an amino acid (e.g., glycine-vinyl, and amine, arginine, lysine, IA &# amino acid or threonine), polyethylene glycol, aspartic acid, aspartic acid Color) acetate aqueous one percent (e.g. PEG400), or in one or more - seed mixtures thereof. Any sugar such as a monodomain, disaccharide, or multidomain, or water-soluble dextran can be used. Non-limiting examples of the use of sugar, chitosan, or succinyl alcohol — rice flour xylose, maltose, lactose, trehalose, dextran, branched chain powder, dextrin, ring Dextrin, Soluble powder, Ethyl starch, Isomethylcellulose, Mannitol, Sorbitol, Inositol, Galactitol, Scopolitol, Xylitol, Arabitol, Glycerin, Propylene], 2 Glycol (propylene glycol), propylene glycol and butanediol. The above k to sugars, alcohols and acid alcohols can be used individually or in combination. There is no fixed limit on the amount used as long as the substance can be dissolved in the liquid preparation and the physical stability of the peptide in the solution is improved. In this regard, please refer to "Remington's 1 Pharmaceutical Science and Practice", 19th edition, 1995. In a specific embodiment of the invention, buffer C contains one or more than one stabilizer of the polyol type. In a specific example of the present invention, the buffer C contains NaC1. One or more than one selected from the group consisting of glycerin (propane-1,2,3-triol), propylene 200529870 glycol (propane-1,2-diol), propylene glycol, propanol (1-propanol), and A stabilizer of the group isopropyl alcohol propanol) was added to the liquid obtained from (χ). In a specific example of the present invention, one or more than one stabilizer selected from the group consisting of glycerol, propylene glycol and propylene], 3-diol is added. In a specific example of the present invention, propylene glycol is added. In yet another embodiment of the present invention, the stabilizer is about 50%. A volume ratio (ν / ν) to about 50% (ν / ν) exists. In yet another specific example, the stabilizer of the liquid alcohol or liquid polyol type is present at a concentration of from: to about 50% (v / v). In yet another specific example, the liquid alcohol or liquid polyol type hafnium agent is present at a concentration of about 20% (v / v). In yet another specific example, the liquid alcohol or liquid polyol type stabilizer is present at a concentration of about 100% (v / v). Fork ^ Ｍ 上 八 上 夕 趿 冼 prolapsed:

The composition of 爰, 爰 冲 / 夜 C changed during the elution. Typically, the concentration of one or more of the components in the washing buffer is increased or decreased during the elution in the case of the working solution c, or a new species i is added to the buffer. The concentration of ingredients will increase during the elution process. This increase or decrease may occur continuously or in separate steps ::: As is well known in the art ... will be separated from the hydroxyapatite color: material. . The eluted substance elutes, and we usually add salt, such as Κ_ρ〇 = aa to buffer c and increase the salt concentration until at least the bound F

= Wide portions are eluted. Determine which part to close.隼: The substance contains 1 day to collect it for further processing, such as excluding U 41 200529870 the beginning of the peptide elution process or too well known to the skilled person. Same = Unnecessary impurities are familiar with this. About the general technique of hydroxyapatite color separation, you 丨 π station 4 · Jin He ^ pre-equilibration, elution time, rinse, cation parent, color layer Analysis of material reorganization, etc. is well known. In a series of specific examples, the use of any or more than one stabilizer in the purification of any or bath of FXI will increase the physical or chemical properties of FXI by at least more than the control group (that is, affected by Physically and / or chemically stable, at least 1%, 50%, or 100% of the FXl) i, which is also treated but does not contain stabilizers. In another series of specific examples, it is used for purifying coffee: the use of _ kinds of _ kinds of stabilizers in any or all solutions will increase the physical and / or chemical stability of FXI at least more than the control group (ie, subject to The physical and / or chemical stability of FXI), which is also treated without stabilizers, is at least twice, five times, ten times, or twenty times. "FXI activation Wild-type human FXI is generally activated by proteolytic cleavage between ArgMG and Ile37G, and this activation is catalyzed by FXIa, FXIIa, or thrombin. If necessary, the FXI used in the present invention Activation can be achieved using FXIa or Fxna (both from Enzyme Research Laboratories, South Bend, Indiana) or thrombin (produced by Sigma). Please refer to Sun et al. (1999), Journal of Biochemistry, No. Issue 51: pages 36373-36373 and Beglia (2003), Journal of Biochemistry, Issue 24: pages 21744-21750. The use of other proteins to activate FXI polypeptides and especially FXI-related polypeptides is also described in the present invention 42 200529870. The method and composition of this volume of Ming / Han are used for the western therapy of FXI using preparations with different degrees of ρχι activation. In some specific examples, the method and composition have not been activated Programmable F magic polypeptide. In some specific examples, FXI or FXI-related polymorphisms present activated FXI or FXI-related polypeptide: ratio of zymogen ( Amount ratio) is between about 99: 1, such as between about 5:95 to about 95: 5; about 10:90 to about 90:10; about 20:80 to about 80:20; approximately 3070; approximately 70:30; approximately 40:60 to approximately 60:40; and approximately 50:50. In some specific examples, the formulation contains no more than FXIa The total FXI is about 5% on a Mohr basis, preferably no more than about 2.5%, even more preferably no more than about 1%, and most preferably no more than about 0.5% or 0.1%. In some specific cases, Example towels, the formulation contains no more than about 0.001-1.05% of FXIa on a mole basis. In some specific examples, the formulation contains no more than about 0.001 on a mole basis · 〇03% of FXIa. The present invention also relates to FXI-related polypeptides that exhibit a unique ability to be activated relative to wild-type FXI polypeptides, such as FXI-related polypeptides that are more easily activated with Fxna than with thrombin, and vice versa ; A polypeptide that is constitutively activated without proteolytic cleavage; a heterodimer in which one of the monomers cannot be activated by proteolysis (because Mutations or chemical modifications); and the like. Moreover, the present invention also relates to FXI-related polypeptides that are resistant to self-activation, that is, the rate of activation by thrombin (and / or FXIIa) relative to the rate of activation by FXla The ratio is higher than that of wild-type FXI variants. 43 200529870 The method and composition of the present invention can also be treated with treatment 1 or with other inhibitors that can inhibit and / or promote activation. For example, ^ ^ ^ is used in combination with FXI to produce KCH. B) Non-limiting examples of agents include C1 esterase inhibition (CDnb) ... 2 anti-plasmin u2AP), α1 • anti-pancreas Protease (αΙΑΤ), protease connexin π, thrombin III; can promote activation, and anti-leg, FXIIa and thrombin. Prescriptive examples of K include pharmaceutical formulations containing FXI: The present invention encompasses pharmaceutical compositions comprising FXI polypeptides or fish FXI-related polypeptide formulations for pouring large θ / dose, with prevention and / or treatment. The pharmaceutical composition of the formulation according to the present invention includes multiple forms of fxi, such as those having a concentration of 0 · _] 00 mg / ml, which is preferably dissolved in a pharmaceutically acceptable carrier, preferably an aqueous solution In short, a pharmaceutical composition suitable for use in accordance with the present invention is a formulation comprising, preferably in purified form, FXI and / or FXI-related polypeptides with an appropriate: an adjuvant and an appropriate carrier Or diluents. Many aqueous carriers can be used, such as water, buffered water, 0.4% saline, 0.3% glycine, sugar, detergents, salts, buffers, glycerol, preservatives, Protease inhibitors, glycols, and the like. The formulations of the present invention can also be formulated using non-aqueous carriers, such as in the form of gels or microlipid formulations for delivery or targeting the injured site. Micro Lipid formulations are generally described in, for example, U.S. Patent Nos. 4,837,028, 4,501,728, and 4,975,282. The composition can be eliminated by conventional, well-known disinfection techniques. 44 200529870 Sterilized water toxicity. The resulting aqueous solution can be packaged for use or dried without drying, and the freeze-dried solution is combined before application. The composition may contain pharmaceutically acceptable auxiliary substances or adjuvants. Agents, including but not limited to pH adjusting and buffering agents, permeability regulators, preservation agents, stabilizers, surfactants, chelating agents, and the like. Familiar with the art; can use the correct method, and according to the accepted Hands-on, such as with «Ray

Ming: "Medical Science" edited by "Gennaro", ⑽publishing company ,: The method disclosed in Easton, Pennsylvania (1990 car, 艉 + 4 丄, 丄 外 outside +) to deploy the composition of the present invention. In a specific example of the present invention ', the medical music composition comprising the Fxi or fxi-related peptide preparation further comprises a pH f weekly agent and a buffer. In a specific example of the invention, the medical composition comprising a G 3 FXI or FXI-related polypeptide preparation further comprises a permeability modifier. In a specific example of the invention

In the pharmaceutical composition containing FXI or FXI-related polypeptide preparation, a preservative is further included. In a specific example of the present invention, the medicinal product M containing the FXI or FXI-related polypeptide preparation further includes a tincture. In the present invention, the pharmaceutical product containing "FXI or FXI-related polypeptide preparations" in the case of "Honghong" also contains a surfactant. In the specific example of the present invention, the pharmaceutical composition of FXI or FXI-related polypeptide preparations is as follows: A non-limiting example of a chelating agent is also included. Acetate buffer, carbonic acid hydrazone, citrate buffer, glycine methylglycine buffer, group h, U, Glycolic acid buffer, lysine buffer, arginine buffer 45 200529870 agent, salt filling buffer (including, for example, sodium dihydrogenate, sodium hydrogenate or sodium acrylate), TRIS [see ( Hydroxymethyl) aminopane] buffer, bis (transethyl) glycine buffer, tricine buffer, malate buffer, succinate buffer, cis Non-limiting examples of medically acceptable preservatives include phenol, o-butyrate, o-fumarate, tartrate, aspartate, and mixtures thereof. -Methoxycresol, m-methoxycresol, p-methoxycresol, chloromethoxy Phenol, butyl p-hydroxybenzoate, 2-phenoxyethanol, phenylethanol, phenethyl alcohol, chlorobutanol, ethyl mercuric sodium thiosalicylate, 2_bromo_2_nitro_propane_ 1 Glycol, phenylarsinic acid, iminourea, chlorhexidine, sodium dehydroacetate, benzoxamine, chlorphenesine (3-p-chlorophenoxypropane, 2 · diol), benzamidine and its Mixture. In yet another specific example of the present invention, the preservative is present at a concentration of 0.1 mg / ml to 20 mg / ml. In the present invention = and / specific examples, the preservative is present at a concentration of 0.1 mg It exists in a concentration of 5 mg / ml to 5 mg / ml. In another specific example of the present invention, the preservative is present in a concentration of 5 mg / ml to 10 mg / ml. In another specific example of the present invention, The preservative is present at a concentration of 10 mg / ml to 20X mg / ml. The use of the preservative in the composition is well known to the cookbook (artist) (please refer to, for example, "Remington's · Medical Sciences and Practical Skills, 19th edition, 1995. Non-permeability regulators (usually incorporated to make the formulation basically Saki Terajima) Restrictive examples include salts (such as sodium gas), sugars, alcohols (such as c ◦, aldol, amino acids (such as glycine, histidine, arginine, etc.) two libraries such as acid, iso46 200529870 Alanine, aspartic acid, tryptophan, or threo PEG400) and mixtures thereof. One% (for example, guanidine, such as early sugar, disaccharide, or doxopolyglucose can be used or water such as ... …, Non-limiting examples of fermenting or aldol substances are fructose, glucose, mannose, f-S-cellulose, xylose, maltose, lactulose, bath sugar, dextran, branch powder, soluble … 1 ethyl acetate, m-methylcellulose *,: =: sorbase, muscle fermentation, galactitol, glycerol-alcohol (propylene glycol di: sugars and alcohols mentioned above can be used individually or in combination use. The use of quantum has long been a fixed limit, as long as the substance is J M / Fenjie in Rong liquid preparations. In a specific example, the permeability adjusts the heart to about 丨 mg / to about! A concentration of 50 mg / ml is present. In still another aspect of the present invention, the permeability adjusting agent is present at a concentration of about 1 mg / ml to about 50, mg / ml, and the permeability adjusting agent is _. In one aspect, the permeability adjusting agent is present at a concentration of about 1 mg / ml to about 150 g / litre. In yet another embodiment of the present invention, the permeability modifier is NaC1 present at a concentration of about 1 mg / ml to about 50 mg / ml. Non-limiting examples of chelating agents include salts of EDTAmidine and aspartic acid and mixtures thereof. In certain embodiments, the integrator is present at a concentration of about 01 mg / ml to about 5 mg / ml; 0 "mg / ml to about 2 mg / ml; or about 2 mg / ml to about 5 mg / ml. The pharmaceutical composition of the present invention may contain a polypeptide as a medical agent. The polypeptide may exhibit the formation of aggregates during storage in a liquid pharmaceutical composition. The term `` 隹 & & >,. ^ / ,,,, ° ''

Physical interaction, 1 ^ / iU "The effect may cause the formation of oligomers that remain soluble, $ # & 4 * 1" ~ ^ Spoon visible aggregates precipitate out of solution. During the storage of betin The word "_" refers to a liquid pharmaceutical composition or formulation that is not immediately administered to a subject once prepared. Instead, the beans τ 4 are prepared, or later reconstituted into a liquid form, or other dried ^ ^ form suitable for the application subject is packaged and stored. The term "dry type" refers to the after-chilling age,…,…, which are left over (meaning freeze-drying, please refer to, for example, Williams and P lli ^ (1984), Journal of Scientific Toiletry, Issue 38:… page], spray drying [please refer;

= 1 rast⑽ (1991), The Handbook of Money Drying '(5th edition, L ° ngman Technology Publishing Company, Weihe, and V 1 AA in Essex, Yangon): pages 491-676;

Broadhead et al. Π 9Q? I \ 7 / ^ # years) '"Journal of R & D Industry and Medicine", 18: 1169-1206 Bai · 丨 v »a,' and Mmnenthaler et al. (1 994)," Journal of Medical Research, No. u · Yi Shi .. Brother 12_2〇] or by air-drying Fa Qingca and Xin Jiao 1988), (Cryogenic Biology 1 1 4 4 Brother 470 pages; and RoSe_l year ), "Biopharmaceuticals", No. 4: pp. 47-53], the liquid-like M 4, ^ Yetiyue music composition or preparation. During the storage of the liquid pharmaceutical composition, the formation of the peptide may affect the biological activity of the peptide, which may adversely affect the biological activity of the peptide, resulting in a decrease in the medical effect of the composition of Saijo. In addition, the formation of aggregates may cause problems such as the use of an infusion system to block the catheter, film, or pump when using a pharmaceutical composition of the peptide. In a specific example of the present invention, the medicinal composition contains a 48 200529870 amine group @ 文 验 丨 li enough to reduce the shelf life of the composition. "Amino acid test" means an amino acid or a combination of amino acids, in which any given amino acid exists in its free test form or in its salt form. When a combination of amino acids is used, all amino acids may be present in their free form, all in the form of their salts, or some in their free form and others in the form of their salts. In a specific example, the amino acids used to prepare the composition of the present invention are those having a charged side bond, such as arginine, lysine, aspartic acid, and glutamic acid. -A special type of amino acid (for example: glycine, sulfanilic acid, histidine, arginine, lysine, alanine, aspartic acid, tryptophan, threonine or one or More than one mixture) of stereoisomers or a combination of these stereoisomers may be present in the pharmaceutical composition of the present invention 'provided that the particular amino acid can exist in its freely identifiable form or in its salt form. In a specific example, the l-stereoisomer is used. The composition of the present invention can also be formulated with these amino acid analogs. "Amine terrestrial analog" Ioka means a natural amino acid derivative which has the effect of reducing the formation of peptide aggregates during storage of the liquid pharmaceutical composition of the present invention. Suitable arginine analogs include, for example, aminoguanidine, ornithine, and N-monoethyl l-spermine, and suitable methionine analogs include ethyl & amine, butyl thiamine Acid, and suitable cysteine analogs include S-methyl-L-cysteine. As with other amino acids, the amino acid analog is incorporated into the composition in its free form or in its salt form. A microphone compound is also considered to be an amino acid analog in accordance with the present invention. Typically, the amino acids and amino acid analogs are used at a concentration sufficient to prevent or delay protein buildup. 49 200529870 In the specific example: When the factor X1 polypeptide is a methionine residue that contains at least this kind of emulsification, the pharmaceutical formulation contains sulfanilide (or another sulfide-containing Amino acids or amino acid analogs) In order to inhibit: oxidation of thiamin residues to their form of maple. "Inhibition of oxidation"-§ means to minimize the accumulation of oxide species (of methionine) over time. Inhibition of the emulsification effect of Sekisho Izuki Secrets from Moon Girl I will cause the peptide to stay longer in the correct molecular form. Any stereoisomer of methionine (L D or DL isomer) or a combination thereof can be used. Added to inhibit the oxidative effect of methionine, and to emulsify it so that the sub-milled form of methionine is acceptable to the Authority. Code m means that the composition contains: more than about 10% to about 30% of the thiomethionine form. This can generally be achieved by adding a certain amount of methionine, which makes the ratio of the added methionine to the methionine residue range from large & 1: 1 to about 1000: 1 'such as 10: 1 to about 100: 1. Non-limiting examples of stabilizers include high molecular weight polymers or low molecular weight compounds such as polyethylene glycol (e.g., PEG3350), polyethylene (PVA), polyethylene viloxain, tether- / warp-based fibers And its derivatives (including HPC, HPC-SL, JJPC1 τ 4 Tj λ λ > r 〇CL and HPMC), cyclodextrin, sulfur-containing substances

Substances such as monothioglycerol, bismuth thiosulfanyl I, thioacetic acid, and f-based thioethanol, various salts (such as sodium chloride), glycerol, propylene glycol, propylene-u-diol, and propanol (1 · propyl Alcohol) and isopropanol (2-propanol). Restrictive examples of surfactants include detergents, ethoxylated flaxseed oil, polyglycolic acid glycerol, acetated monoglycerol, sorbitan fatty acid esters, polypropylene-polyoxyethylene Segment polymer (such as polyoxy 50 200529870 ethylene polychloroprene copolymers such as Pluronic @ F68, polyoxyethylene polychloroprene copolymers 188 and 407, TritonX-100), polyoxyethylene sorbitan fatty acid esters, Polyoxyethylene and polyethylene derivatives such as alkylated and alkoxylated derivatives ("Tweens", such as ween-2O, Tween-40, Tween-80 and Bnj-3 5) Esters and their ethoxylated derivatives, diglycerides, and their polyoxyethylene derivatives, alcohols, glycerol, exogenous coagulins, and phospholipids (eg, phospholipids, serine, phospholipids) Choline, phospholipids ethanolamine, phospholipids inositol, diphospholipids glycerol, and sphingomyelin), derivatives of stone fats (such as dipalmitinyl phosphatidic acid), and dissolved phospholipids (such as Luru Palmitoyl dissolves o-lipidylserine and ethanolamine, choline, serine or threonine1 -Fluorenyl_sn-glycerol-3—filler ester) and alkoxy- (alkyl_) derivatives derived from solubilizers and phosphatidylcholine choline) and alkoxy_ • (alkyl_) derivatives And the polar end-modifying groups, that is, choline, ethanolamine, discontinol, serine, threonine, glycerol, inositol, and positively charged DQDAC, DOTMA, DCP, BISHOP, solubilizer Amidoserine and Dissolved Lithosalbumin Threonine and Glycerol Phospholipids (such as cerebrolysin), Glycoglycolipids (such as galactopyranoside), Sphingolipids (such as ceramide, nerve Runodeosin), choline dodecyl phosphate, dissolved lecithin in eggs, derivatives of fusidic acid (such as sodium bovine dihydrofusidic acid, etc.), long-chain fatty acids [such as C6-C12 Fatty acids (such as oleic or octanoic acid)] and their salts, fluorenyl carnitine and its derivatives, lysine, arginine, and histamine-derived N α -fluorinated derivatives, lysine and spermine Acid side chain-fluorenated derivative, dipeptide N α-fluorinated derivative-which contains lysine, arginine and histidine with a neutral or Any combination of amino acids, tripeptides, N α -fluorenyl group 51 200529870 biology-it contains any combination of a neutral amino acid and two charged amino acids, DSS (Docusate Sodium, CAS registration number [577-1 1-7], Docusate's CAS registration number [128-49-4], Docusate potassium, CAS registration number [749 1 -09-0], SDS (ten Sodium bisulfate), sodium caprylate, cholic acid and its derivatives, bile acids and their derivatives, a linker with glycine or taurine, ursodeoxycholic acid, monoacid salts, taurocholic acid Acid salt, sodium glycine cholate. N-deca /, N-N, N-dimethyl-3-ammonium-1-propanoic acid, anionic Calcium salt) Monovalent surfactant, Zwitterionic surfactant (such as N-alkyl-N, N-dimethylammonium-1-propanesulfonate, 3-chloroamido-1 -propyldiamine Methylammonium _ 丨 _propanesulfonic acid, cationic surfactants (quaternary ammonium bases such as cetyl bromide trimethylammonium chloride, cetylpyridinium chloride), non-ionic surfactants (eg ten Diyl_々_D_glucopyranoside), and polyoxyethylene polychloride Ethylenediamine derivatives of ethylene copolymers (such as Tetronic's), that is, copolymers derived from the continuous addition of four functional group blocks of ethylene oxide and ethylene oxide to ethylene diamine; Surfactants of groups of derivatives, or mixtures thereof. In a specific example, the pharmaceutical composition contains a surfactant at a concentration of about 0.01 mg / ml to about mg / ml. In a specific example, the pharmaceutical formulation comprises TW⑽-80. In a specific example, the pharmaceutical formulation comprises a polyoxyethylene-polychloropropylene copolymer 188. In a specific example, the pharmaceutical formulation comprises an electrolyte. In a specific example, the pharmaceutical formulation comprises an electrolyte, and in a specific embodiment, the pharmaceutical formulation comprises an electrolyte such as KC1 052 200529870 In the specific example, when Tween 80 is used as a stabilizer, an electrolyte such as NaCI is used, and the concentration is such as 50 mM. In one specific example, agglomeration after storage is avoided. In a series of specific examples, the use of one or more stabilizers in pharmaceutical formulations for use in formulations containing FXI or FXI-related polypeptides results in the physical and / or chemical stability of FXI being greater than that of the control group (ie, FXI is subjected to the same treatment but without stabilizers) and the physical and / or chemical stability is increased by at least or 100%. In another series of specific examples, the use of one or more stabilizers in pharmaceutical formulations containing FXI or FXI-related polymorphic formulations will result in physical and / or chemical stability of FXI compared to the control group ( That is, the physical and / or chemical stability of FXI subjected to the same treatment but without stabilizers is increased at least 2 times, 5 times, 10 times, or 20 times. The following table provides non-limiting examples of suitable formulations. When this formulation contained 1.7 mg / milliliter FXI, no agglomeration was observed after at least one month at ambient temperature. 53 200529870

ID pH buffering agent isotonicity regulator stabilizer antimicrobial preservative A1 8.5 50mM gins- (hydroxymethyl) aminoformamidine (TRIS), pH 8.5 na na na A2 8.5 50mM gins- (hydroxyamidino) amine Nail Na (NaCl na na A3) 8.5 50 mM 50% gins- (hydroxymethyl) aminomethane (TRIS), pH 8.5 na 0.001% w / v Tween80 na A4 8.5 50 mM ana- (hydroxymethyl) Amino methane (TRIS), pH 8.5 na 0.1% w / v Tween80 na A5 8.5 50 mM para- (hydroxymethyl) amino methane (TRIS), pH 8.5 150 mM NaCl 0.1% w / v Tween 80 na A6 8.5 50 mM Ginseng- (hydroxymethyl) aminomethane (TRIS), pH 8.5 16.0mg / ml Glycerol na 0.5w / v0 / 〇A7 8.5 50mM Ginseng- (hydroxymethyl) aminomethane (TRIS), pH8 .5 na 5w / v% via propyl-yS-cyclodextrin na A8 8.5 50mM gins- (hydroxymethyl) aminomethane (TRIS), pH 8.5 na 0.1w / v% human serum albumin na A9 8.5 50mM gins- (hydroxymethyl) aminotrioxane (TRIS), pH 8.5 na 0.5M sucrose na A10 8.5 50mM ana- (hydroxymethyl) aminomethane (TRIS), pH8.5 na 0.3w / v % Poloxamer 188 na All 8.5 50mM para- (hydroxymethyl) aminomethane (TRIS)? PH 8.5 na 18.6mg / ml EDTA

54 200529870 B1 8.0 50 mM gins- (hydroxymethyl) aminomethyl (TRIS), pH 8.0 na na na B2 8.0 50 mM gins- (hydroxymethyl) amino methane (TRIS)? PH 8.0 150mM NaC] na na B3 8.0 50 mM para- (hydroxymethyl) aminomethane (TRIS)? PH8.0 na 0.001% w / v Tween80 na B4 8.0 50mM para- (hydroxymethyl) aminomethane (TRIS)? PH8.0 na 0.1% w / v Tween80 na B5 8.0 50mM p- (hydroxyfluorenyl) aminomethane (TRIS), pH8.0 150mM NaCl 0.1% w / v Tween80 na B6 8.0 50mM p- (hydroxymethyl) aminomethane ( TRIS), pH8.0 16.0mg / ml glycerol na 0.5w / v〇 / 〇 hope B7 8.0 50mM para- (hydroxy fluorenyl) aminomethane (TRIS), pH8.0 na 5w / v ° / 〇 -/ 3-cyclodextrin na B8 8.0 50 mM gins- (hydroxymethyl) aminopane (TRIS), pH 8.0 na 0 · 1 w / v% human serum albumin na B9 8.0 50 mM s- (hydroxy Methyl) aminomethane (TRIS), pH 8.0 na 0.5M sucrose na BIO 8.0 50mM Phenyl- (hydroxymethyl) aminomethane (TRIS), pH 8.0 na 0.3w / v ° / 〇 Poloxamer 188 na Bll 8.0 50mM Phenyl- (hydroxymethyl) aminomethane (TRIS)? PH8.0 na 18.6mg / ml EDTA B12 6.1 Histidine 1.36mg / ml, pH6.1 40mg / ml mannitol 0.3 w / v% Poloxamer 188 6mg / ml g Cl 7.4 50mM salt filling, pH 7.4 na na na C2 7.4 50mM acid salt, pH 7.4 150mM NaCl na na C3 7.4 50mM phosphate, pH7.4 na 0.001% w / v Tween80 na C4 7.4 50mM phytate, pH7.4 na 0.1 ° / 〇w / v Tween80 na

55 200529870 C5 7.4 50 mM phosphate, pH 7.4 150 mM NaCl 0.1% w / v Tween80 no C6 7.4 50 mM phosphate, pH 7.4 16.0 mg / ml glycerol ---- — — na C7 7.4 50 mM phosphate, pH 7. 4 na 5w / v% via propyl- · cyclodextrin C8 7.4 50mM phosphate, pH7.4 na 0.1 w / v% human serum albumin • — upper id —-— C9 7.4 50mM phosphate, pH7.4 na 0.5M sucrose — ^^. Na CIO 7.4 50mM phosphate, pH7.4 na 0.3w / v% Poloxamer 188 na Cll 7.4 50mM phosphate, pH7.4 na 18.6mg / ml EDTA na C12 7.7 50mM phosphate, pH7. 7 14mg / ml propylene glycol na ~~~~~ ----- 5 * 5mg / mi phenol D1 6.0 50mM citrate, pH6.0 na na ------ na D2 6.0 50mM citrate, pH6.0 150 mM NaCl na na D3 6.0 50 mM citrate, pH 6.0 na 0.001% w / v Tween80 na D5 6.0 50 mM citrate, pH 6.0 150 mM NaCl 0.1% w / v Tween80 na D6 6.0 50 mM citrate, pH 6 .0 16.0mg / ml glycerol na 0.5 · ν / ν% D D7 6.0 50mM citrate, pH6.0 na 5 w / v% via propyl- / 3-cyclodextrin na D8 6.0 50mM citric acid Salt, pH6.0 na 0.1w / v% human serum albumin na D9 6.0 50mM citrate, pH6.0 na 0.5M sucrose na DIO 6.0 50mM citrate, pH6.0 na 0.3w / v% Poloxamer 188 na Dll 6.0 50mM citrate, pH6.0 na 18.6mg / ml EDTA E2 10.0 50mM glycine, ρΗΙΟ.Ο 150mMNaCl na na E3 10.0 50mM glycine, ρΗΙΟ.Ο na 0.001% w / v Tween80 na E4 10.0 50mM glycine, ρΗΙΟ.Ο na 0.1% w / v Tween80 na E5 10.0 50mM glycine, ρΗΙΟ.150 150mMNaCl 0.1% w / v Tween80 na E6 10.0 50mM glycine, ρΗΙΟ.Ο 16.0mg / ml glycerol na 0.5w / vo / 〇 酉 分 E7 10.0 50mM glycine, ρΗΙΟ.Ο na 5 w / v% via propyl-yS-cyclo Dextrin na E8 10.0 50 mM glycine, ρ ΙΟ.Ο na 0.1 w / v% human serum albumin na E9 10.0 50 mM glycine, ρ ΙΟ.Ο na 0.5 M sucrose na E10 10.0 50 mM glycine, ρ ΙΟ.Ο na 0.3w / v% Poloxamer 188 na FI 7.0 50mM phosphate, pH7.0 na na na F2 7.0 50mM phosphate, pH7.0 150mM NaCl na na F3 7.0 50mM phosphate, pH7.0 na 0.001% w / v Tween80 na F5 7.0 50mM phosphate, pH7.0 150mM NaCl 0.1% w / v Tween80 na F6 7.0 50mM phosphate, pH7.0 16.0mg / ml glycerol na 0.5w / v% S points F7 7.0 50mM phosphate pH7.0 na 5 w / v% hydroxypropyl- / 3-cyclodextrin na F8 7.0 50 mM phosphate, pH 7.0 na 0.1 w / v% human serum albumin na F9 7.0 50 mM phosphate, pH 7. 0 na 〇.5M sucrose na F10 7.0 50mM phosphate, pH7.0 na 0.3w / v% Poloxamer 188 na

56 200529870

G12 50mM Glycinyl Glycine ρΗ7 · 6 ρΗ3.0 150mM NaCl na 0.05mg / ml Tween20 0.21mg / mi phenol H2 3.0 50mM citrate na na H4 3.0 50mM citrate ρΗ3.0 0.1% w / v Tween80 na H5 3.0 50 mM citrate pH 3.0 0 mM NaCl 0.1% w / v Tween80 na H6 3.0 50 mM citrate pH 3.0 16.0 mg / ml glycerol na 0.5 w / v 0 / 〇 fen H7 3.0 50 mM citric acid Salt ρΗ3.0 na 5w / v% via propyl- / 3-cyclodextrin H8 3.0 50mM citrate ρΗ3.00 na 0.1w / v% human serum albumin H9 3.0 50mM citrate ρΗ3.0 0.5M sucrose na H10 3.0 50 mM citrate, ρΗ3.0 na 0.3w / v% Poloxamer 188 na H12 7.3 5.7 mM phosphate, ρΗ7.3 · 137 mM NaCl 5.4 mM KC1 In a non-limiting specific example, allow after freeze-drying Suitable formulations for recovery of active FXI include: FXI concentration: 0.2 mg / ml buffer: 20 mM buffer (histidine or TRIS) (pH 5.5, 6.5 or 7.4), 25 mg / ml mannitol (filler), 2.5 mg / ml NaCl (filler), and 0.01% Tween 80. Preferably, the pharmaceutical composition is administered parenterally, i.e. intravenously, subcutaneously, or intramuscularly; most preferably, it is intravenously. It can also be applied by continuous or pulsed infusion. I should be aware that any effective method of administering FXI polypeptides can be applied, including, for example, mucosal or inhaled administration methods. Local delivery of the formulations of the present invention, such as topical application, can be achieved by, for example, spraying, infusion, double balloon catheters, stents, vascular grafts or 57 200529870: hydrogels for covering balloon balloons, Incorporate gauze materials or other well-established methods. 2. The pharmaceutical composition of the present invention can be applied in various dosage forms, such as * suspension, emulsion, micro-emulsion, multiple emulsion ... night, howl, paste, 'wound cream, ointment, tablet, menstrual pack Covered tablet ointment lotion, Sheng capsule (such as hard gelatin capsules, soft Ming Sheng :, Er Run II :: Gen, agent, eye ointment, eye lotion, vaginal support, vaginal II ,: Daolezhuang injection In situ transformation solution (such as in situ gels: = sedimentation effect, in situ sinks or in situ crystals used in fluids or implants. '/ Huo Lun The pharmaceutical composition of the present invention You can also combine or combine or link = covalent, hydrophobic or electrostatic interactions) to a drug load Availability, increase solubility, decrease by-products 1: reach mature f chronic treatments well known to the artisan, and / or increase patients: mf. Vehicles, animal transport systems and advanced drug transport systems include but are limited to: Polymers such as cellulose and its derivatives, others such as dextran Its derivatives), poly (vinyl alcohol) = polymers of vinegar and methacrylic acid vinegar, polylactic acid and polyacetic acid and diethylene glycol, and co-conductors, polyethylene glycol, and proteins (such as white Protein) knees (eg, thermogelation systems, such as block copolymerization as known to those skilled in the art: systems), micelles, liposomes, microspheres, nanocrusts, liquid crystals, and others: politics, familiarity Lipids_Detailed by the artist of water system # PHASE AND ITS DISPERSION58 200529870 substances, polymer micelles, multiple emulsions (customization and self-emulsification), cyclodextrin and its derivatives and dendrimers polymer. The selenium composition containing factor μ polymorphs prepared by the method according to the present invention is suitable for pulmonary administration ^ ^ ^, U 骽, + solid, powder and bath formulations, the pulmonary administration system uses Examples are known devices such as dosimetric aspiration, dry powder inhalers or nebulizers. Jin Youdu-Heat study by the artist: 1 = Two of the factor χι polypeptide prepared by the method of the invention = used for controlled release, continuous release, extended release use4, and then Preparations for the fun transport system. Using the method according to the present invention ^ ^ ^ ^ prepared medicine containing factor XI polypeptide (two preparations ek, release and sustained release system ',,,, and the first sentence lead to a decrease in the number of applications ff W 1 & ° Midnight Eve), the application master for the heat treatment of this technique to control the release of "w those who have been known, such as for the subcutaneous application of the classics do make restrictions Γ continuous release The system of the present invention is not intended to be used for the controlled release and sustained release system and group of spheres and nano-grains of the present invention. The liquid crystal, polymer micelles, micro-use Factor XI peptides prepared by containing the pharmaceutical composition according to the present invention > P 4 spoon, including: but not limited to, crystal crystallization, primary method, and auxiliary crystallization Method, Shen-dispersion method, age-assisted precipitation method, emulsification method, political process, pressure homogenization method, chemical method, seismic repair method, spray-drying method, microcapsule 'breaking I method, phase separation remote extrusion Method, solvent evaporation method, compaction method that produces microspheres by over-exposure and wide-ranging.-General reference The literature is found in "Medicine 59 200529870 Release Handbook" (ed. Wlse, D.L., ed. __Ekker 5th edition 2000) and "Pharmaceutical and Medical Sciences", 9th White Matter Preparations and Ships (MacNa E • Egg

Called as company publishing, _year). ···, flat, New York MarceI Parenteral administration can be performed subcutaneously, intramuscularly, or intravenously using a syringe (for example, within: ^.) Alternatively, non-administrative administration can be performed using infusion: (Night = two cylinders)-the composition containing the factor χι = =, or in the form of a suspension liquid prepared by the method according to the present invention is also applied in the form of a liquid mixture. Another item, Deng Zhe said, “Choose a spray on the nose or lungs containing a factor XI m. The pharmaceutical composition of the u-magic polypeptide prepared according to the method of the present invention can be applied, for example, using ^, instead, it is applicable. In transdermal patches) or by transmucosal (for example, frequent) application methods (into the iontophoresis ::: clear-in a specific example, according to the present invention and more than 3 years: The product is stable in the use period of more than 6 weeks and the flat storage period. In another aspect of the present invention, the preparation of apricots includes Yin + XI 22, which is expected according to the method of the present invention, and companions of more than 3 years. : The shelf life of the oxalox composition is stable for more than 4 weeks when the cattle are used. Therefore, in another specific example, the method according to the present invention includes a factor χι_ 万 沄 lifetime and more than 2 vehicles. ~ The product is stable for more than 4 weeks [4 2 years of shelf life. There is still a specific method prepared in the present invention which contains the factor: Λ 'according to the formula of the present invention The pharmaceutical composition is stable over a period of more than 2 weeks of 200529870 and a shelf life of more than 2 years. In some specific examples, the polypeptide formulation has a p Η value from about 4.0 to about 1 0.0. In the specific examples, the FXI polypeptide formulation has a pH from about 4.0 to about 8 D from, · Value. In some specific examples, the fxj polypeptide formulation has a ρ value from about 4 • to about 7. 0. In some specific examples, the FXI polypeptide formulation has a Yamaha / A gate that has a range from about 4.0. To a pH value of about 6.5. In some specific examples & FXl polypeptide formulation has a pH value from about 4.0 to about 6.0. Ligan & in some specific examples the FXI polypeptide formulation has about 6. 5 or Below 6.5, for example ^^ ι ^ 1 歹 J such as between about 5.0 and about 6.5; such as between about 5.5 and 6.5?

Fxi medical administration method ... The present invention provides a method for preventing and treating bleeding using FXI. Bleeding is the extravasation of gold fluid from any component of the circulatory system. Infrequent incidents of business include undesired 'uncontrolled or frequent excess bleeding associated with surgery, trauma, or other forms of tissue damage, as well as unwanted bleeding in subjects with bleeding disorders. The occasional event of hemorrhage can be born in a subject with a substantially normal coagulation system, which has been experienced (temporarily white blood vessels, and subjects with congenital or acquired coagulation or bleeding disorders. In patients with platelet dysfunction In subjects, the bleeding tends to be caused by hemophilia 'because its hemostatic system is lacking or has normal essential coagulation "compounds" (such as platelets or von Willebrand factor proteins in extensive hemorrhagic conditions) Corruption, for example, on a subject associated with surgery or a large number of traumas, the normal stop 61 200529870 mechanics ^ was overwhelmed by the need for immediate hemostasis, and despite its normal hemostatic mechanism before surgery or pretrauma), H =: has been produced. Such subjects are also often multiple transfusion recipients who develop an egg-blood disease due to bleeding and lysis (lowering platelet count). The outflow of blood occurs in organs such as the brain, inner ear area, and eyes; ^: :: treats the restricted area of hemostasis and thus achieves a satisfactory hemostatic effect ::

=. Similar problems may occur during the biopsy of various organs (liver, lung, tumor tissue, gastrointestinal tract), and during laparotomy and post-bone prostate eradication. The same for these conditions is that it is difficult to provide hemostasis through hemostasis techniques (sutures, clips, etc.), and the condition (such as hemorrhagic gastritis and a large number of uterine ash) is also difficult. Out: It may also occur in a subject undergoing anticoagulant therapy, where the subject's treatment introduces an impaired hemostatic effect; these bleedings are often acute and large. Anticoagulant therapy is often administered to prevent blood clots and diseases. This type of alpha therapy may include heparin, other forms of peptone, or a form of vitamin kappa antagonist, a coagulation protein inhibitor, and A ', Jin Inhibitors of pelvic U platelet aggregation, such as antibodies or other inhibitors of supine activity. Bleeding can also be caused by a so-called thrombolytic therapy: the treatment includes the use of an antiplatelet agent (such as acetic acid), an anti-blood d (such as heparin), and a fibrinolytic agent (such as tissue plasminogen) 'Tongue' tPA) for combined treatment. Incidents of bleeding also intentionally include but are not limited to those occurring in acute joint hematoma (bleeding at the joint site), chronic bleeding arthropathy, hematoma, (eg, muscular, retroperitoneal, sublingual 62 200529870 spear) Unrelated surgery or trauma in subjects with bleeding from other tissues, hematuria (renal channel bleeding), brain bleeding, surgery (eg liver resection), dental removal, and gastrointestinal bleeding (eg UGI bleeding) Control and excessive bleeding. This occasional hemorrhagic event can be related to inhibitors of factor m; hemophilia A; hemophilia and inhibitors; hemophilia B; lack of factor vπ; lack of factor XI; thrombocytopenia, lack of von wiuebrand factor (Von Wdlebrand's disease); severe tissue destruction, severe trauma, surgery, laparotomy; acidosis, hemodilution, wasting coagulopathy, excessive fibrinolysis, hypothermia, hemorrhagic gastritis, biopsy, anticoagulation Treatment, upper gastrointestinal bleeding, Tao Guang or stem cell transplantation. Contingent hemorrhagic events can be massive uterine bleeding; those that occur in organs and have a limited possibility of mechanically stopping bleeding; those that occur in the brain; those that occur in the area; or those that occur in the eye. The lower number or activity of platelets refers to the number of blood platelets (thrombocytes) present in the subject's plasma and the biological and coagulation-related activities of platelets. The lower number may seem different because, for example, the blood in the spleen is increased and the production of platelets is reduced. The edges of the plate are larger than the normal platelet portion. For example, thrombocytopenia is defined as the number of platelets per microliter to the heart; the upper limit of the number of normal platelets-generally =: per micrometer ... . " 5., _ platelets: is a known method. Two workers who are skilled in this art include but are not limited to thrombocytopenia, m group package includes but is not limited to blood small = 隼, disease. Platelets Aspects of activity include aggregation, adhesion, and coagulation activity. Reduced activity may be due to, for example, abnormal glycoproteins, incorrect membrane-cytoskeleton interactions, abnormal platelet particles, abnormal platelet coagulation activity, and signaling Transmission and secretion are abnormal. Platelet activity, including aggregation, adhesion, and coagulation activity, are measured using standard methods known to those skilled in the art. Please refer to Platelets, Practical Methods, edited by SPWatson and KSAuthi: Clinical Perspectives ”(KJ clemets〇n), No. 15: pp. 299-3 18, 1996. Published by Oxford Press; William's Hematology, 6th edition, edited by Beutler, Lichtman, ⑽er, Kipps * sewn 'Published by McGraw in 2001. Symptoms caused by low platelet activity include, but are not limited to, ⑴ buckle coffee plus ⑽bathenis, Bernard_suouHer syndrome Storage investigations of disease, anticoagulant therapy, and thrombolytic therapy. ~ In the context of the present invention, treatment includes the prevention of bleeding including, but not limited to, the prevention of unanticipated bleeding. This is expected to occur during or immediately after the surgical procedure, and adjustments have occurred, such as those occurring in the injury, to suppress or minimize bleeding. The birth was born in a recognized site Or not in a preparation that has not yet been tested and included in the therapeutic application package ™ continued to be included ^ In some specific examples, a normal human patient, that is, a person with congenital FXI deficiency ' A polypeptide of " = may be administered in an amount equal to approximately 5 mg to approximately 5.0 mg, wild 31 FXI polypeptide per day or per kilogram of Ping Jin # 丄 锅 毛 Event, such as for one bit 70 about 1 milligram to about _ milligram, or for example about 64 200529870 14 grams to about 175 grams per day or every bleeding incident as a loading and maintenance dose, the administered dose depends on the subject's weight and condition And the condition Severity depends. In some specific cases, blood has been obtained from a patient in need of FXI peptide treatment and a test (before FXI peptide administration) is performed to evaluate one or more of the following ... (I) the plasma concentration of FXI; (ii) the ratio of activated FXI: FXI zymogen; and / or (iii) the concentration of FXI that needs to be added exogenously to restore effective coagulation, based on the results of the test, using a pre-measurement Appropriate amounts of FXI polypeptides are administered by biopsy. Any appropriate test can be predicted using, including, for example, ELISA or colloid-based methods. Appropriate calibration standards are used to allow comparison of the measured concentration to the normal concentration in human plasma (30 nM). Typically, I would like to supplement the fxi concentration to at least about 5 nM, preferably at least about ιηηM, more preferably at least about 15 nM, still more preferably at least about 20 nM, and most preferably at least about 30 nM FXI. When we use a peptide associated with FXI to supplement the fxj activity in a patient ', the FXI associated polypeptide will exhibit at least one special concentration of FXI biological activity and blood. The goal of this treatment is to provide a pre-determined wild-type FXI Equal amount of biologically active amount (ie "effective fxi plasma concentration"). In certain specific examples, the invention encompasses medically administering FXI polypeptides to patients having a plasma concentration below about 3 nM; 5 nM or 1 OnM. Combined Therapy 65 200529870 The present invention also encompasses methods and compositions that provide a combined therapy in which the FXI polypeptide is administered with a non-Factor VII / Factor VIIA coagulant. Suitable non-Factor VII / Factor VIIA coagulants include, but are not limited to, Factor XIII (see, for example, WO 01/85 198), inhibitors of tissue factor pathway inhibitors (TFPI inhibitors) (see, for example, WO 01/85199); Factor IX (please refer to eg WO 02/062376); fibrinolytic inhibitor (TAFI) which can be activated by thrombin (please refer to eg PCT / DK02 / 00734); PAI-1 (please refer to eg PCT / DK02 / 00735 ); Factor V (please refer to eg PCT / DK02 / 00736); protein C inhibitor (please refer to eg PCT / DK02 / 00737); thrombomodulin (please refer to eg PCT / DK02 / 00738); protein S inhibitor (please (Refer to PCT / DK02 / 00739); tissue plasminogen activator inhibitor (please refer to PCT / DK02 / 00740); a2-plasmin (please refer to PCT / DK02 / 00741); Aprotinin (please refer to eg PCT / DK02 / 00742); tranexamic acid (please refer to eg PCT / DK02 / 0075 1); ε-aminocaproic acid (please refer to eg PCT / DK02 / 00752); prothrombin Thrombin; Factor VII; Factor X and Fibrinogen. The following is a list of specific examples of the present invention: Specific Example 1: A method of treating an incidental bleeding event, the method comprising administering to a patient in need of the treatment a preparation containing Factor XI (FXI) or a polypeptide associated with FXI The amount is effective for this treatment. 66 200529870 Specific Example 2: The method as defined in Specific Example 1, wherein the administration causes a decrease in the coagulation time of the patient. Specific example 3: The method as defined in specific example 1 or specific example 2, wherein the administration improves hemostasis of the patient. Specific Example 4: The method as defined in Specific Examples 1 to 3, wherein the administration causes an increase in the blood clot dissolution time of the patient. Specific Example 5: The method as defined in Specific Examples 1 to 4, wherein the administration causes an increase in the blood clot strength of the patient. Specific example 6: The method as defined in specific examples 1 to 5, wherein the application causes the patient's overall blood clot quality (OCQ) to increase. 0 Specific example 7: The method as defined in specific examples 1 to 6, in which After administration, the patient showed an effective FXI plasma concentration of at least about 5 nM. Specific example 8: The method as defined in specific example 7, wherein the effective FXI plasma concentration is at least about 1 OnM. Specific Example 9 The method as defined in Specific Example 8 wherein the effective FXI plasma concentration is at least about 30 nM. Specific Example 10: The method as defined in any one of Specific Examples 1 to 9, wherein the FXI and the FXI-associated polypeptide comprise the sequence of SEQ ID NO: 1 or it retains at least one biological activity associated with FXI Fragment. Specific Example 11: The method as defined in any one of Specific Examples 1 to 9, wherein the FXI and the FXI-associated polypeptide 67 200529870 Specific Example 12: a sequence comprising SEQ ID NO: 2 or retaining at least one type of FXI Related biologically active fragments. The method as defined by any of the specific examples 1 to 11 in which the patient does not suffer from a congenital FXI deficiency. Specific Example Specific Example 13: The method as defined in any one of Specific Examples i to 12, wherein the bleeding incident is subordinate to a condition selected from the group consisting of: surgery, dental procedure, trauma, or hemodilution. 14: The method defined by any one of the specific examples ", " 3 which also includes before the administration ... (a) taking a sample from a patient; (13) measuring at least one of the following: FXn agronomy, ratio of FXI: FXIa, the amount of exogenous FXI necessary to restore coagulation; and ((〇 According to the result of (b) ', determine the amount of fxi that is effective for treatment. Specific examples 15: a A method of treating an incidental hemorrhagic event, the method comprising administering to the patient a first amount of a formulation comprising an FXI polymorph and (ii) a second amount of a formulation comprising a non-Factor VII / Factor Vila coagulant, wherein the first Combining one amount with a second amount is effective for such treatment. 16: The method as defined in specific example 15, wherein the non-factor VII / factor Vila coagulant is selected from the group consisting of: factor III, tissue factor pathway inhibitor (TFPI) inhibitors; Factor lx; Thrombin may be active 68 200529870 Specific examples Specific examples Specific examples Specific examples Specific examples Specific examples Specific examples 17 ·· Specific example 25:, Dairy protein dissolution inhibitor (TAFI); Fiber Lysozyme Inhibitors] (P AI)); factor V; protein c / medium preparation; protein S inhibitors; and tissue plasminogen activator (tPA) inhibitors. As defined in Specific Example 15 or Specific Example 16 Fang ', Zhonghai Zhiyong caused the patient's coagulation time to decrease 0 18: The method as defined in specific examples 15 to 17, wherein the administration enhances the patient's hemostasis. 19: as in specific examples 15 to 18 A method as defined, wherein the administration causes an increase in blood clot dissolution time in the patient. 20 · A method as defined in specific examples 15 to 19, wherein remote application causes an increase in the blood clot strength of the patient. 21: as in specific example 15 to The method as defined in 20, wherein the administration causes the patient's overall blood clot quality (0CQ) to strengthen the mouth. 22: As defined in specific examples 15 to 21, after the administration, 'the patient shows at least about 5nM effective FXI plasma concentration. 23: The method as defined in specific example 22, wherein the effective FXI blood destruction concentration is at least about 10 nM. 24: the method as defined in specific example 23, wherein the effective FXI is as slurry The concentration is at least about 30 nM. Example 1. Method 69 200529870 as defined in any one of 5 to 14, wherein the FXI and the polymorphism associated with FXI comprise a sequence of SEQ m N0: 1, or it is retained until 乂 'an organism associated with FXI Active fragment. Specific Example 2 6 Specific Example 27: Specific Example 28: Specific Example 29: The method as defined in any one of Specific Examples 15 to 24, wherein the ™ and the polymorphisms associated with FXI include SEQ ID NO : The sequence of 2, or at least one of its

FXI related biologically active fragment. Any one of the examples 15 to 26, where the patient does not have congenital FXI

A method as defined by any of the specific examples 15 to 27, wherein the incidental bleeding event is subordinate to a condition selected from the group consisting of: surgery, dental procedures, trauma, or hemodilution. The method as defined in any one of the specific examples ^ 28, which further comprises prior to the administration: 取得 obtaining a sample from a patient; ⑻ measuring at least one of the following: ™ concentration, ratio of coffee, necessary to restore coagulation The amount of exogenous, FXI, and ⑷ based on the result of (b) * 'Measure the amount of FXI effective for treatment 0 Specific Example 30: Specific Example 3 1: The method defined by the specific example, where the party does not A clotting agent for factor 3 VJI / factor VHa. A kind of medicinal preparation containing isolated recombination; FXI polypeptide and (i) a carrier or excipient which can be used as a medicine in a redundant manner 70 200529870 Specific Example 32: 物. FXI peptides are used to treat incidental bleeding episodes. Specific example 33: According to specific example ^ Use of ancient example 32 ', wherein the hemorrhagic incident is subordinate to the conditions of the following groups: surgery, oblique and obstetric order, trauma or hemodilution. Specific example 34: According to the use of specific% 32 + "32 or specific example 33, wherein δH haemorrhagic 値 ^. Incidental events are not treated with a factor VII / factor Vila coagulant. Specific Example 35: FXI polypeptide is used to enhance the hemostatic use of patients in 1 and 2 hospitals. Specific Example 36: The use of a polypeptide for increasing the dissolution time of blood clots of a patient required by Rose Mail 9. Specific Example 37: Use of FXI polypeptide for increasing the blood clot strength of a desired patient. Specific example 38 ········································································ Specific Example 39: Use of FXI peptides to reduce the clotting time of a patient in need thereof. Specific Example 40: According to the use of any one of Specific Examples 32 to 39, in which the & FXI in the patient's body is increased to at least about 5 nM. Specific example 41. «Use of specific example 40, wherein the effective ρ2005 20057070 plasma concentration is increased to J main · > about 10 nM. Specific example 42: The use according to specific example 41, wherein the effective FXI plasma concentration is increased to ~ 30 nM of the main 5. Specific Example 43: The use according to any one of Specific Examples 32 to J 42, wherein the patient is not treated with a blood clotting agent due to early childhood VII / factor Vila. Specific Example 44: The use of FXI polypeptides for the preparation, preparation, and application of Pharmacotherapy for the treatment of occasional bleeding incidents. Specific Example 45 · The use according to Specific Example 44, the use of H, wherein the bleeding incident is subordinate to a condition selected from the group consisting of surgery, dental procedure, trauma, or hemodilution. Specific example 46: According to specific example 44 and the use of specific example 45 in addition to 1 H A, wherein the bleeding incident is not treated with a factor vπ / factor Vila coagulant. Specific Example 47: ™ is more useful for the preparation of a pharmaceutical formulation that improves hemostasis of a patient in need thereof. Specific example 48: Use of FXI peptides for the preparation of a pharmaceutical formulation that increases the dissolution time of the blood clot required by a patient. Specific Example 49: Use of FXI peptides for the preparation of a pharmaceutical formulation that increases the blood clot strength of a patient in need thereof. Specific Example 50: Use of FXI polypeptide for preparing a pharmaceutical formulation that increases the overall blood clot quality (OCQ) of a patient in need thereof. Specific Example 51: Use of FXI polypeptide for preparing a pharmaceutical formulation that reduces the coagulation time of a patient in need thereof. 72 200529870 Specific Examples Specific Examples Specific Examples Specific Examples Specific Examples Specific Examples 52 ·· According to the use of any one of specific examples 47 to 51, wherein the effective FXi plasma concentration in the patient is increased to at least about 5nM. 53. Use according to specific example 52, wherein the effective FXI plasma concentration is increased to at least about 10 nM. 54: The use according to specific example 53, wherein the effective positive plasma concentration is increased to at least about 30 nM. 55. The use according to any one of specific examples 44 to 54, wherein the patient is not treated with a factor VII / factor VIIa coagulation agent. 56: According to the use of any one of the specific examples 32 to 55,

The patients to be treated did not suffer from congenital FXI deficiency. 57: The use according to any one of specific examples 32 to 56, wherein the FXI and the FXI-associated polypeptide comprise the sequence of SEQ ID NO ·· 1, or a fragment that retains at least one of the biological activities associated with FXI . 57: The use according to any one of specific examples 32 to 56, wherein the FXI and the FXI-associated polypeptide comprise a sequence of SEQ ID NO. · 2, or a fragment that retains at least z of biological activities associated with FXI. 5δ: The use according to any one of specific examples 32 to 57, wherein the FXI polymorphism is administered in combination with a non-factor γη / factor coagulant. 73 200529870 Specific example 59: The use according to specific example 58, wherein the non-factor VII / factor Vila coagulant is selected from the following group: factor π, tissue factor pathway inhibitor (TFpi) inhibitor; factor IX; thrombin may Activated Fibrinolysis Inhibitor (TAFI); Plasminogen Activator Inhibitor-UPAM); Factor V; Protein c Inhibitor. Protein S Inhibitor; and Tissue Plasminogen Activator (tPA) Inhibitor. Specific example 60: specific example 61: specific example 62: specific example 63: specific example 64: specific example 65: specific example 66:

A «Method for purifying FXI polypeptide from biological substance f 'This method includes analyzing the material in a cation exchange color layer by continuous color analysis, a color layer analysis material that interacts with K, and hydroxyphosphorus Graystone color analysis is done on the material. Polypeptide FXI Polymorphism

The method according to specific example 60 or specific example 61, wherein the ™ polypeptide is a human ρχι. : According to the method of specific example 60 or specific example 6i ^ The polymorphic system is a dimer. According to the method of specific example 63 ... the ρχ1 peptide is a dimer of the human subunit. : According to any one of specific examples 60 to 64, = the biological substance is a biological liquid. The method according to specific example 65, wherein the Sheng 4 74 200529870 body is a supernatant of mammalian cells. Specific example 67: The method according to specific example 66, wherein the biological fluid is a supernatant of CHO cells. Specific example 68: The method according to any one of specific examples 60 to 67, wherein the method includes the following steps: (a) applying the biological substance including the FXI polypeptide to a first cation exchange color layer analysis material, the color layer The analysis includes: (i) applying the biological substance to the first cation exchange chromatography analysis material; (ii) using buffer A to elute unbound substances from the first cation exchange chromatography analysis material, Buffer A is suitable for eluting substances that fail to bind to the first cation exchange chromatography material; and (Hi) Use buffer A to remove unbound substances from the first cation exchange chromatography material. Elution, buffer A 'is suitable for eluting substances that fail to bind to the first cation exchange chromatographic analysis material; and (iv) by buffer a, the FXI polypeptide is removed from the first cation Buffer A ”is suitable for eluting FXI peptides from the first cation exchange chromatographic analysis material. 75 200529870 Specific examples Specific examples Specific examples (b ) Causing the eluate from step (iv) or the liquid prepared using the eluate from step (iv) to be processed by tomography, which uses a hydrophobic interaction of the tomographic analysis material, the tomography includes Ο) Apply the eluate from step (iv) or the liquid prepared in step (lv) to the hydrophobic interaction color layer to analyze the material; (VI) use buffer B to remove unbound material from The hydrophobic parent interaction color layer analysis material elutes, and the buffer solution A is suitable for eluting a substance that fails to bind with the hydrophobic interaction color analysis material; and (νϋ) uses a buffer solution B , The FXI polypeptide is eluted from the hydrophobic interaction color analysis material, and the buffer B is suitable for eluting the FXI polypeptide from the hydrophobic parent interaction color analysis material. According to the specific implementation The method of Example 68, wherein the buffer a comprises one or more stabilizers capable of increasing the stability of the FXI polypeptide. 70. The method according to the specific example 69, wherein the buffer a comprises a stabilizer, and the stabilizer is A sugar, alcohol, or alcohol The method according to the specific example 70, wherein the buffer A contains a stabilizer, the stabilizer is a sugar, 76 200529870 specific example specific example specific example specific example specific example specific example specific example specific example c4-c8 alcohol or jun Alcohol. 72: The method according to the specific example 71, wherein the buffer a contains a stabilizer, the stabilizer is a polyhydric alcohol. 73: The method according to the specific example 72, wherein the buffer a contains a member selected from glycerol, propylene glycol, propylene _ 丨, 3_ diol, propanol and isopropanol stabilizers.

74: The method according to specific example 73, wherein buffer A comprises a stabilizer selected from the group consisting of glycerin, propylene glycol, and propylene glycol. 75: The method according to any one of the specific examples 72 to 74, wherein the stabilizer is present at a concentration of about 5% (v / v) to about 50% (v / v). 76. The method according to the specific example 75, wherein the stabilizer is present at a concentration of about 10% (ν / ν) to about 50% (v / v). 77: The method according to the specific example 76, wherein the stabilizer is present at a concentration of about 10% (Wv) to about 20% (v / v). 78: The method according to the specific example 77, wherein the stabilizer is present at a concentration of about 10% (v / v). 79: The method according to the specific example 78, wherein the stabilizer is present at a concentration of about 20% (v / v). 80: The method according to any one of the specific examples 68 to 79, 77 200529870, wherein the pH of the buffer A is between about 6.5 and about 9. Specific examples Specific examples Specific examples Specific examples Specific examples Specific examples Specific examples Specific examples 81: The method according to specific example 80, wherein the pH of the buffer solution A is between about 7 and about 9. 82: The method according to the specific example 81, wherein the pH of the buffer A is about 8. 83: according to any one of specific examples 6 8 to 8 2, wherein the conductivity of the buffer solution A is less than about 50 mS / cm 〇 84: method according to any one of specific examples 60 to 83, wherein The hydrophobic interaction color layer analysis material uses butyl or phenyl as the ligand. 85: The method according to the specific example 84, wherein the hydrophobic interaction color analysis material is a phenyl agarose high performance and high substitution material. 86 ·· Method according to the specific example 84 Interactive color layer analysis material High performance replaces the material. 87: Gen County

Its _ edge hydrophobicity is high in butyl agarose

Wherein, the pH of the buffer solution B is between about 8 and about 9. According to the method of specific example 87, the pH of B is about 8. Among them, the buffer solution 89 ·· According to the method of any of the specific examples 68 to 88, 78 200529870 specific example 90 specific example 91 specific example 92: specific example 93: specific example 94: specific example 95: specific example 96: The conductivity of the buffer B is greater than 50 Qin. The method according to the specific example 89, wherein the conductivity of the buffer B is greater than 70 mS / cm. According to the method of any one of specific examples 68 to 90, the second method uses the following method to treat the eluate obtained from stage (vii) or the liquid prepared by using the eluate obtained from stage ii), which The steps include: (1) adding one or more stabilizers capable of increasing the stability of the F polypeptide, the addition amount is effective to significantly improve its stability, and / or (2) adjusting the phase (Wi) The pH of the eluate, or liquid prepared using the eluate from stage (vii), to a pH between about 7 and about 9. The method according to the specific embodiment 91, wherein the stabilizer used in the step is a sugar, an alcohol or an acid: alcohol. The method according to the specific example 92, wherein the stabilizer used in step (1) is a sugar, a CfC8 alcohol or an aldol. The method according to the specific example 93, wherein the stabilizer used in the step is a polyhydric alcohol. The method according to specific example 94, wherein the stabilizer used in step (i) is selected from the group consisting of glycerin, propylene glycol, propylene β 1,3-diol, propanol, and isopropanol. The method according to specific example 95, wherein the stabilizer used in step (1) is selected from the group consisting of glycerin, propylene glycol, and propylene_1 3_79 200529870 diol. Specific example 97: According to any one of specific examples 94 to 96 + 丄% <ten thousand, wherein the stabilizer for step (1) is added to about 5% (ν / ν) to about 50% (ν / v). Specific example 98: The method according to specific example 97, wherein the stabilizer for step (1) is added to a concentration of about 10% (ν / ν) to about 50% (ν / ν). Specific Example 99: The method according to Specific Example 98, wherein the stabilizer for step (i) is added to a concentration of about 10% (v / v) to about 20% (v / v). Specific Example 10 0. The method according to any one of Specific Examples 60 to 99, wherein the method further comprises using an eluate obtained from hydrophobic interaction chromatography, or using hydrophobicity The substance prepared by the eluate of the parent interaction chromatography is subjected to chromatography using a hydroxyapatite chromatography analysis material. Specific example 101 · The method according to any one of specific examples 68 to 100, wherein the method further comprises a step of: eluting from phase (vii) or using elution from phase (vii) The liquid prepared by the product is subjected to color layer analysis processing on the hydroxyapatite color layer analysis material. The color layer analysis includes: (V111) the eluate (diluted and pH adjusted) obtained from stage (vii) or Apply the liquid prepared using the 200529870 eluate from stage (vii) to the hydroxyapatite color layer analysis material;

(ix) using buffer c to elute the unbound material from the hydroxy 4 gray layer analysis material, and buffer C is suitable for eluting the material that fails to bind to the hydroxy phosphor gray layer analysis material; and (X) Use buffer solution C to elute the FXI polypeptide from the hydroxyphosphorus gray layer analysis material, and buffer solution C, which is suitable for the FXI polypeptide that is combined with the hydroxyphosphorus gray layer analysis material in step (viii) Eluted down. Specific example 102: The method according to specific example 101, wherein the buffer solution c 彳 or 、, the wash solution c 'contains one or more than one stabilizer capable of increasing the stability of the FXI polypeptide. Specific example 103: The method according to specific example 101, in which a

A stabilizer is added to a portion of the collection containing FXI, and the stabilizer is a sugar, alcohol, or aldol. Specific example 104: According to the specific method of [103], a stabilizer is added, and the stabilizer is sugar, c4_c8_alcohol or aldol. Specific example 105: The method according to specific example 104, wherein a stabilizer is added, and the stabilizer is a polyhydric alcohol. Specific Example 106: The method according to Specific Example 105, wherein a stabilizer selected from the group consisting of glycerin, propylene glycol, propylene-13-diol, propanol, and isopropanol is added. Specific Example 107: The method according to specific example 106, wherein a stabilizer selected from 81 200529870 containing glycerin, propylene glycol, and propane-1,3-diol is added. Specific Example 108 · The method according to any one of Specific Examples 105 to 107, wherein the stabilizer is added to a concentration of about 5% (v / v) to about 50% (v / v). Specific example 109: The method according to specific example 108, wherein the stabilizer is added to a degree of about 10% (ν / ν) to about 50% (v / v). Specific example 110: The method according to specific example 109, wherein the stabilizer is added to a degree of about 10% (ν / ν) to about 20% (ν / ν). Specific Example 111. The method according to Specific Example 110, wherein the stabilizer is added to a concentration of about 10% (v / v). Specific example 112: The method according to any one of specific examples 101 to 111, wherein the buffer solution C and / or C has a pH of about 5.8 to about 7.8. Specific example 113: The method according to any one of specific examples 101 to 112, wherein the buffer solution C and / or C has a pH of about 6.0. Specific Example 114: A pharmaceutical composition comprising the FXI polypeptide prepared by the method of any one of Specific Examples 60 to 103. The following is intended as a non-limiting example of the present invention. Example 82 200529870 Real male case j___: Operation of FXI on hemostasis in patients with palpitations. Cardiopulmonary bypass was performed before and after surgery to obtain blood from 5 patients undergoing cardiac surgery. RoTEG (rotary thromboelastography) was used to evaluate the effect of FXI on blood clot formation and stability using the methods of Vig Temple ‘(2001)’ “Blood Coagulation &amp; Fibrinolysis”, No. 12 ·· 555. In short, the coagulation is achieved by adding Innovin (final dilution 1: 50,0000) (produced by Dade Behring) and CaCl2 (final concentration · 5ηΜ) in FXI (2.5, 10 or 25nM) (HTI / enzyme Jiu Bei inspected, Essen) started in the presence or absence. Fiber _ proteolysis is by adding 4nM tPA (produced by American Diagnostica). The measurement was performed using a ROTEG-04 whole blood hemostasis system with a rotary coaguloelastography device (produced by Pentapharm GmBH). The overall clot quality (OCQ) was calculated as: (Max vel / tmax rel) x (tmin rel -tmax rel) The OCQ was then normalized relative to the control sample (incubated without any hemostatic agent).

… The results are shown in the picture! Being. FXI considerably enhances the formation of overall blood clots and the blood clots formed in the presence of FXI have increased resistance to fibrinolytic effects. Role in normal blood history The diarrhea was obtained from four normal subjects, and FXI was used to evaluate the formation of blood clots using rOTeg as described in Example 丨. Figure 2 illustrates that FXI causes an increase in OCQ in normal blood. 83 200529870 Example 3: FXI maggot-like activity disrupted by saccharification. FXI variants containing the following substituents were constructed using standard methodology and expressed in HEK293 cells after metastatic infection. From 37. (: Cells cultured for 96 hours Collect crude cell culture supernatants. FXI activity was measured using ROTEG as described in Example 1. The results are shown in the table below. FXI activity NHP (normal Human plasma) (3 InM FXI) 106 FXI N72Q-1, 2nM 42 FXIN108Q-1, 3nM 62 FXI N335Q-0, 4nM 75 FXI N432Q-1? 2nM 33 FXI N473Q-0, 6nM 83 Example 4: FXI formulation Storage stability The following FXI solution was prepared and stored at 5 ° C for 5 weeks, and then the activity of FXI was determined by the method described in Example 1. 1.3 84nM FXI was dissolved in 4mM acetate, 150mM NaC, pH 5.4 2.190 nM FXI is dissolved in 50 mM acetate buffer, 150 mM NaCn, pH 5.4 3.190ηΜ FXI is dissolved in 50 mM acetate buffer, 150 mM NaC ρ 5.4, ImM CaCl2 4.190 nM FXI is dissolved in 50 mM acetate buffer, 75 mM NaC pH 5.4, 300 mg / ml sucrose

5.190nM FXI in 50mM MES buffer, ρΗ6.5, 150mM

NaCl 6.190ηΜ FXI dissolved in 50mM MES buffer, pH 6.5, 150mM NaCJ, ImM CaCl2 200529870

7.190nM FXI in 50nm MES buffer, pH 6 5, 75mM

NaCl ′ 300 ^: g / zengshengyan sugar The results are not shown in Figure 3. Binding peptides The following experiments were performed to identify peptides that bind to FXI. 1. Synthesis of peptide libraries ... The following libraries were synthesized using Fmoc solid-phase peptides from Rapp

Polymere (Germany) on Tentagei resin beads. Three different peptide libraries were used for screening. Their names are BU2i, BUM, and BL123. The format of library BL 1 2 1 is ... 〇] -〇2-〇3-〇4-〇5-〇6-〇7-〇8-〇9-0 ^ 〇1 "0" 2_〇] 3_ 〇】 4

Tentagel resin, where On is L-amino acid and η = ι, 2 and n, 12 can be any ethylamine which produces protein except methionine and cysteine

The library BL122 is in the form of 14 and can be L-amine in addition to formaldehyde I

Lipids, where On is L-amino acid and may be any L-amino acid that produces proteins other than methionine and cysteine. 2. Screen the recombinant factor χι of the mitochondrial library and purchase it from a Hematology Technology Company (Heamatiogic Techi_g㈣ method) according to standard laboratory biotinylation methods. 85 200529870 Then 5 microliters of Factor XI (1,2 // M) and 1 microliter of streptavidin-alkaline phosphatase (lmg / ml, produced by Sigma) were added to the three synthetic peptide libraries BL121, BL122 and BL124, and allowed to incubate for about 2-3 hours. The incubation buffer is 15mM TRIS-HCH, ρ = 7.4, 0.15M NaCl, 0.5% bovine serum albumin (BSA), and 0.05% Tween20. Rinse buffer (M TRIS-HC1, pH = 7.4, 0.15M NaCl and 0.05% Tween20) After washing, add BCIP and NBT to the coloring buffer (50mM TRIS-HC1, ρΗ = 8 · 8, 0.15M NaCl and 0.05% Tween20 And 15 mM MgCl2) and allow coloration for 30 minutes to 1.5 hours. 3. Sequence determination Remove the active blue beads from the library and sequence them with an Edman sequencer (Procise, manufactured by Applied Biosystems). Results: In the libraries BL121, BL122 and BL123 according to the invention Now the specific factor XI and factor XI-like binding peptides include peptides comprising the amino acid sequence of an amine of the article as follows:

Sequence found in BL121 SEQ ID NO: 03: SRWPWSVFPDFPD SEQ ID NO: 04: DVWDYVVFDDFPS SEQ ID NO: 05: QRWVPYDDFPSLRS SEQ ID NO: 06: RHFHVFPDFPFVH SEQ ID NO: 07: HHFPPFSHFPDLPQ 200529870

SEQ ID NO: 08: RRLPLSRLPDFP SEQ ID NO: 09: HPFFRGYPDFPD SEQ ID NO: 10: HPWHLVYPDFPS SEQ ID NO: l 1: HDWLVRWPDFPS SEQ ID NO: 12: SHFWRQWPDFSD SEQ ID NO: 13: PQLRWHDFPDFGS SEQ ID NO: 14: VVWRHWQDFDQFVVV SEQ ID NO: 15: VDWQWSRFDDFPS SEQ ID NO: 16: HPWFDDFPHLFLFQ Sequence from library BL122 SEQ ID NO: 17: YKWIHHDDFPLV SEQ ID NO: 18: FDRKRVHPDFPH SEQ ID NO: 19: DVWDYVVFDDFPS SEQ ID NO: 20: QQPIQRFPDFP SEQ ID NO: 21: QAIFTRFPDFPN SEQ ID NO: 22: EWFPDFPEGSDG SEQ ID NO: 23: HTHAFPDFPPH SEQ ID NO: 24: LVKGFPDFPNHN SEQ ID NO: 25: GPFPYAYEDFPE SEQ ID NO: 26: FYLKTRYYDFPE SEQ ID NO: 27: FQARHTIGDFPA SEQ ID NO : 28: RIKDFPSDSNTV SEQ ID NO: 29: IWESHKVIEDFP SEQ ID NO: 30: QWFSVSRYQDFD 87 200529870

SEQIDNO: 31: QKDFHWRILPDF SEQ ID NO: 32: KIVKFPHTFPDL SEQ ID NO: 33: HLYDFDLDNEY SEQ ID NO: 34: KTILGDVDFDI SEQ ID NO: 35: RQLHPFHHFHG SEQ ID NO: 36: RSWLRYGYGH SEQ ID NO: 37: FNWNNVDEYYDW SEQ ID NO : 38: DQWDWEDYDEAW SEQ ID NO: 39: YDIYDDYEIWA sequence found in library BL124 SEQ ID NO: 40: YPKHIYADFPSTRL SEQ ID NO: 41: YPRHIYPDFPTDTT SEQ ID NO: 42: YLKHAWPDFPKLQQ SEQ ID NO: 43: YVRHRFEDFPTALP SEQ ID NO: 44: FPWHKYEDFPSPRT SEQ ID NO: 45: QPAHRYPDFPRNNH SEQ ID NO: 46: LPKTRFLDFPHVSF SEQ ID NO: 47: LPPARYPDFPAAKK SEQ ID NO: 48: IPKNRFSDFPDAQG SEQ ID NO: 49: LPSFRFPDFPATKT SEQ ID NO: 50: RVLNRYPDFPTTNQ SEQ ID NO : FFKKTYADFPTSQT SEQ ID NO: 52: IFKKTYEDFPRFVY SEQ ID NO: 53: VLHNKYDDFPRVKK 88 200529870

SEQ ID NO: 54: KVKHRFNDFPVWGN It is concluded that useful FXI-binding peptides include those having an Asp-Phe-Pro core amino motif. Example 6 Separate cells from mammalian cell cultures with supernatant by centrifugation or filtration. Add benzyl fat and EDTA to a final concentration of ImM. Example 7: Obelix ST CIEX (Cat. No. 1 1-0010), the first positive ion exchange chromatography method, was used to equilibrate the Ob el ix matrix with 5 column volumes (cv) of buffer A. And a load equal to 150 ml of the supernatant / per ml of the packed column was applied to the column. The column was washed with 4 cv of Buffer A (30 mM Tris, ρΗ8 · 0), and then washed with 5 cv of Buffer A '(50 mM Tris 50% glycerol 87%, ρΗ9 · 0). Then, 5 cv of buffer A '(50 mM Tris 50% glycerol 87%, 1M NaCl, ρΗ9 · 0) was used for dissociation. The flow rate was 16 cv / hour and the temperature was 0-10 ° C. The column was regenerated with 1M NaOH. Collect a portion of the collection at approximately 50% of the peak height. Discard the first peak eluent but the next main peak contains FXI. C4 Jupiter Phenomonex (Cat. No. OUG-4167-EO, 4.6x250mm) and SDS-PAGE were used for MOPS electrophoresis on NUpage4-12% Bis / Tris colloid (produced by Invitrogen) using HPI (νζ · &amp; k / M). A running buffer was used to analyze a portion of the FXI polypeptide-containing collection under reducing conditions. Benzyl fat and EDTA were added to a partial collection (to ImM) containing FXI polypeptide and kept at approximately 4 in the freezer. (:, Or refrigerate at -80 ° C until it can be used in step 89 200529870. For Obelix ST CIEX, the alternative is Streamline Direct CST (Amersham's catalog number 1 7-5266-03). (Figure 4 'Chromatographic analysis drawing for preparation.) Example 8 The chromatographic analysis of hydrophobic interactions using butyl galactose with high performance and high substitution (catalog number 17-3100) will contain 1.5M volume containing 2M NaCl, 40 mM Tris pH 8 buffer was added to the pooled partial collection containing FXI polypeptide from Example 7 'adjust the pH to 8.2 if it is not already between 8.0 and 8.4. Use 3 cv of buffer B (1 M NaCH (20 mM Tris, ρΗ8 · 0) equilibrate the high-performance butyl lipobromide-substitute matrix and equilibrate a load equivalent to 1 mg / nii onto the I column. Then rinse the column with 2 cv buffer b, then Gradient elution was performed, from buffer B to 100% elution buffer B (20 mM Tris pH 80) exceeding 20 CV, followed by 100% elution buffer B with 2 cv. The flow rate was 12 cv / Hr, the temperature is 0_1 (rc. Fractions are collected after about 10 cv dissolution The aggregates were collected until 15 cv. MOPS electrophoresis buffer (running buffer) was used on CPI Juxer Phenomonex (Cat. No. 〇〇g_4167_e〇, sDS-PA〇E, NUpage, 12% Bls ^ company) using HPI (νΰ ^ / νβ) ) Analyze partial collections containing FXI polypeptide under reducing conditions. Immediately add M volumes of propylene glycol to the pool of partial collections containing t FXI polypeptide to a final concentration of myocardium.) Propylene glycol. The collection is stored at about 1 generation, or cold rolled until further use. 200529870 For the high performance and high substitution of butyl agarose, the substitute is phenyl agarose with high performance. This kind of alternative Substituting the matrix will cause it to elute later (Figure 5, chromatographic analysis drawing for preparation). Example 9 Lilly Ϊ Apatite (_BJ ^ Rad 1 Division Catalog Number 157-0.0g0) Scaly graystone analysis adjusted the pH of the pool of partial collections containing FXI peptides obtained in Example 8 to 6.0, and added a volume of water to a conductivity below 20 mS / cm. Use type 6cv buffer c to make type I hydroxyphosphorus 20 μηι matrix was equilibrated, and then a load equivalent to 5 mg / ml of colloid was applied to the column. The column was then washed with 15 cv of buffer c (20 mM KP04, pH 60), and 95% buffer C and 5 were used. % Buffer c, (20 mM K_po4, 2M NaCn 'ρΗ6.0 ·) buffer was used as a washing step. A gradient elution of 5% to 100% C 'was performed and the FXI polypeptide was eluted in a small fraction of the collection. The conductivity of the aggregate containing the partial collection of FXI polypeptides was about 60 mS / cm and the pH was about 6.0. C4 Jupiter Phenomonex (Cat. No. 0〇g_4167_e〇, 46 × 250mm) and SDS-PAGE were used for hPLC (W &amp; K plus) on NUpage 4-12% Bis / Tris colloid (manufactured by Invhrogen) using Mops An electrophoresis buffer (buffer) was used to analyze a portion of the FXI polypeptide-containing collection under reducing conditions. The purity of HPLC was> 97% and the concentration of FXI was about 12 mg / mi. The #XI high purity FXI-containing fraction collection was collected and propylene glycol was added to a final concentration of 10% ν / ν, and stored below -20. 〇 (Figure 6, chromatographic drawing for preparation). 91 200529870 Example: The HPLC analysis program uses C4 Jupiter Phenomonex (Cat. No. 00-4167-EO, 4.6x250mm) and uses the following buffer solution for high performance liquid chromatography (HPLC; please refer to the above examples) 7_9): Buffer 1: 0.1% TFA in water Buffer 11: 〇.〇7% TFA in CH3CN

Equilibrate the column with a mixture of 750 / 〇 (ν / ν) buffer and 25% (v / v) buffer π for 5 minutes (flow rate is 丨 ml / min). Use 75% Buffer I and 25% Buffer π to 39% Buffer! The column was eluted with a gradient of 61% buffer solution over 18 minutes (flow rate 1 ml / min). The publication uses the column to rinse all the documents from 2 to 2 in detail. The patent application [figure 1 of the whole solution of the liquid 2 100% buffer solution Ⅱ rinse for 2 minutes (flow rate of 0.05 ml / min Nansheng. Institute The detection wavelength used is 214 nm. A sample with a temperature of 50 ° C and 50 micrograms is loaded on the column. The patents, patent applications mentioned in this specification, and

For its complete content and in this manual. Many changes to the Ming Dynasty can be suggested to the artist in the light of the Ming Dynasty. Such obvious changes are included in the complete scope of the desired scope. Brief description of the formula] It is a graphic illustration of the effect of increasing the amount of FXI on the quality of blood clots before and after surgery. This is a graphical representation of the effect of increased amounts of FXI on the quality of a subject's blood. Figure 3 is a graphical representation of the biological activity of different FXI formulations after 50% storage for 5 days. Figure 4 is a chromatogram for the preparation of the fraction containing the factor XI polypeptide from the first cation exchange chromatograph using Obelix STCIEx (catalog number 11-GG1G) as described in Example 7. FIG. 5 is a hydrophobic interaction chromatography analysis using Buty Sepharose High Perfance (Cat. No. 17-3100) as described in Example 8. Chromatographic analysis diagram for the preparation of the fraction containing the factor XI polypeptide. Figure 6 is obtained from hydroxyapatite using CHT hydroxyapatite Digong Pear BioRad (catalog number 157-0020) as described in Example 9. Chromatographic analysis chart for the preparation of the part containing the factor XI polypeptide by stone chromatographic analysis. Knife [Description of the main component symbols]

93 6762 Ή 0529870 Sequence Listing &lt; 110> Norfolk Nondick Corporation 5 &lt; 120 &gt; Therapeutic Use of Factor XI &lt; 130 &gt; 6762 10 &lt; 160 &gt; 2 &lt; 170> Patentln version 3.2 15 &lt; 210 &gt; 1 &lt; 211 &gt; 607 &lt; 212 &gt; PRT &lt; 213 &gt; Plasma &lt; 400 &gt; 1 20

Glu Cys Val Thr Gin Leu Leu Lys Asp Thr Cys Phe Glu Gly Gly Asp 15 10 15 25 lie Thr Thr Val Phe Thr Pro Ser Ala Lys Tyr Cys Gin Val Val Cys 20 25 30

Thr Tyr His Pro Arg Cys Leu Leu Phe Thr Phe Thr Ala Glu Ser Pro 30 35 40 45

Ser Glu Asp Pro Thr Arg Trp Phe Thr Cys Val Leu Lys Asp Ser Val 50 55 60 35 2 672

Thr Glu Thr Leu Pro Arg Val Asn Arg Thr Ala Ala lie Ser Gly Tyr 65 70 75 80

Ser Phe Lys Gin Cys Ser His Gin lie Ser Ala Cys Asn Lys Asp lie 85 90 95 10 Tyr Val Asp Leu Asp Met Lys Gly Gong le Asn Tyr Asn Ser Ser Val Ala 100 105 110

Lys Ser Ala Gin Glu Cys Gin Glu Arg Cys Thr Asp Asp Val His Cys 15 115 120 125

His Phe Phe Thr Tyr Ala Thr Arg Gin Phe Pro Ser Leu Glu His Arg 130 135 140 20

Asn lie Cys Leu Leu Lys His Thr Gin Thr Gly Thr Pro Thr Arg lie 145 150 155 160 25

Thr Lys Leu Asp Lys Val Val Ser Gly Phe Ser Leu Lys Ser Cys Ala 165 170 175 30 Leu Ser Asn Leu Ala Cys Arg Asp lie Phe Pro Asn Thr Val Phe 180 185 190

Ala Asp Ser Asn lie Asp Ser Val Met Ala Pro Asp Ala Phe Val Cys 35 195 200 205 3 67 529 870

Gly Arg lie Cys Thr His His Pro Gly Cys Leu Phe Phe Thr Phe Phe 210 215 220 5

Ser Gin Glu Trp Pro Lys Glu Ser Gin Arg Asn Leu Cys Leu Leu Lys 225 230 235 240 10

Thr Ser Glu Ser Gly Leu Pro Ser Thr Arg lie Lys Lys Ser Lys Ala 245 250 255 15 Leu Ser Gly Phe Ser Leu Gin Ser Cys Arg His Ser lie Pro Val Phe 260 265 270

Cys His Ser Ser Phe Tyr His Asp Thr Asp Phe Leu Gly Glu Glu Leu 20 275 280 285

Asp lie Val Ala Ala Lys Ser His Glu Ala Cys Gin Lys Leu Cys Thr 290 295 300 25

Asn Ala Val Arg Cys Gin Phe Phe Thr Tyr Thr Pro Ala Gin Ala Ser 305 310 315 320 30

Cys Asn Glu Gly Lys Gly Lys Cys Tyr Leu Lys Leu Ser Ser Asn Gly 325 330 335 4 6 529 870

Ser Pro Thr Lys lie Leu His Gly Arg Gly Gly lie Ser Gly Tyr Thr 340 345 350 5 5 Leu Arg Leu Cys Lys Met Asp Asn Glu Cys Thr Thr Lys lie Lys Pro 355 360 365

Arg lie Val Gly Gly Thr Ala Ser Val Arg Gly Glu Trp Pro Trp Gin 10 370 375 380

Val Thr Leu His Thr Thr Ser Pro Thr Gin Arg His Leu Cys Gly Gly 385 390 395 400 15

Ser lie lie Gly Asn Gin Trp lie Leu Thr Ala Ala His Cys Phe Tyr 405 410 415 20

Gly Val Glu Ser Pro Lys lie Leu Arg Val Tyr Ser Gly lie Leu Asn 420 425 430 25 Gin Ser Glu lie Lys Glu Asp Thr Ser Phe Phe Gly Val Gin Glu lie 435 440 445 lie lie His Asp Gin Tyr Lys Met Ala Glu Ser Gly Tyr Asp lie Ala 30 450 455 460

Leu Leu Lys Leu Glu Thr Thr Val Asn Tyr Thr Asp Ser Gin Arg Pro 465 470 475 480 35 5 6720tT529870 lie Cys Leu Pro Ser Lys Gly Asp Arg Asn Val lie Tyr Thr Asp Cys 485 490 495 5

Trp Val Thr Gly Trp Gly Tyr Arg Lys Leu Arg Asp Lys lie Gin Asn 500 505 510 10 Thr Leu Gin Lys Ala Lys lie Pro Leu Val Thr Asn Glu Glu Cys Gin 515 520 525

Lys Arg Tyr Arg Gly His Lys lie Thr His Lys Met lie Cys Ala Gly 15 530 535 540

Tyr Arg Glu Gly Gly Lys Asp Ala Cys Lys Gly Asp Ser Gly Gly Pro 545 550 555 560 20

Leu Ser Cys Lys His Asn Glu Val Trp His Leu Val Gly lie Thr Ser 565 570 575 25

Trp Gly Glu Gly Cys Ala Gin Arg Glu Arg Pro Gly Val Tyr Thr Asn 580 585 590 30 Val Val Glu Tyr Val Asp Trp lie Leu Glu Lys Thr Gin Ala Val 595 600 605 &lt; 210 &gt; 2 35 &lt; 211 &gt; 553 6 6720 (Τ529870 &lt; 212 &gt; PRT &lt; 213> Platelets &lt; 4〇〇 &gt; 2 5

Glu Cys Val Thr Gin Leu Leu Lys Asp Thr Cys Phe Glu Gly Gly Asp 15 10 15 10 lie Thr Thr Val Phe Thr Pro Ser Ala Lys Tyr Cys Gin Val Val Cys 20 25 30

Thr Tyr His Pro Arg Cys Leu Leu Phe Thr Phe Thr Ala Glu Ser Pro 15 35 40 45

Ser Glu Asp Pro Thr Arg Trp Phe Thr Cys Val Leu Lys Asp Ser Val 50 55 60 20

Thr Glu Thr Leu Pro Arg Val Asn Arg Thr Ala Ala lie Ser Gly Tyr 65 70 75 80 25

Ser Phe Lys Gin Cys Ser His Gin lie Ser Asn lie Cys Leu Leu Lys 85 90 95 30 His Thr Gin Thr Gly Thr Pro Thr Arg lie Thr Lys Leu Asp Lys Val 100 105 110

Val Ser Gly Phe Ser Leu Lys Ser Cys Ala Leu Ser Asn Leu Ala Cys 35 115 120 125 6720ίΤ529870 lie Arg Asp lie Phe Pro Asn Thr Val Phe Ala Asp Ser Asn lie Asp 130 135 140

Ser Val Met Ala Pro Asp Ala Phe Val Cys Gly Arg lie Cys Thr His 145 150 155 160 10

His Pro Gly Cys Leu Phe Phe Thr Phe Phe Ser Gin Glu Trp Pro Lys 165 170 175 15 Glu Ser Gin Arg Asn Leu Cys Leu Leu Lys Thr Ser Glu Ser Gly Leu 180 185 190

Pro Ser Thr Arg lie Lys Lys Ser Lys Ala Leu Ser Gly Phe Ser Leu 20 195 200 205

Gin Ser Cys Arg His Ser lie Pro Val Phe Cys His Ser Ser Phe Tyr 210 215 220 25

His Asp Thr Asp Phe Leu Gly Glu Glu Leu Asp lie Val Ala Ala Lys 225 230 235 240

Ser His Glu Ala Cys Gin Lys Leu Cys Thr Asn Ala Val Arg Cys Gin 245 250 255 30 8 Spirit 29870

Phe Phe Thr Tyr Thr Pro Ala Gin Ala Ser Cys Asn Glu Gly Lys Gly 260 265 270 5 Lys Cys Tyr Leu Lys Leu Ser Ser Asn Gly Ser Pro Thr Lys lie Leu 275 280 285

His Gly Arg Gly Gly lie Ser Gly Tyr Thr Leu Arg Leu Cys Lys Met 10 290 295 300

Asp Asn Glu Cys Thr Thr Lys lie Lys Pro Arg lie Val Gly Gly Thr 305 310 315 320 15

Ala Ser Val Arg Gly Glu Trp Pro Trp Gin Val Thr Leu His Thr Thr 325 330 335 20

Ser Pro Thr Gin Arg His Leu Cys Gly Gly Ser lie lie Gly Asn Gin 340 345 350

25 Trp lie Leu Thr Ala Ala His Cys Phe Tyr Gly Val Glu Ser Pro Lys 355 360 365 lie Leu Arg Val Tyr Ser Gly lie Leu Asn Gin Ser Glu lie Lys Glu 30 370 375 380

Asp Thr Ser Phe Phe Gly Val Gin Glu lie lie lie His Asp Gin Tyr 385 390 395 400 35 9

6762.204-WO 200529870

Lys Met Ala Glu Ser Gly Tyr Asp lie Ala Leu Leu Lys Leu Glu Thr 405 410 415 5

Thr Val Asn Tyr Thr Asp Ser Gin Arg Pro lie Cys Leu Pro Ser Lys 420 425 430 10 Gly Asp Arg Asn Val lie Tyr Thr Asp Cys Trp Val Thr Gly Trp Gly 435 440 445

Tyr Arg Lys Leu Arg Asp Lys lie Gin Asn Thr Leu Gin Lys Ala Lys 15 450 455 460 lie Pro Leu Val Thr Asn Glu Glu Cys Gin Lys Arg Tyr Arg Gly His 465 470 475 480 20

Lys lie Thr His Lys Met lie Cys Ala Gly Tyr Arg Glu Gly Gly Lys 485 490 495

25

Asp Ala Cys Lys Gly Asp Ser Gly Gly Pro Leu Ser Cys Lys His Asn 500 505 510 30 Glu Val Trp His Leu Val Gly lie Thr Ser Trp Gly Glu Gly Cys Ala 515 520 525

Gin Arg Glu Arg Pro Gly Val Tyr Thr Asn Val Val Glu Tyr Val Asp 35 530 535 540 72ΰίΤ§29870

Thr Gin 550

Trp lie Leu Glu Lys 545

Claims (1)

200529870 10. Scope of patent application: 1. A method for treating episodic hemorrhagic incidents, which includes treating the needy heart; ^ Use the therapeutically effective amount of the factor χι (ρχΐ) or FXI related polymorphism Of preparations. The method according to item 1, wherein the application enables the method according to item 01, wherein the application promotes the method according to item 1, wherein the application enables the method according to item 1, wherein the application causes 2. The coagulation time in the patient is reduced 3. The hemostasis of the patient is advanced according to the scope of the patent application. 4 _ According to the scope of the patent application, the blood clot dissolution time of the patient is increased. 5. According to the scope of the patent application, the blood clot strength of the patient is increased. 6, 6. The method according to item 1 of the scope of patent application, wherein the administration will increase the overall blood clot quality (ovenU quality, 0cq) of the sick heart. The method according to item 1 of the scope of patent application, wherein after application, Patients will show an effective ρχι plasma concentration of at least about 5 nM. 8. The method according to item 7 of the scope of patent application, wherein the effective FXI blood polymerisation concentration is at least about 10 nM. 9. The method according to item 8 of the scope of patent application, wherein the effective FXI blood aggregation concentration is at least about 30 nM. 1__ The method according to item 1 of the scope of patent application, wherein the FXI or FXI sequence includes the sequence of SEQ ID NO · 1, or it retains at least one biologically active fragment associated with FXI. 94 200529870 The related polypeptide comprises 歹 J of SEQ ID NO: 2 or a fragment thereof retained to / | y, a biological activity associated with FXI. 12. According to the scope of patent application Di Luo suffers from a congenital deficiency of FXI. Methods The patient was not 13. According to the total scope of the patent application, the total number of smoke paintings, "Λ Η Wan Fa" in which the hemorrhagic couple: the event system belongs to a condition selected from the group consisting of surgery, sequence, Trauma or blood thinning. Wang 14. The method according to item 丨 of the scope of patent application before its application: ⑷ Obtain a blood sample from the patient; (b) Determine at least: 'one · FXI concentration, FXTa · fyt + l is the ratio of fX.之外, 之外 outside of what is needed to restore coagulation = ⑷; and ⑷ determine the therapeutic FXI content based on the result of step (b). n ... α-a method for treating incidental bleeding incidents' The patient administers ⑴-amount of package + FXI polypeptide preparation and ⑻Second = preparation containing non-Factor factor VIIa coagulant = Chu-Dan A / , Τ niandi one measure pandi one li and is effective for this type of treatment. vn / I6. The method according to the scope of application patent 帛 15 ', wherein the non-factory Vila coagulant is selected from the group consisting of the following factors ... Factor Η έ = sub-pathway inhibitor (TFPI) inhibitor; because + Ιχ; Thrombin can be active = fibrinolytic inhibitor (TAFI); plasminogen activity 'irPAT κ primary inhibitor-(AW); factor v; protein c inhibitor; protein &quot; and tissue plasminogen activator (TpA) inhibitor. 17. According to the scope of patent application! Item method 'wherein the method does not include 3% factor VII / factor Vila coagulant. 95 200529870 U Pharmaceutical Substance ', which comprises a pharmaceutically acceptable carrier) 19. A recombinant FXI polymorph of FXI polypeptide and (Π) a pharmaceutically acceptable carrier or tablet. k It is used for the treatment of bleeding incidents 20. According to the scope of the patent application, incident incidents belong to the use selected from the group consisting of 19 items, in which the condition of the group below bleeding is · surgery, dental procedures, trauma or Blood thinning. 21. According to the scope of application for patent No. 19 or 2, the contingent bloody event is not the factor VII / factor viia I ', medium. 22 · A hemorrhage of FXI polypeptide and 23 · —blood lysis time of a FXI polypeptide. Coagulant treatment. Use 'is used to promote the use of a patient in need, it is used to increase the variety of FXI in a patient in need, and it is used to require the blood clot strength. 25.-FX: [Polymorphic use, which is used in patients in need to improve their overall blood clot quality (0CQ). 26. The clotting time of a FXI polypeptide. Patients for enhancement use, which is used for patients in need to reduce 27. The use according to any one of claims 19 to 25, wherein the effective FXI plasma concentration in the patient's body is increased to at least About 5 nM 28. The use according to item% of the patent application range, wherein the effective ρχι plasma concentration is increased to at least about 10 nM. 96 200529870 2 9 · According to the application of Shen Shizhu_ Jiaming Patent Qianwei No. 27, the plasma concentration in the middle and right is increased by 刭 s 丨 丄 /, and the effective FXI is added to at least about 30nM. • According to any one of items 19 to 28 in the patent scope of the application, 6 the ulcers to be treated * * The invertebrate is treated with inverse, when μm is not used VI Huzi VIIa coagulant is treated. Use of a FXI polypeptide, which is used for preparing medicine for treating incidents 32. The use according to item 31 of the scope of patent application, wherein the hemorrhagic galvanic event is subordinate to a condition selected from the group consisting of surgery, trauma, or hemodilution. 33. The use according to item 31 or 32 of the scope of the patent application, wherein the bleeding event is not treated with a factor VII / factor Vila coagulant. 34.—The use of a FXI polypeptide for the preparation of a pharmaceutical formulation for use in a patient in need to promote hemostasis. 35. The use of a FXI polypeptide, which is used to prepare a pharmaceutical formulation for use in a patient in need to increase the clot dissolution time. 36. The use of a FXI polypeptide, which is used to prepare a pharmaceutical formulation for use in a patient in need to increase the strength of his blood clot. 37. The use of a FXI polypeptide, which is used to prepare a pharmaceutical formulation for use in a patient in need to improve its overall blood clot quality (OCQ). 38. The use of a FXI polypeptide for the preparation of a pharmaceutical formulation for use in a patient in need to reduce clotting time. 39. The use according to any one of claims 31 to 38 in the scope of the patent application, wherein the effective plasma concentration of FXI in a patient is increased to at least about 5 nM. 97 200529870 40. The use according to item 39 of the scope of patent application, wherein the effective ρχι plasma concentration is increased to at least about 10 nM. 41. The use according to item 40 of the scope of the patent application, wherein the effective ρχιη table / degree is increased to at least about 30 nM. 42. According to the patent application of any one of items 31 to 41, the patient to be treated has not been treated with a factor VII / factor VlIa coagulant. 43. According to the use of any one of claims 19 to 42 in the scope of the patent application, wherein the patient to be treated does not suffer from a congenital ρχι deficiency. 44. The use according to any one of items 19 to 43 of the Chinese Patent Application, wherein the FXI polymorphic sequence 3 SEQ ID NO: 1 or it retains at least one biological activity associated with FXI Fragment. 4 5 · The use of any one of items 19 to 43 according to the patent application, where the FXI polypeptide comprises the sequence of SEQ ID NO: 2 or it retains at least one species and FXI related biologically active fragment. 46. According to the application of any one of the 19th to 45th paragraphs of the patent application J, the FXI polypeptide is a combination of a clotting agent for 7VII, and a factor VII / factor Vila. 47. According to 46 uses of the scope of patent application, the non-Factor VII / Factor Vila coagulant is from the following groups: Factor III, tissue factor inhibitor (TFPj) inhibits seizure, and envy 4 Tablets, factor IX; thrombin-activated fibrin> glutinase inhibitors (TAFIV total wi, t / synthase activator inhibitor-1 (PAI_1); factor V; protein c inhibits mm ^ η, ' Protein shell S inhibitor; and tissue fibrinolytic activator of fegen activator (tPA). 98