TFPI inhibitors and methods of use

Abstract

The present invention relates to inhibitors of tissue factor pathway inhibitor (TFPI) and methods of use. More specifically, the present invention provides peptides that bind to TFPI, including TFPI-1 inhibitory peptides, and compositions thereof. The peptides can be used to inhibit TFPI, increase thrombin formation in subjects with coagulation factor deficiency, increase blood clot formation in subjects, and / or treat coagulation disorders in subjects.

Description

[0001] This application is a divisional application of international application number PCT / US2009 / 069060, with international application date December 21, 2009, entering the Chinese national phase on August 10, 2011, application number 200980156547.8, and invention name “TFPI inhibitors and methods of use”. Technical Field

[0002] The present invention generally relates to peptides that bind tissue coagulation factor pathway inhibitor (TFPI) and their uses.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0004] This application claims priority to U.S. Provisional Patent Application No. 61 / 139,272, filed December 2008, the disclosure of which is incorporated by reference in its entirety. Background Art

[0005] Hemostasis depends on a complex coagulation cascade, in which a series of events mediated by coagulation factors cause prothrombin to change to thrombin. Activation of coagulation factor X (FX) is the central event of the endogenous and exogenous pathways of the coagulation cascade. Exogenous pathways have been proposed as the main activating factors of the coagulation cascade (Mackman et al., Arterioscler. Thromb. Case. Biol, 27, 1687-1693 (2007)). Circulating tissue coagulation factor (TF) interacts with activated coagulation factor VII (FVIIa) to form an "exogenous complex" mediating FX activation. The coagulation cascade is amplified by the endogenous pathway, during which the continuous activation of coagulation factors XII, XI, IX and VIII leads to the formation of an "endogenous" FIXa-FVIIIa complex mediating FX activation. Activated FX promotes thrombin formation, and thrombin formation is required for the body to produce fibrin and effectively stop bleeding.

[0006] Severe bleeding disorders, such as hemophilia, are caused by the destruction of the coagulation cascade. Hemophilia A is the most common type of hemophilia, originating from a deficiency of coagulation factor VIII, while hemophilia B is associated with a deficiency of coagulation factor IX (FIX). Hemophilia C is caused by a deficiency of coagulation factor XI (FXI) (Cawthern et al., Blood, 91 (12), 4581-4592 (1998)). Currently, hemophilia and other coagulation disorders cannot be cured. Coagulation factor replacement therapy is the most common treatment for coagulation disorders. However, coagulation factors are typically removed from the bloodstream shortly after administration. In order to be effective, patients must frequently receive intravenous infusions of plasma-derived or recombinant coagulation factor concentrates, which are uncomfortable, require a clinical setting, and are expensive and time-consuming. In addition, the efficacy of coagulation factor replacement therapy is sharply weakened after the formation of inhibitory antibodies. Approximately 30% of patients with severe hemophilia A develop inhibitory antibodies that neutralize coagulation factor (FVIII) (Peerlinck and Hermans, Haemophilia, 12, 579-590 (2006)). For patients with anti-coagulation factor antibodies, there are few treatment options.

[0007] Therefore, there is a need in the art for compositions and methods for treating coagulation disorders. The present invention provides such compositions and methods. Summary of the invention

[0008] The present invention provides peptides that bind to tissue factor pathway inhibitor (TFPI), including TFPI antagonist peptides that have the ability to regulate the coagulation cascade. For example, the present invention provides a peptide comprising the amino acid sequence X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 (SEQ ID NO: 3109), wherein

[0009] X7 is selected from L, P, K, S, W, V, N, and Q;

[0010] X8 is selected from L, R, N, F, and I;

[0011] X9 is selected from Y, V, P, and C;

[0012] X 10 Selected from F, L, and G;

[0013] X 11Selected from L, W, V, A, M, T, and S;

[0014] X 12 Selected from T, F, V, R, A, D, L, E, S, and Y;

[0015] X 13 Selected from I, M, G, Q, D, and R;

[0016] X 14 Selected from G, W, Y, L, M, and H;

[0017] X 15 Selected from N, P, F, H, K, and Y;

[0018] X 16 Selected from M, D, E, V, G, and K;

[0019] X 17 Selected from G, I, R, S, T, and L;

[0020] X 18 Selected from M, K, L, and I;

[0021] X 19 Selected from Y, G, R, and S;

[0022] X 20 is selected from A, E, S, C, and Y; and

[0023] X 21 Selected from A, V, K, and E.

[0024] In one aspect, the peptide comprises one or more N-terminal amino acids directly linked to X7, wherein the N-terminal amino acids comprise an amino acid sequence selected from the group consisting of

[0025] X6,

[0026] X5X6,

[0027] X4X5X6,

[0028] X3X4X5X6 (SEQ ID NO: 3110),

[0029] X2X3X4X5X6 (SEQ ID NO: 3111), and

[0030] X1X2X3X4X5X6 (SEQ ID NO: 3112), wherein

[0031] X1 is selected from T and G; X2 is selected from F and V; X3 is selected from V, W, Y and F; X4 is selected from D, Q and S; X5 is selected from E, T, N and S; and X6 is selected from R, H, K and A.

[0032] Alternatively or additionally, the peptide comprises one or more 21 The C-terminal amino acid connected to the

[0033] X 22 ,

[0034] X 22 X 23 ,

[0035] X 22 X 23 X 24 ,

[0036] X 22 X 23 X 24 X 25 (SEQ ID NO:3113),

[0037] X 22 X 23 X 24 X 25 X 26 (SEQ ID NO:3114), and

[0038] X 22 X 23 X 24 X 25 X 26 X 27 (SEQ ID NO:3115), wherein

[0039] X 22 is selected from Q, I, E, W, R, L, and N; X 23 Selected from L, V, M, and R; X 24 Selected from K, L, A, and Y; X 25 It is F;X 26 is G; and X 27 It's T.

[0040] In one aspect, the present invention provides a peptide comprising an amino acid sequence as shown in SEQ ID NOs: 1-7, for example, a peptide comprising an amino acid sequence as shown in any one of JBT0132, JBT0303, JBT0193, JBT0178, JBT0120 and JBT0224, which inhibits TFPI activity in the coagulation cascade. The present invention also provides a peptide that binds to TFPI, comprising an amino acid sequence that is at least 60% identical to the sequence Phe-Gln-Ser-Lys-Gly-Asn-Val-Phe-Val-Asp-Gly-Tyr-Phe-Glu-Arg-Leu-Arg-Ala-Lys-Leu (FQSKGNVFVDGYFERLRAKL) (SEQ ID NO: 32).

[0041] In addition, the present invention provides a peptide that binds TFPI, wherein the peptide comprises the structure of formula (I): X1001-X1002-X1003-X1004-X1005-X1006-X1007-X1008-X1009-X1010-X1011-X1012-X1013-X1014-X1015-X1016-X1017-X1018-X1019-X1020 (SEQ ID NO: 3116). In formula (I),

[0042] X1001 is an amino acid selected from the group consisting of Bhf, C, D, F, G, H, I, K, L, M, N, Nmf, Q, R, T, V, W, and Y;

[0043] X1002 is an amino acid selected from the group consisting of G, K and Q;

[0044] X1003 is an amino acid selected from the group consisting of A, Aib, Bhs, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;

[0045] X1004 is an amino acid selected from the group consisting of A, Aib, Bhk, C, D, E, F, G, H, I, K, K, L, M, N, Nmk, P, Q, R, S, T, V, W, and Y;

[0046] X1005 is an amino acid selected from a, A, Aib, Bal, C, D, d, E, F, G, H, K, k, L, M, N, Nmg, p, Q, R, S, T, V, W, and Y;

[0047] X1006 is an amino acid selected from the group consisting of A, Aib, Btq, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y;

[0048] X1007 is an amino acid selected from the group consisting of A, F, G, I, K, L, Nmv, P, Q, S, V, W, and Y;

[0049] X1008 is an amino acid selected from the group consisting of F, H, K, W and Y;

[0050] X1009 is an amino acid selected from the group consisting of A, Aib, f, I, K, S, T, and V;

[0051] X1010 is an amino acid selected from the group consisting of A, Aib, C, D, E, F, G, H, I, K, L, M, N, Nmf, P, Q, R, S, T, V, W, and Y;

[0052] X1011 is an amino acid selected from the group consisting of Aib, C, K, G and Nmg;

[0053] X1012 is Y;

[0054] X1013 is an amino acid selected from the group consisting of A, Aib, C, E, F, G, H, K, L, M, Q, R, W and Y;

[0055] X1014 is an amino acid selected from the group consisting of A, Aib, Bhe, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;

[0056] X1015 is an amino acid selected from (ω-methyl)-R, D, E, K and R;

[0057] X1016 is L;

[0058] X1017 is an amino acid selected from the group consisting of (ω-methyl)-R, A, Aib, Bhr, C, Cha, Cit, D, Dab, Dap, E, Eag, Eew, F, G, H, Har, Hci, Hle, I, K, L, M, N, Nle, Nva, Opa, Orn, Q, R, S, T, V, W, and Y;

[0059] X1018 is an amino acid selected from the group consisting of A, Bal, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y;

[0060] X1019 is an amino acid selected from the group consisting of Bhk, K, R, and V; and

[0061] X1020 is present or absent, and when X1020 is present, it is an amino acid selected from Aib, Bhl, C, F, G, H, I, K, L, Nml, Q, R, S, T, V, W and Y.

[0062] In one aspect, the peptide that binds TFPI comprises the structure of formula (III): X1001-Q-X1003-X1004-X1005-X1006-I / V-X1008-V-X1010-GYC / F-X1014-RL-X1017-X1018-KK / L (III) (SEQ ID NO: 3117). In formula (III), X1001, X1003, X1004, X1005, X1006, X1008, X1010, X1014, X1017 and X1018 are each independently selected from any amino acid.

[0063] The present invention also provides a TFPI-binding peptide comprising a structure of formula (V): X2001-X2002-X2003-X2004-X2005-X2006-[X2007-X2008-X2009-X2010-X2011-X2012-X2013-X2014-X2015-X2016-X2017-X2018]-X2019-X2020-X2021-X2022-X2023 (V) (SEQ ID NO: 3118). In formula (V), X2001, X2002 and X2023 are independently present or absent. When present, X2001 is an amino acid selected from A, D, E, F, G, H, I, K, L, P, R, S, T, V, and W; and X2002 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, P, R, S, T, V, and W.

[0064] in addition,

[0065] X2003 is an amino acid selected from the group consisting of A, F, I, K, L, R, S, T, V, W, and Y;

[0066] X2004 is an amino acid selected from the group consisting of A, D, E, F, G, I, K, L, R, S, T, V, and W;

[0067] X2005 is W;

[0068] X2006 is an amino acid selected from the group consisting of F, H, I, K, L, R, V, and W;

[0069] X2007 is an amino acid selected from C, Hcy, Dap and K, preferably from C and Hcy;

[0070] X2008 is an amino acid selected from the group consisting of A, G, R, S and T;

[0071] X2009 is an amino acid selected from the group consisting of a, A, I, K, L, M, m, Nle, p, R, and V;

[0072] X2010 is an amino acid selected from the group consisting of A, G, I, K, L, P, R, S, T, and V;

[0073] X2011 is an amino acid selected from the group consisting of D, E, G, S and T;

[0074] X2012 is an amino acid selected from the group consisting of A, a, D, d, E, e, F, f, G, I, K, k, L, I, M, m, Nle, nle, P, p, R, r, S, s, T, t, V, v, W, and w;

[0075] X2013 is an amino acid selected from the group consisting of A, D, d, E, e, F, G, I, K, L, R, S, s, T, V, and W;

[0076] X2014 is an amino acid selected from the group consisting of A, D, E, F, G, I, K, L, M, R, S, T, V, and W;

[0077] X2015 is an amino acid selected from the group consisting of A, D, E, F, G, I, K, L, M, Nle, R, S, T, V, and W;

[0078] X2016 is an amino acid selected from the group consisting of A, D, E, F, I, K, L, M, Nle, R, S, T, V, W, and Y;

[0079] X2017 is an amino acid selected from the group consisting of A, D, E, F, G, I, K, L, R, S, T, V, W, and Y;

[0080] X2018 is an amino acid selected from C and D (preferably X2018 is C);

[0081] X2019 is an amino acid selected from the group consisting of A, F, I, L, S, T, V, and W;

[0082] X2020 is an amino acid selected from F and W;

[0083] X2021 is an amino acid selected from I, L and V; and

[0084] X2022 is an amino acid selected from A, D, E, F, G, I, K, L, P, R, S, T, V, and W.

[0085] When X2023 is present in the peptide, X2023 is an amino acid selected from A, D, E, F, G, I, K, L, R, S, T, V, W and Y. The peptide comprises a cyclic structure created by the bond between X2007 and X2018, indicated by brackets in formula (V).

[0086] The present invention also provides a peptide that binds TFPI, wherein the peptide comprises a structure of formula (VI): X2001-X2002-F / YKWF / H-[C-X2008-M / V-X2010-D-X2012-X2013-GI / T-X2016-S / TC]-A / VWV-X2022-X2023 (VI) (SEQ ID NO: 3119). In formula (VI), X2001, X2002 and X2023 are each independently present or absent. X2008, X2010, X2012, X2013, X2016 and X2022, and X2001, X2002 and X2023, when present, are each independently selected from any amino acid. The peptide comprises a cyclic structure resulting from the bond between X2007 and X2018, indicated by brackets in formula (VI).

[0087] In one aspect, the invention provides a TFPI-binding peptide, wherein the peptide comprises a structure of formula (VIII): X3001-X3002-X3003-X3004-X3005-X3006-X3007-X3008-X3009-X3010-X3011-X3012-X3013-X3014-X3015-X3016-X3017-X3018-X3019-X3020-X3021 (VIII) (SEQ ID NO: 3120). In formula (VIII), X3001 and X3002 are each independently present or absent. When present, X3001 is an amino acid selected from A, C, D, F, G, I, K, L, M, N, P, Q, R, S, T, W, E, H and Y; and X3002 is an amino acid selected from A, C, D, F, H, K, M, N, P, R, S, T, W, Y, G, I and L. As for the remainder of Formula (VIII),

[0088] X3003 is an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, and Y;

[0089] X3004 is an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y, and P;

[0090] X3005 is an amino acid selected from the group consisting of C, D, F, G, H, I, K, L, M, N, P, R, S, T, V, W, and Y;

[0091] X3006 is an amino acid selected from the group consisting of A, W, C, K, P, R and H;

[0092] X3007 is an amino acid selected from the group consisting of Q, A, C, F, G, H, I, K, L, N, R, S, T, W, and Y;

[0093] X3008 is an amino acid selected from the group consisting of A, C, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, Y, and I;

[0094] X3009 is an amino acid selected from the group consisting of A, C, F, G, H, I, L, M, R, S, T, V, W, Y, and K;

[0095] X3010 is an amino acid selected from the group consisting of A, C, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;

[0096] X3011 is an amino acid selected from the group consisting of A, G, I, K, L, M, N, Q, R, S, T, V, W, Y, C, F, and H;

[0097] X3012 is an amino acid selected from the group consisting of A, C, H, I, K, L, and R;

[0098] X3013 is an amino acid selected from the group consisting of A, C, F, G, H, K, L, M, R, S, V, W, Y, and I;

[0099] X3014 is an amino acid selected from the group consisting of A, C, F, G, H, I, L, M, N, Q, R, S, T, V, W, Y, and K;

[0100] X3015 is an amino acid selected from A, K and R; X3016 is an amino acid selected from A, F, K and R;

[0101] X3017 is an amino acid selected from the group consisting of A, C, F, G, I, K, L, N, Q, R, S, T, V, W, Y, H, A, and M;

[0102] X3018 is an amino acid selected from the group consisting of A, C, F, I, K, L, M, Q, R, V, W, and Y;

[0103] X3019 is an amino acid selected from the group consisting of A, C, D, E, F, G, H, K, L, N, P, Q, R, V, W, Y, and I;

[0104] X3020 is an amino acid selected from the group consisting of A, C, F, G, H, K, L, M, N, Q, R, V, W, Y, I, and P; and

[0105] X3021 is an amino acid selected from A, C, H, I, K, L, M, N, P, Q, R, T, V, W, Y, F and G.

[0106] In addition, the present invention provides a TFPI-binding peptide comprising the structure of formula (IX): X3001-X3002-X3003-X3004-X3005-X3006-X3007-X3008-X3009-X3010-X3011-H-X3013-X3014-K / RR-X3017-X3018-X3019-X3020-X3021 (IX) (SEQ ID NO:3121), wherein X3001, X3002, X3003, X3004, X3005, X3006, X3007, X3008, X3009, X3010, X3011, X3013, X3014, X3017, X3018, X3019, X3020 and X3021 are each independently selected from any amino acid. In addition, the present invention includes peptides that bind to TFPI, wherein the peptide comprises an amino acid sequence that is at least 60% identical to the sequence of formula (X): Ac-GYASFPWFVQLHVHKRSWEMA-NH2 (SEQ ID NO:223). Within the scope of the disclosure, any peptide encompassed by any one of formulas (I) to (X) and any TFPI-binding peptide described herein are also referred to as "peptides of the present invention" and "peptides described herein".

[0107] In some embodiments, the peptides of the invention bind TFPI-I (eg, TFPI-1α) and, optionally, improve TFPI-regulated thrombin generation in the absence of FVIII, FIX, and / or FXI. Compositions (eg, pharmaceutical compositions) comprising the peptides are also provided.

[0108] In addition, the present invention provides methods of using the peptides of the present invention. For example, the present invention provides methods of inhibiting TFPI, the methods comprising contacting TFPI with a peptide as described herein. The present invention also provides methods for increasing thrombin formation in subjects with coagulation factor deficiencies, methods for increasing clot formation in subjects, and methods for treating coagulation disorders in subjects. The methods are referred to herein as, for example, "methods of the present invention" in their entirety. The methods include administering the peptides provided herein to subjects in an amount effective to increase thrombin formation, an amount effective to increase clot formation, or an amount effective to treat coagulation disorders in subjects. Unless expressly stated to the contrary, the descriptions of a peptide or a method of the present invention provided herein apply to each and all peptides and methods of the present invention, respectively. Other aspects of the present invention include the use of the peptides of the present invention in the manufacture of medicaments, methods for targeting cells that display TFPI, methods for treating or diagnosing subjects with a disease or at risk of disease, and methods for purifying TFPI. The foregoing methods are also referred to herein as, for example, "methods of the present invention" in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] Figure 1 is a schematic diagram of the coagulation cascade.

[0110] Figure 2 is a schematic diagram of the secondary structure of tissue factor pathway inhibitor-1.

[0111] Figure 3 Schematic diagram of the formation of a quaternary complex comprising tissue factor, coagulation factor Xa (FXa), coagulation factor VIIa (FVIIa) and TFPI.

[0112] Figure 4 is a listing of the amino acid sequences of various TFPI inhibitory peptides, with the amino acid substitutions (bold and underlined) of the reference peptide JBT0293 indicated.

[0113] Figure 5 Schematic diagram of mRNA display screening of TFPI inhibitory peptides.

[0114] Fig. 6A YesEC 50 Schematic diagram of binding ELISA, Figure 6B It is the IC described in Example 1 50 Schematic diagram of ELISA.

[0115] Figure 7 Binding ELISA curve of % OD (y-axis) versus concentration [nM] (x-axis) of biotinylated peptide JBT0132.

[0116] Figures 8A-8D Competition ELISA curves of % OD (y-axis) versus concentration [nM] (x-axis) of exemplary peptides of the invention.

[0117] Fig. 9A and 9B The induction graphs of peptides JBT0120 and JBT0132 are plotted in RU (y-axis) versus time (seconds) (x-axis).

[0118] Fig. 10A and 10B The induction graph of peptide JBT0120 interacting with tissue factor pathway inhibitor-1 and tissue factor pathway inhibitor-2 is plotted in RU (y-axis) versus time (seconds) (x-axis).

[0119] Fig.11A and 11B is a graph of thrombin generation (nM) (y-axis) versus time (minutes) (x-axis) for peptide JBT0120 and peptide JBT0132 in a plasma-based assay.

[0120] Figure 12-18 The table below lists the amino acid sequences of various TFPI inhibitory peptides; the EC values ​​of TFPI observed in the FXa inhibition assay are50 and percentage inhibition; EC of TFPI observed in exogenous tenase inhibition assay 50 and percentage of inhibition; and equivalent activity (mU / mL) of FEIBA, Factor VIII (FVIII) Immunate or Factor IX (FIX) in a plasma-based assay. "*" indicates a negative control.

[0121] Figure 19-21 The table lists the BIAcore analysis results of several TFPI-binding peptides. "*" indicates a negative control.

[0122] Figure 22-30 The table lists the amino acid sequences of various TFPI-binding peptides; the EC values ​​of TFPI observed in the FXa inhibition assay are 50 and percentage inhibition; EC of TFPI observed in exogenous tenase inhibition assay 50 and percentage of inhibition; and equivalent activity (mU / mL) of FEIBA, FVIII Immunate or FIX in a plasma-based assay. "*" indicates a negative control.

[0123] Fig.31 is a comparison of the pharmacokinetic profile of a PEGylated TFPI-binding peptide (peptide concentration (y-axis) versus time post-dose (x)) with that of the same peptide lacking PEG. The peptide was administered intravenously to C57B16 mice at a dose of 10 mg / kg. Three biological samples were analyzed for the presence of the peptide at each time point.

[0124] Figure 32-39 The table lists the amino acid sequences and IC 50 or EC 50 "*" indicates negative control. DETAILED DESCRIPTION

[0125] The present invention provides peptides that block tissue factor pathway inhibitor-1 (herein referred to as TFPI) in the coagulation cascade. After vascular injury, the tissue factor (TF) complex forms an "exogenous complex" or "exogenous tenase complex" with coagulation factor VIIa, which activates coagulation factors IX and X ( Figure 1 TFPI is the major natural regulator of the activity of the TF / FVIIa exogenous complex and, by extension, plays a role in controlling thrombin generation (Panteleev et al., Eur. J. Biochem., 249, 2016-2031 (2002)). TFPI is a 43 kDa serine protease inhibitor containing three Cooney-type inhibitory domains ( Figure 2TFPI's Cooney domain 1 binds FVIIa and Cooney domain 2 binds FXa, enabling the inhibitor to form a quaternary FXa-TFPI-FVIIa-TF complex that blocks the activity of the TF / FVIIa exogenous complex ( Figure 3 TFPI binding to FXa also downregulates the common pathway of the coagulation cascade, during which FXa converts prothrombin to thrombin (Audu et al., Anesth. Analg., 103(4), 841-845 (2006)). The present invention provides, for example, TFPI inhibitory peptides that block the inhibitory effect of TFPI on the coagulation cascade, thereby increasing thrombin formation.

[0126] The amino acid sequences of several TFPI-binding peptides are provided herein. Conventional amino acids are identified according to their standard one-letter or three-letter codes, as shown in Table 1.

[0127] Table 1

[0128]

[0129]

[0130] Unconventional amino acids and other peptide building blocks are identified according to the three-letter codes found in Table 2 (except Ttds, which are conventional four-letter abbreviations).

[0131] Table 2

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] The amino acid sequences of the peptides provided herein are described in the form of typical peptide sequences known to those of ordinary skill. For example, the three-letter code or single-letter code of conventional amino acids, or the three-letter code or abbreviation of unconventional amino acids, indicate that the amino acid is present in a specified position in the peptide sequence. The code of each unconventional amino acid is connected to the code of the next and / or previous amino acid in the sequence by a hyphen. Adjacent amino acids are connected by a chemical bond (typically an amide bond). The formation of the chemical bond is when the amino acid is located on the left side of the adjacent amino acid (e.g., Hle-adjacent amino acid), a hydroxyl group is removed from the amino acid, and when the amino acid is located on the right side of the adjacent amino acid (e.g., adjacent amino acid-Hle), a hydrogen is removed from the amino group of the amino acid. It is to be understood that both modifications are applicable to the same amino acid and to adjacent conventional amino acids present in the amino acid sequence where no hyphen is explicitly shown. In the case where the amino acid contains more than one amino and / or carboxyl group in the amino acid side chain, 2- or 3-amino and / or 1-carboxyl groups are generally used to form peptide bonds. For unconventional amino acids, a 3-letter code is used, in which the first letter indicates the stereochemistry of the C-α-atom. For example, an uppercase initial letter indicates the presence of an L-form amino acid in the peptide sequence, while a lowercase initial letter indicates the presence of the corresponding amino acid in the D-form in the peptide sequence. When a single-letter code is used, lowercase letters represent D-amino acids, while uppercase letters represent L-amino acids. Unless otherwise indicated, amino acid sequences are presented herein in an N- to C-terminal direction.

[0139] The C-terminus of several TFPI-binding peptide sequences described herein are explicitly indicated by abbreviations containing OH, NH2, or a specific terminal amine linked to the C-terminal amino acid code by a hyphen. The N-terminus of several peptides described herein are explicitly indicated by abbreviations containing hydrogen (for a free N-terminus) or a specific terminal carboxylic acid or other chemical group linked to the N-terminal amino acid by a hyphen.

[0140] The present invention provides a novel amino acid sequence X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 (SEQ ID NO:3109), wherein (using the single letter code for amino acids)

[0141] X7 is selected from L, P, K, S, W, V, N, and Q;

[0142] X8 is selected from L, R, N, F, and I;

[0143] X9 is selected from Y, V, P, and C;

[0144] X 10 Selected from F, L, and G;

[0145] X 11 Selected from L, W, V, A, M, T, and S;

[0146] X 12 Selected from T, F, V, R, A, D, L, E, S, and Y;

[0147] X 13 Selected from I, M, G, Q, D, and R;

[0148] X 14 Selected from G, W, Y, L, M, and H;

[0149] X 15 Selected from N, P, F, H, K, and Y;

[0150] X 16 Selected from M, D, E, V, G, and K;

[0151] X 17 Selected from G, I, R, S, T, and L;

[0152] X 18 Selected from M, K, L, and I;

[0153] X 19 Selected from Y, G, R, and S;

[0154] X 20 is selected from A, E, S, C, and Y; and

[0155] X 21 Selected from A, V, K, and E.

[0156] In addition to the core structure X7-X shown above 21 Specifically contemplated are other structures in which one or more additional amino acids are combined with the core structure X7-X 21 Thus, the present invention includes a peptide comprising a core structure and further comprising one or more N-terminal amino acids, wherein the N-terminal amino acids comprise an amino acid sequence selected from the group consisting of:

[0157] X6,

[0158] X5X6,

[0159] X4X5X6,

[0160] X3X4X5X6 (SEQ ID NO: 3110),

[0161] X2X3X4X5X6 (SEQ ID NO: 3111), and

[0162] X1X2X3X4X5X6(SEQ ID NO:3112);

[0163] wherein X6 is directly connected to X7 of the core structural amino acid sequence, and

[0164] X1 is selected from T and G;

[0165] X2 is selected from F and V;

[0166] X3 is selected from V, W, Y and F;

[0167] X4 is selected from D, Q and S;

[0168] X5 is selected from E, T, N and S; and

[0169] X6 is selected from R, H, K and A.

[0170] In one aspect, the peptide of the invention comprises or consists of the amino acid sequence QSKKNVFVFGYFERLRAK (SEQ ID NO: 1).

[0171] In another embodiment, the peptide of the present invention comprising a core structure comprises one or more C-terminal amino acids comprising an amino acid sequence selected from the group consisting of:

[0172] X 22 ,

[0173] X 22 X 23 ,

[0174] X 22 X 23 X 24 ,

[0175] X 22 X 23 X 24 X 25 (SEQ ID NO:3113),

[0176] X 22 X 23 X 24 X 25 X 26 (SEQ ID NO:3114), and

[0177] X 22 X 23 X24 X 25 X 26 X 27 (SEQ ID NO:3115),

[0178] Where X 22 X with the core structure amino acid sequence 21 Direct connection, and

[0179] X 22 Selected from Q, I, E, W, R, L, and N;

[0180] X 23 Selected from L, V, M, and R;

[0181] X 24 Selected from K, L, A, and Y;

[0182] X 25 It is F;

[0183] X 26 is G; and

[0184] X 27 It's T.

[0185] In one aspect, the peptides of the invention comprise or consist of the amino acid sequence VIVFTFRHNKLIGYERRY (SEQ ID NO: 4). It is also contemplated that the peptides of the invention comprise additional amino acids at both the N-terminus and the C-terminus of the core structure. In this case, the peptides comprise or consist of the following amino acid sequences: TFVDERLLYFLTIGNMGMYAAQLKF (SEQ ID NO: 3), GVWQTHPRYFWTMWPDIKGEVIVLFGT (SEQ ID NO: 5), KWFCGMRDMKGTMSCVWVKF (SEQ ID NO: 6), or ASFPLAVQLHVSKRSKEMA (SEQ ID NO: 7).

[0186] The present invention also includes a peptide comprising the following amino acid sequence: X3X4X5-F-X7-NVF-X 11 X I2 -GY-X 15 X 16 -RLRAK-X 22 (SEQ ID NO: 2), wherein X3 is Y or F; X4 is Q or S; X5 is N or S; X7 is K, N or Q; X 11 is V, A, S or T; X 12 is F, A, D, L, Q, S or Y; X 15 is F, K or Y; X 16 is E or D; and X22 It is L or N.

[0187] In addition, the present invention provides a peptide that binds TFPI, wherein the peptide comprises the structure of formula (I): X1001-X1002-X1003-X1004-X1005-X1006-X1007-X1008-X1009-X1010-X1011-X1012-X1013-X1014-X1015-X1016-X1017-X1018-X1019-X1020 (SEQ ID NO: 3116). In formula (I),

[0188] X1001 is an amino acid selected from the group consisting of Bhf, C, D, F, G, H, I, K, L, M, N, Nmf, Q, R, T, V, W, and Y;

[0189] X1002 is an amino acid selected from the group consisting of G, K and Q;

[0190] X1003 is an amino acid selected from the group consisting of A, Aib, Bhs, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;

[0191] X1004 is an amino acid selected from the group consisting of A, Aib, Bhk, C, D, E, F, G, H, I, K, K, L, M, N, Nmk, P, Q, R, S, T, V, W, and Y;

[0192] X1005 is an amino acid selected from a, A, Aib, Bal, C, D, d, E, F, G, H, K, k, L, M, N, Nmg, p, Q, R, S, T, V, W, and Y;

[0193] X1006 is an amino acid selected from the group consisting of A, Aib, Btq, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y;

[0194] X1007 is an amino acid selected from the group consisting of A, F, G, I, K, L, Nmv, P, Q, S, V, W, and Y;

[0195] X1008 is an amino acid selected from the group consisting of F, H, K, W and Y;

[0196] X1009 is an amino acid selected from the group consisting of A, Aib, f, I, K, S, T, and V;

[0197] X1010 is an amino acid selected from the group consisting of A, Aib, C, D, E, F, G, H, I, K, L, M, N, Nmf, P, Q, R, S, T, V, W, and Y;

[0198] X1011 is an amino acid selected from the group consisting of Aib, C, K, G and Nmg;

[0199] X1012 is Y;

[0200] X1013 is an amino acid selected from the group consisting of A, Aib, C, E, F, G, H, K, L, M, Q, R, W and Y;

[0201] X1014 is an amino acid selected from the group consisting of A, Aib, Bhe, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;

[0202] X1015 is an amino acid selected from (ω-methyl)-R, D, E, K and R;

[0203] X1016 is L;

[0204] X1017 is an amino acid selected from the group consisting of (ω-methyl)-R, A, Aib, Bhr, C, Cha, Cit, D, Dab, Dap, E, Eag, Eew, F, G, H, Har, Hci, Hle, I, K, L, M, N, Nle, Nva, Opa, Orn, Q, R, S, T, V, W, and Y;

[0205] X1018 is an amino acid selected from the group consisting of A, Bal, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y;

[0206] X1019 is an amino acid selected from the group consisting of Bhk, K, R and V.

[0207] X1020 is present or absent in formula (I) (i.e., in some cases, the peptide of the invention comprises the following structure: X1001-X1002-X1003-X1004-X1005-X1006-X1007-X1008-X1010-X1011-X1012-X1013-X1014-X1015-X1016-X1017-X1018-X1019 (SEQ ID NO: 3116). When X1020 is present, it is an amino acid selected from Aib, Bhl, C, F, G, H, I, K, L, Nml, Q, R, S, T, V, W and Y.

[0208] For example, the peptides of the present invention comprise the structure of formula (I), wherein X1001 is an amino acid selected from C, F, I, K, L, Nmf, V, M, W and Y; X1002 is Q; X1003 is an amino acid selected from A, C, D, E, H, K, M, I, N, Q, R, S, T and V; X1004 is an amino acid selected from A, Aib, C, D, E, G, H, F, I, K, k, L, M, N, Nmk, P, Q, R, S, V, W and Y; X1005 is an amino acid selected from a, A, Aib, Bal, C, d, E, D, F, G, H, K, k, L, M, N, Nmg, p, Q, R, S, T and Y; X1006 is an amino acid selected from A, Btq, C, D, G, I, K, H, L, M, N, Q, R, S, V and Y; X1007 is an amino acid selected from I, K, L, Q, V and Y; X1008 is an amino acid selected from F, H and Y; X1009 is an amino acid selected from f, I and V; X1010 is an amino acid selected from A, D, E, F, G, H, K, L, M , N, P, Q, R, S, T, V, W and Y; X1011 is an amino acid selected from G and Nmg; X1012 is Y; X1013 is an amino acid selected from Aib, C, F, H, L, W and Y; X1014 is an amino acid selected from A, Aib, Bhe, C, D, E, H, I, K, L, M, N, Q, R, S, T, V, W and Y; X1015 is an amino acid selected from E and R; X1016 is L; X1017 is an amino acid selected from (ω-methyl)-R, A, Aib, wherein X1018 is an amino acid selected from the group consisting of A, C, D, E, F, I, K, L, M, N, Q, R, V and W; X1019 is an amino acid selected from the group consisting of K and R; and X1020 is an amino acid selected from the group consisting of Aib, Bhl, F, K, L, R and W (when X1020 is present in the peptide).

[0209] In one aspect, a peptide of the invention comprises a structure of formula (I), wherein X1001 is an amino acid selected from F, L, Y and M; X1002 is Q; X1003 is an amino acid selected from M, Q, R, S, T and C; X1004 is an amino acid selected from Aib, K, L, P, R, E, G, I, Y, M and W; X1005 is an amino acid selected from a, Aib, D, d, G, H, K, k, N, Nmg, p, Q, R, A, E, C and M; X1006 is an amino acid selected from A, C, D, G, H, K, N, Q, R, S and M; X1007 is an amino acid selected from I and V; X1008 is an amino acid selected from F, H and Y; X1009 is V; X1010 is an amino acid selected from the group consisting of A, D, E, K, M, N, Q, R, F, H, P, S, V, W and Y; X1011 is G; X1012 is Y; X1013 is C or F; X1014 is an amino acid selected from the group consisting of A, C, D, E, K, L, M, N, Q, R, T, V and Aib; X1015 is R; X1016 is L; X1017 is an amino acid selected from the group consisting of A, Aib, C, Cha, Dab, Dap, Eag, Eew, H, Har, Hci, Hle, K, Nle, Nva, Opa, Orn, R, I, L, S and M; X1018 is an amino acid selected from the group consisting of A, L, N, M and R; X1019 is K; and X1020 is K or L.

[0210] When amino acid X1020 is absent from formula (I), the peptide of the invention in one aspect further comprises amino acid X1000 at the N-terminus of formula (I), such that the peptide comprises or consists of the structure of formula (II): X1000-X1001-X1002-X1003-X1004-X1005-X1006-X1007-X1008-X1009-X1010-X1011-X1012-X1013-X1014-X1015-X1016-X1017-X1018-X1019 (II) (SEQ ID NO: 3122). When X1000 is present in the peptide, X1000 is an amino acid selected from A, E and P, and the amino acids of X1001-X1019 are as defined above.

[0211] In another aspect, the TFPI-binding peptide of the present invention comprises a structure of formula (III): X1001-Q-X1003-X1004-X1005-X1006-I / V-X1008-V-X1010-GYC / F-X1014-RL-X1017-X1018-KK / L (III) (SEQ ID NO: 3117). In formula (III), X1001, X1003, X1004, X1005, X1006, X1008, X1010, X1014, X1017 and X1018 are each independently selected from any amino acid. For example, in formula (III),

[0212] X1001 is optionally an amino acid selected from Bhf, C, D, F, G, H, I, K, L, M, N, Nmf, Q, R, T, V, W, and Y, e.g., an amino acid selected from C, F, I, K, L, Nmf, V, M, W, and Y (e.g., an amino acid selected from F, L, Y, and M);

[0213] X1003 is optionally an amino acid selected from A, Aib, Bhs, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, e.g., an amino acid selected from A, C, D, E, H, K, M, I, N, Q, R, S, T, and V (e.g., the amino acid is M, Q, R, S, T, or C);

[0214] X1004 is optionally an amino acid selected from A, Aib, Bhk, C, D, E, F, G, H, I, K, k, L, M, N, Nmk, P, Q, R, S, T, V, W, and Y, e.g., an amino acid selected from A, Aib, C, D, E, G, H, F, I, K, k, L, M, N, Nmk, P, Q, R, S, V, W, and Y (e.g., an amino acid selected from Aib, K, L, P, R, E, G, I, Y, M, and W);

[0215] X1005 is optionally an amino acid selected from a, A, Aib, Bal, C, D, d, E, F, G, H, K, k, L, M, N, Nmg, p, Q, R, S, T, V, W, and Y, e.g., an amino acid selected from a, A, Aib, Bal, C, d, E, D, F, G, H, K, k, L, M, N, Nmg, p, Q, R, S, T, and Y (e.g., the amino acid is a, Aib, D, d, G, H, K, k, N, Nmg, p, Q, R, A, E, C, or M);

[0216] X1006 is optionally an amino acid selected from A, Aib, Btq, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y, such as an amino acid selected from A, Btq, C, D, G, I, K, H, L, M, N, Q, R, S, V, and Y (e.g., an amino acid selected from A, C, D, G, H, K, N, Q, R, S, and M);

[0217] X 1008 is optionally an amino acid selected from F, H, K, W and Y, for example an amino acid selected from F, H and Y;

[0218] X1010 is optionally an amino acid selected from A, Aib, C, D, E, F, G, H, I, K, L, M, N, Nmf, P, Q, R, S, T, V, W, and Y, e.g., an amino acid selected from A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, and Y (e.g., an amino acid selected from A, D, E, K, M, N, Q, R, F, H, P, S, V, W, and Y);

[0219] X1014 is optionally an amino acid selected from A, Aib, Bhe, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, e.g., an amino acid selected from A, Aib, Bhe, C, D, E, H, I, K, L, M, N, Q, R, S, T, V, W, and Y (e.g., A, C, D, E, K, L, M, N, Q, R, T, V, or Aib);

[0220] X1017 is optionally an amino acid selected from the group consisting of (ω-methyl)-R, A, Aib, Bhr, C, Cha, Cit, Dab, Dap, Eag, Eew, F, G, H, Har, Hci, Hle, I, K, L, M, N, Nle, Nva, Opa, Orn, Q, R, S, T, V, W, and Y, e.g., an amino acid selected from the group consisting of (ω-methyl)-R, A, Aib, Bhr, C, Cha, Cit, Dab, Dap, Eag, Eew, F, H, Har, Hci, Hle, I, K, L, M, N, Nle, Nva, Opa, Orn, R, S, T, V, and Y (e.g., an amino acid selected from the group consisting of A, Aib, C, Cha, Dab, Dap, Eag, Eew, H, Har, Hci, Hle, K, Nle, Nva, Opa, Orn, R, I, L, S, and M); and / or

[0221] X1018 is optionally an amino acid selected from A, Bal, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W and Y, e.g., an amino acid selected from A, C, D, E, F, I, K, L, M, N, Q, R, V and W (e.g., an amino acid selected from A, L, N, M and R).

[0222] In some embodiments, the peptides of the invention comprise one or more additional amino acid residues connected to the N- or C-terminus of the amino acid sequence. For example, in some embodiments, the peptide comprising any one of the structures of formula (I)-(III) further comprises one or more N-terminal amino acids directly connected to X1001, wherein the N-terminal amino acid comprises an amino acid sequence selected from the group consisting of:

[0223] X1000,

[0224] X999-X1000,

[0225] X998-X999-X1000,

[0226] X997-X998-X999-X1000 (SEQ ID NO:3123),

[0227] X996-X997-X998-X999-X1000 (SEQ ID NO:3124),

[0228] X995-X996-X997-X998-X999-X1000 (SEQ ID NO:3125),

[0229] X994-X995-X996-X997-X998-X999-X1000 (SEQ ID NO:3126),

[0230] X993-X994-X995-X996-X997-X998-X999-X1000 (SEQ ID NO:3127),

[0231] X992-X993-X994-X995-X996-X997-X998-X999-X1000 (SEQ ID NO:3128),

[0232] X991-X992-X993-X994-X995-X996-X997-X998-X999-X1000 (SEQ ID NO: 3129), and

[0233] X990-X991-X992-X993-X994-X995-X996-X997-X998-X999-X1000 (SEQ ID NO: 3130).

[0234] When the peptide comprises one or more N-terminal amino acids, X1000 is A or K; X999 is V or K; X998 is Q or K; X997 is L or K; X996 is R or K; X995 is G or K; X994 is V or K; X993 is G or K; X992 is S or K; X991 is K; and X990 is K.

[0235] In addition to the core structures shown in formulas (I)-(III), specifically contemplated other structures are those in which one or more additional amino acids are attached to the C-terminus of the core structure, which in turn is directly attached to X1020. For example, the C-terminal appendage optionally comprises an amino acid sequence selected from the group consisting of X1021, X1021-X1022, X1021-X1022-X1023, and X1021-X1022-X1023-X1024 (SEQ ID NO: 3131), wherein X1021 is T or K; X1022 is S or K; and X1023 and X1024 are K.

[0236] The present invention also includes a TFPI-binding peptide comprising or consisting of an amino acid sequence having at least 60% identity (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identity) to the amino acid sequence Ac-FQSK-Nmg-NVFVDGYFERL-Aib-AKL-NH2 (Formula IV) (SEQ ID NO: 164). As described herein, in some cases, the peptide comprises or consists of an amino acid sequence of any one of Formulas (I)-(III). The present invention also includes peptides containing or consisting of an amino acid sequence selected from SEQ ID NOs: 8-978 (for example, peptides containing or consisting of an amino acid sequence selected from SEQ ID NOs: 8-741 and 962-972 (for example, SEQ ID NOs: 8-741, 962-968, 971 or 972) and / or selected from 742-961 (for example, SEQ ID NOs: 744-961) and / or selected from SEQ ID NOs: 973-978).

[0237] In some cases, the peptides of the present invention contain intramolecular disulfide bonds. In this case, the peptides containing the structures of formula (I)-(III) contain at least two cysteine ​​residues (e.g., the peptides contain two cysteine ​​residues) separated by at least three amino acid residues, so that the cysteines form intramolecular disulfide bonds. In some cases, the cysteines are separated by more than three amino acid residues. For example, in the peptides containing the structures of formula (I), (II) or (III), any two of X1000, X1001, X1003, X1004, X1005, X1006, X1010, X1011, X1013, X1014, X1017, X1018, X1020 and X1021 are optionally cysteines capable of forming disulfide bridges. Thus, in some aspects, the peptide comprises two cysteine ​​residues: one of X1000, X1005, X1010 and X1014 is cysteine, and one of X1006, X1010, X1017 and X1021 is cysteine. The present invention contemplates all possible combinations of cysteine ​​pairs, such as X1000 and X1006 are C; X1000 and X1010 are C; X1000 and X1017 are C; X1005 and X1017 are C; X1010 and X1017 are C; X1010 and X1021 are C; or X1014 and X1021 are C.

[0238] The present invention also provides a TFPI-binding peptide, the peptide comprising a structure of formula (V): X2001-X2002-X2003-X2004-X2005-X2006-[X2007-X2008-X2009-X2010-X2011-X2012-X2013-X2014-X2015-X2016-X2017-X2018]-X2019-X2020-X2021-X2022-X2023 (V) (SEQ ID NO: 3118), wherein the peptide forms a cyclic structure through a bond, such as a disulfide bond, between X2007 and X2018 (represented as brackets within formula (V)). In formula (V), X2001, X2002, and X2023 are independently present or absent. When present, X2001 is an amino acid selected from A, D, E, F, G, H, I, K, L, P, R, S, T, V, and W; X2002 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, P, R, S, T, V, and W; and X2023 is an amino acid selected from A, D, E, F, G, I, K, L, R, S, T, V, W, and Y. Additionally,

[0239] X2003 is an amino acid selected from the group consisting of A, F, I, K, L, R, S, T, V, W, and Y;

[0240] X2004 is an amino acid selected from the group consisting of A, D, E, F, G, I, K, L, R, S, T, V, and W;

[0241] X2005 is W;

[0242] X2006 is an amino acid selected from the group consisting of F, H, I, K, L, R, V, and W;

[0243] X2007 is an amino acid selected from the group consisting of C, Hcy, Dap, and K (e.g., C or Hcy);

[0244] X2008 is an amino acid selected from the group consisting of A, G, R, S and T;

[0245] X2009 is an amino acid selected from a, A, I, K, L, M, m, Nle, p, R, Sem, and V;

[0246] X2010 is an amino acid selected from the group consisting of A, G, I, K, L, P, R, S, T, and V;

[0247] X2011 is an amino acid selected from the group consisting of D, E, G, S and T;

[0248] X2012 is an amino acid selected from the group consisting of A, a, D, d, E, e, F, f, G, I, K, k, L, I, M, m, Nle, nle, P, p, R, r, S, s, Sem, T, t, V, v, W, and w;

[0249] X2013 is an amino acid selected from the group consisting of A, D, d, E, e, F, G, I, K, L, R, S, s, T, V, and W;

[0250] X2014 is an amino acid selected from the group consisting of A, D, E, F, G, I, K, L, M, R, S, T, V, and W;

[0251] X2015 is an amino acid selected from the group consisting of A, D, E, F, G, I, K, L, M, Nle, R, S, T, V, and W;

[0252] X2016 is an amino acid selected from the group consisting of A, D, E, F, I, K, L, M, Nle, R, S, Sem, T, V, W, and Y;

[0253] X2017 is an amino acid selected from the group consisting of A, D, E, F, G, I, K, L, R, S, T, V, W, and Y;

[0254] X2018 is an amino acid selected from C and D (e.g., X2018 is C);

[0255] X2019 is an amino acid selected from the group consisting of A, F, I, L, S, T, V, and W;

[0256] X2020 is an amino acid selected from F and W;

[0257] X2021 is an amino acid selected from I, L and V; and

[0258] X2022 is an amino acid selected from the group consisting of A, D, E, F, G, I, K, L, P, R, S, T, V and W.

[0259] In some cases, the peptides of the invention comprise the structure of formula (V),

[0260] X2001 is optionally an amino acid selected from A, D, F, G, H, K, L, P and S, for example an amino acid selected from A, D, F, G, H, K, L and S (when X2001 is present);

[0261] X2002 is optionally an amino acid selected from A, D, F, G, H, K, L, P, R and S, such as an amino acid selected from A, F, H, K, L, M, R and S (e.g., H, F, M or R) (when X2002 is present);

[0262] X2003 is optionally an amino acid selected from A, F, K, L, S and Y, such as an amino acid selected from F, S and Y (e.g., F or Y);

[0263] X2004 is optionally an amino acid selected from A, D, F, G, K, L and S (eg, K);

[0264] X2005 is optionally W;

[0265] X2006 is optionally an amino acid selected from F, H, K and L (eg, F or H);

[0266] X2007 is optionally an amino acid selected from C and HcY (eg, X2007 is C);

[0267] X2008 is optionally an amino acid selected from A, G and S;

[0268] X2009 is optionally an amino acid selected from a, A, K, L, V, M, m, Nle, Sem and p, such as an amino acid selected from M, Nle, p and V (e.g., M, Sem or V);

[0269] X2010 is optionally an amino acid selected from A, G, K, L, P, R and S, such as an amino acid selected from A, K, L, P, R and S (eg, K, P or R);

[0270] X2011 is optionally an amino acid selected from D, G and S (eg, D or S);

[0271] X2012 is optionally an amino acid selected from A, a, D, d, F, f, G, K, k, L, I, M, m, Nle, P, S, and s, such as an amino acid selected from D, d, F, f, G, K, k, L, I, M, Nle, P, S, and Sem (e.g., an amino acid selected from F, L, I, Sem, and M);

[0272] X2013 is optionally an amino acid selected from A, D, d, F, G, K, L, S and s, such as an amino acid selected from A, D, F, G, K, L and S (e.g., D, G, K or S);

[0273] X2014 is optionally an amino acid selected from D, F, G, K, L and S (eg, D or G);

[0274] X2015 is optionally an amino acid selected from A, D, F, G, I, K, L, M, Nle, S and T (e.g., I or T);

[0275] X2016 is optionally an amino acid selected from D, F, K, L, M, Nle, S and Y, such as an amino acid selected from D, F, K, L, M, Nle, S, Sem and Y (e.g., D, F, M, Sem or Y);

[0276] X2017 is optionally an amino acid selected from A, D, F, G, K, L, S, T and Y (eg, S or T);

[0277] X2018 is optional C;

[0278] X2019 is optionally an amino acid selected from A, F, L, S and V (eg, A or V);

[0279] X2020 is optionally an amino acid selected from F and W (eg, W);

[0280] X2021 is optionally an amino acid selected from L and V (eg, V);

[0281] X2022 is optionally an amino acid selected from A, D, F, G, K, L, P, R, S and W, e.g., an amino acid selected from A, F, G, K, L, P, R, S and W (e.g., an amino acid selected from F, L, K, R, P and W); and

[0282] X2023 is optionally an amino acid selected from A, D, F, G, K, L, M, S and Y, such as an amino acid selected from A, D, F, G, L, M, S and Y (e.g., an amino acid selected from A, D, F, M, S and Y) (when X2023 is present).

[0283] The present invention also includes a TFPI-binding peptide, wherein the peptide comprises a structure of formula (VI): X2001-X2002-F / YKWF / H-[C-X2008-M / V-X2010-D-X2012-X2013-GI / T-X2016-S / TC]-A / VWV-X2022-X2023 (VI) (SEQ ID NO: 3119). In the peptide comprising the structure of formula (VI), X2001, X2002 and X2023 are each independently present or absent. If X2001, X2002, and / or X2023 are present, any one of X2001, X2002 and X2023 is independently selected from any amino acid. In addition, X2008, X2010, X2012, X2013, X2016 and X2022 are each independently selected from any amino acid.

[0284] In certain cases, in the peptide of formula (VI),

[0285] X2001 is optionally an amino acid selected from A, D, E, F, G, H, I, K, L, P, R, S, T, V and W, e.g., an amino acid selected from A, D, F, G, H, K, L, P and S (e.g., an amino acid selected from A, D, F, G, H, K, L and S) (when X2001 is present);

[0286] X2002 is optionally an amino acid selected from A, D, E, F, G, H, I, K, L, M, P, R, S, T, V and W, e.g., an amino acid selected from A, D, F, G, H, K, L, M, P, R and S (e.g., an amino acid selected from A, F, H, K, L, M, R and S, e.g., H, F, M or R) (when X2002 is present);

[0287] X2008 is optionally an amino acid selected from A, G, R, S and T, for example an amino acid selected from A, G and S;

[0288] X2010 is optionally an amino acid selected from A, G, I, K, L, P, R, S, T and V, such as an amino acid selected from A, G, K, L, P, R and S (e.g., an amino acid selected from A, K, L, P, R and S, such as K, P or R);

[0289] X2012 is optionally an amino acid selected from A, a, D, d, E, e, F, f, G, I, I, K, k, L, I, M, m, Nle, nle, P, p, R, r, S, s, Sem, T, t, V, v, W, and w, such as an amino acid selected from A, a, D, d, F, f, G, K, k, L, I, M, m, Nle, P, S, s, and Sem (e.g., an amino acid selected from D, d, F, f, G, K, k, L, I, M, Nle, P, S, and Sem, such as F, L, I, Sem, or M);

[0290] X2013 is optionally an amino acid selected from A, D, d, E, e, F, G, I, K, L, R, S, s, T, V, and W, e.g., an amino acid selected from A, D, d, F, G, K, L, S, and s (e.g., an amino acid selected from A, D, F, G, K, L, and S, e.g., D, G, K, or S);

[0291] X2016 is optionally an amino acid selected from A, D, E, F, I, K, L, M, Nle, R, S, Sem, T, V, W and Y, such as an amino acid selected from D, F, K, L, M, Nle, S, Sem and Y (e.g., an amino acid selected from D, F, K, L, M, Nle, S, Sem, such as F, Sem or M);

[0292] X2022 is optionally an amino acid selected from A, D, E, F, G, I, K, L, P, R, S, T, V and W, e.g., an amino acid selected from A, D, F, G, K, L, P, R, S and W (e.g., an amino acid selected from A, F, G, K, L, P, R, S and W, e.g., F, L, K, R, P or W); and / or

[0293] X2023 is optionally an amino acid selected from A, D, E, F, G, I, K, L, R, M, S, T, V, W and Y, e.g., an amino acid selected from A, D, F, G, K, L, M, S and Y (e.g., an amino acid selected from A, D, F, G, L, M, S and Y, e.g., A, D, F, M, S or Y) (when X2023 is present).

[0294] In one aspect, the TFPI-binding peptide of the invention comprises an amino acid sequence that is at least 60% identical (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identical) to the sequence of Formula VII: Ac-FYYKWH[CGMRDMKGTMSC]AWVKF-NH2(VII) (SEQ ID NO: 1040). Optionally, the peptide comprises or consists of an amino acid sequence of Formula (V)-(VII) as defined herein. The present invention also includes peptides comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1001-1293 (for example, peptides comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1001-1212 and 1290-1291 (for example, SEQ ID NOs: 1001-120, 1290 or 1291) and / or selected from SEQ ID NOs: 1213-1289 and / or selected from 1292 and 1293).

[0295] The present invention also provides a TFPI-binding peptide, the peptide comprising the structure of formula (VIII): X3001-X3002-X3003-X3004-X3005-X3006-X3007-X3008-X3009-X3010-X3011-X3012-X3013-X3014-X3015-X3016-X3017-X3018-X3019-X3020-X3021 (VIII) (SEQ ID NO: 3120). In formula (VIII), X3001 and X3002 are independently present or absent in the peptide. If present, X3001 is an amino acid selected from A, C, D, F, G, I, K, L, M, N, P, Q, R, S, T, W, E, H, and Y; and X3002 is an amino acid selected from A, C, D, F, H, K, M, N, P, R, S, T, W, Y, G, I, and L. Additionally,

[0296] X3003 is an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, and Y;

[0297] X3004 is an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y, and P;

[0298] X3005 is an amino acid selected from the group consisting of C, D, F, G, H, I, K, L, M, N, P, R, S, T, V, W, and Y;

[0299] X3006 is an amino acid selected from the group consisting of A, W, C, K, P, R and H;

[0300] X3007 is an amino acid selected from the group consisting of Q, A, C, F, G, H, I, K, L, N, R, S, T, W, and Y;

[0301] X3008 is an amino acid selected from the group consisting of A, C, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, Y, and I;

[0302] X3009 is an amino acid selected from the group consisting of A, C, F, G, H, I, L, M, R, S, T, V, W, Y, and K;

[0303] X3010 is an amino acid selected from the group consisting of A, C, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;

[0304] X3011 is an amino acid selected from the group consisting of A, G, I, K, L, M, N, Q, R, S, T, V, W, Y, C, F, and H;

[0305] X3012 is an amino acid selected from the group consisting of A, C, H, I, K, L, and R;

[0306] X3013 is an amino acid selected from the group consisting of A, C, F, G, H, K, L, M, R, S, V, W, Y, and I;

[0307] X3014 is an amino acid selected from the group consisting of A, C, F, G, H, I, L, M, N, Q, R, S, T, V, W, Y, and K;

[0308] X3015 is an amino acid selected from the group consisting of A, K and R;

[0309] X3016 is an amino acid selected from the group consisting of A, F, K and R;

[0310] X3017 is an amino acid selected from the group consisting of A, C, F, G, I, K, L, N, Q, R, S, T, V, W, Y, H, A, and M;

[0311] X3018 is an amino acid selected from the group consisting of A, C, F, I, K, L, M, Q, R, V, W, and Y;

[0312] X3019 is an amino acid selected from the group consisting of A, C, D, E, F, G, H, K, L, N, P, Q, R, V, W, Y, and I;

[0313] X3020 is an amino acid selected from the group consisting of A, C, F, G, H, K, L, M, N, Q, R, V, W, Y, I, and P; and

[0314] X3021 is an amino acid selected from A, C, H, I, K, L, M, N, P, Q, R, T, V, W, Y, F and G.

[0315] In some aspects of the invention, the peptide comprises a sequence of formula (VIII), wherein

[0316] X3001 is optionally an amino acid selected from A, C, D, G, I, K, L, M, N, P, Q, R, S, T, W, E, H and Y, for example, an amino acid selected from A, C, D, G, K, L, M, N, P, R, S, T, E, H and Y (when X3001 is present);

[0317] X3002 is optionally an amino acid selected from C, F, H, K, R, S, W, Y, G, I and L, such as an amino acid selected from C, K, R, W, Y, G, I and L (when X3002 is present);

[0318] X3003 is optionally an amino acid selected from A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T and W, for example, an amino acid selected from A, C, G, H, I, K, L, M, R, S, T and W;

[0319] X3004 is optionally an amino acid selected from A, C, D, G, H, I, K, L, M, N, R, S, T, V, and P, for example, an amino acid selected from A, C, G, H, I, K, L, M, N, R, S, T, and P;

[0320] X3005 is optionally an amino acid selected from C, F, H, I, K, M, R, T, W and Y, for example an amino acid selected from C, F, H, K, R and W;

[0321] X3006 is optionally an amino acid selected from P, H and A;

[0322] X3007 is optionally an amino acid selected from C, G, R, W, A and L, such as an amino acid selected from L, C, R and W;

[0323] X3008 is optionally an amino acid selected from A, C, F, G, H, K, L, M, N, Q, R, T, V, W, Y and I, for example, an amino acid selected from A, C, F, H, K, R, V, W, Y and I;

[0324] X3009 is optionally an amino acid selected from C, I, R, V and K, for example an amino acid selected from C, R, V and K;

[0325] X3010 is optionally an amino acid selected from A, C, G, H, I, K, L, M, Q, R, S and T, for example an amino acid selected from A, C, K, L, Q, R and S;

[0326] X3011 is optionally an amino acid selected from A, I, K, L, M, R, S, V, W, C, F and H, for example, an amino acid selected from I, K, L, M, R, V, W, C, F and H;

[0327] X3012 is optionally an amino acid selected from H and R (eg, H);

[0328] X3013 is optionally an amino acid selected from C, F, K, L, M, R, V and I, for example an amino acid selected from C, K, R, V and I;

[0329] X3014 is optionally an amino acid selected from A, M, C, F, H, I, L, N, R, S, V, W, and K, such as an amino acid selected from A, S, C, F, H, I, R, and K;

[0330] X3015 can be K or R;

[0331] X3016 can be K or R;

[0332] X3017 is optionally an amino acid selected from A, C, F, G, I, K, L, N, Q, R, S, T, V, W, H, A, and M, for example, an amino acid selected from C, G, I, K, L, N, Q, R, S, T, V, H, A, and M;

[0333] X3018 is optionally an amino acid selected from A, K, C, I, L, R and W (e.g., K, C, I, R or W);

[0334] X3019 is optionally an amino acid selected from A, C, E, H, K, N, Q, R and I, for example an amino acid selected from C, E, H, K, R and I;

[0335] X3020 is optionally an amino acid selected from C, H, L, M, R, V, I and P (e.g., C, M, I or P); and

[0336] X3021 is optionally an amino acid selected from A, C, H, I, K, L, M, N, Q, R, V, W, Y, F and G, for example, an amino acid selected from A, C, H, I, K, L, M, N, Q, R, V, W, F and G.

[0337] The present invention also provides a TFPI-binding peptide comprising a structure of formula (IX): X3001-X3002-X3003-X3004-X3005-X3006-X3007-X3008-X3009-X3010-X3011-H-X3013-X3014-K / RR-X3017-X3018-X3019-X3020-X3021 (IX) (SEQ ID NO: 3121). In formula (IX), X3001 and X3002 are independently present or absent in the peptide. If present, X3001 and / or X3002 are independently selected from any amino acid. Likewise, X3003, X3004, X3005, X3006, X3007, X3008, X3009, X3010, X3011, X3013, X3014, X3017, X3018, X3019, X3020, and X3021 are each independently selected from any amino acid. When present, X3001 is optionally an amino acid selected from A, C, D, F, G, I, K, L, M, N, P, Q, R, S, T, W, E, H, and Y, such as an amino acid selected from A, C, D, G, I, K, L, M, N, P, Q, R, S, T, W, E, H, and Y (e.g., an amino acid selected from A, C, D, G, K, L, M, N, P, R, S, T, E, H, and Y). Likewise, when present, X3002 is optionally an amino acid selected from A, C, D, F, H, K, M, N, P, R, S, T, W, Y, G, I, and L, such as an amino acid selected from C, F, H, K, R, S, W, Y, G, I, and L (e.g., an amino acid selected from C, K, R, W, Y, G, I, and L). Also with respect to formula (IX),

[0338] X3003 is optionally an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, and Y, e.g., an amino acid selected from A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, and W (e.g., an amino acid selected from A, C, G, H, I, K, L, M, R, S, T, and W);

[0339] X3004 is optionally an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y, and P, e.g., an amino acid selected from A, C, D, G, H, I, K, L, M, N, R, S, T, V, and P (e.g., an amino acid selected from A, C, G, H, I, K, L, M, N, R, S, T, and P);

[0340] X3005 is optionally an amino acid selected from C, D, F, G, H, I, K, L, M, N, P, R, S, T, V, W, and Y, e.g., an amino acid selected from C, F, H, I, K, M, R, T, W, and Y (e.g., an amino acid selected from C, F, H, K, R, and W);

[0341] X3006 is optionally an amino acid selected from A, W, C, K, P, R and H, such as an amino acid selected from P, H and A;

[0342] X3007 is optionally an amino acid selected from Q, A, C, F, G, H, I, K, L, N, R, S, T, W, and Y, e.g., an amino acid selected from C, G, R, W, A, and L (e.g., L, C, R, or W);

[0343] X3008 is optionally an amino acid selected from A, C, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, Y, and I, e.g., an amino acid selected from A, C, F, G, H, K, L, M, N, Q, R, T, V, W, Y, and I (e.g., an amino acid selected from A, C, F, H, K, R, V, W, Y, and I);

[0344] X3009 is optionally an amino acid selected from A, C, F, G, H, I, L, M, R, S, T, V, W, Y, and K, such as an amino acid selected from C, I, R, V, and K (e.g., C, R, V, or K);

[0345] X3010 is optionally an amino acid selected from A, C, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, e.g., an amino acid selected from A, C, G, H, I, K, L, M, Q, R, S, and T (e.g., an amino acid selected from A, C, K, L, Q, R, and S);

[0346] X3011 is optionally an amino acid selected from A, G, I, K, L, M, N, Q, R, S, T, V, W, Y, C, F, and H, e.g., an amino acid selected from A, I, K, L, M, R, S, V, W, C, F, and H (e.g., an amino acid selected from I, K, L, M, R, V, W, C, F, and H);

[0347] X3013 is optionally an amino acid selected from A, C, F, G, H, K, L, M, R, S, V, W, Y, and I, e.g., an amino acid selected from C, F, K, L, M, R, V, and I (e.g., C, K, R, V, or I);

[0348] X3014 is optionally an amino acid selected from A, C, F, G, H, I, L, M, N, Q, R, S, T, V, W, Y, and K, e.g., an amino acid selected from A, M, C, F, H, I, L, N, R, S, V, W, and K (e.g., an amino acid selected from A, S, C, F, H, I, R, and K);

[0349] X3017 is optionally an amino acid selected from A, C, F, G, I, K, L, N, Q, R, S, T, V, W, Y, H, A, and M, e.g., an amino acid selected from A, C, F, G, I, K, L, N, Q, R, S, T, V, W, H, A, and M (e.g., an amino acid selected from C, G, I, K, L, N, Q, R, S, T, V, H, A, and M);

[0350] X3018 is optionally an amino acid selected from A, C, F, I, K, L, M, Q, R, V, W, and Y, e.g., an amino acid selected from A, K, C, I, L, R, and W (e.g., K, C, I, R, or W);

[0351] X3019 is optionally an amino acid selected from A, C, D, E, F, G, H, K, L, N, P, Q, R, V, W, Y, and I, e.g., an amino acid selected from A, C, E, H, K, N, Q, R, and I (e.g., C, E, H, K, R, or I);

[0352] X3020 is optionally an amino acid selected from A, C, F, G, H, K, L, M, N, Q, R, V, W, Y, I, and P, e.g., an amino acid selected from C, H, L, M, R, V, I, and P (e.g., C, M, I, or P); and / or

[0353] X3021 is optionally an amino acid selected from A, C, H, I, K, L, M, N, P, Q, R, T, V, W, Y, F, and G, e.g., an amino acid selected from A, C, H, I, K, L, M, N, Q, R, V, W, Y, F, and G (e.g., an amino acid selected from A, C, H, I, K, L, M, N, Q, R, V, W, F, and G).

[0354] In some cases, the TFPI-binding peptides of the invention comprise an amino acid sequence that is at least 60% identical (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identical) to the sequence of formula (X): AC-GYASFPWFVQLHVHKRSWEMA-NH2(X) (SEQ ID NO:223). Optionally, the peptide comprises or consists of an amino acid sequence of formula (VIII)-(IX) as defined herein. As used herein, "at least 60% identity" and similar terms include any integer from, for example, 60% to 100%, such as 60%, 61%, 62%, etc. In addition, the term "at least [percent] identity" includes any percentage greater than or equal to the number of identical amino acids divided by the total number of amino acids in the peptides of the invention ([at least percent identity] ≥ [number of identical amino acids] / [total number of amino acids in the peptides of the invention]).

[0355] The present invention also includes peptides containing an amino acid sequence selected from SEQ ID NOs: 2001-2498 or consisting of the said sequence (for example, peptides containing an amino acid sequence selected from SEQ ID NOs: 2001-2296 and 2498 (for example, SEQ ID NOs: 2001-2126, 2128-2296 or 2498) and / or selected from SEQ ID NOs: 2297-2497 (for example, SEQ ID NOs: 2298-2497) or consisting of the said sequence). The present invention also provides peptides containing or consisting of an amino acid sequence selected from SEQ ID NOs: 3001-3108 (for example, peptides containing or consisting of an amino acid sequence selected from SEQ ID NOs: 3001-3064 (for example, SEQ ID NOs: 3001-3048, 3051-3053, 3055 or 3057-3064) and / or selected from SEQ ID NOs: 3065-3084 (for example, SEQ ID NOs: 3066-3084) and / or selected from SEQ ID NOs: 3085-3108).

[0356] The peptides of SEQ ID NOs: 1-7 in some cases further comprise one or more amino acids linked to the N- or C-terminus of SEQ ID NOs: 1-7. For example, the present invention includes peptides comprising or consisting of the following amino acid sequences: JBT0047, JBT0051, JBT0055, JBT0131, JBT0132, JBT0133, JBT0155, JBT0158, JBT0162, JBT0163, JBT0164, JBT0166, JBT0169, JBT0170, JBT0171, JBT0174, JBT0175 or JBT0293, all of which comprise the amino acid sequence SEQ ID NO: 1. Exemplary peptides comprising the amino acid sequence of SEQ ID NO: 2 include peptides comprising or consisting of the following amino acid sequences: JBT0294, JBT0295, JBT0296, JBT0297, JBT0298, JBT0299, JBT0300, JBT0301, JBT0302, JBT0303, JBT0304, JBT0305, JBT0306, JBT0307, ​​JBT0308, JBT0309, JBT0310, or JBT0311. Exemplary peptides comprising the amino acid sequence of SEQ ID NO: 3 include or consisting of the following amino acid sequences: JBT0049, JBT0053, JBT0057, JBT0190, JBT0193, or JBT0197. The present invention also includes peptides comprising or consisting of the following amino acid sequences: JBT0050, JBT0054, JBT0058, JBT0129, JBT0130, JBT0205, JBT0208, JBT0211, JBT0212, JBT0217, JBT0218 or JBT0219, all of which contain the amino acid sequence SEQ ID NO: 4. Exemplary peptides comprising the amino acid sequence SEQ ID NO: 5 include peptides comprising or consisting of the following amino acid sequence: JBT0101, JBT0052, JBT0103, JBT0178 or JBT0182. The present invention also includes peptides comprising or consisting of the following amino acid sequences: JBT0120, JBT0124, JBT0247, JBT0248, JBT0251 or JBT0252, each of which contains the amino acid sequence SEQ ID NO: 6. The present invention also provides a peptide comprising the amino acid sequence SEQ ID NO: 7, for example, a peptide comprising or consisting of the following amino acid sequence: JBT0122, JBT0126, JBT0221, JBT0224, JBT0225, JBT0226, JBT0228, JBT0232 or JBT0233. The peptides described herein are shown in Table 5 of Example 1 and Figure 12-18 middle.

[0357] In certain embodiments, the peptides of the invention comprise or consist of the amino acid sequence JBT0047, JBT0049, JBT0101, JBT0120 or JBT0122, or any of the peptides of the invention described herein, or any variants of the foregoing (e.g., a peptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1-3108, for example, a peptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 8-741, 744-968, 971-978, 1001-1210, 1213-1289, 1290-1293, 2001-2126, 2128-2296, 2298-2498, 3001-3048, 3051-3053, 3055, 3057-3064, and 3067-3108). "Variant" refers to a peptide comprising one or more amino acid substitutions, amino acid deletions or amino acid additions relative to a parent amino acid sequence. Variants include, but are not limited to, peptides having an amino acid sequence that is at least 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any amino acid sequence provided herein, while retaining the ability to bind to TFPI and / or inhibit TFPI activity. In one embodiment, the peptide comprises or consists of the amino acid sequence JBT0132, JBT0303, JBT0193, JBT0178, JBT0120 or JBT0224.

[0358] In one aspect, the peptides of the invention are composed of less than 40 amino acids, such as less than 35 amino acids. Optionally, the peptides of the invention are composed of less than 25 amino acids, or less than 10 amino acids. In various embodiments, the peptides comprise 15-35 amino acid residues (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 amino acid residues). However, it is also contemplated that the peptides described herein that comprise one or more deletions are also suitable for the scope of the present invention, as long as the peptides block TFPI inhibition of the coagulation cascade and optionally bind to TFPI. In one aspect, the peptides comprising one or more deletions are suitable for the scope of the present invention, as long as the peptides bind to TFPI and optionally block TFPI inhibition of the coagulation cascade. In some cases, the amino acids are taken from within the amino acid sequence, at the N-terminus and / or at the C-terminus. Such peptide fragments can contain 3-14 amino acid residues (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid residues).

[0359] Optionally, the peptides of the invention comprise one or more amino acid substitutions that do not disrupt the ability of the peptide to bind and / or inhibit TFPI (cf. any amino acid sequence provided herein). For example, a peptide comprising or consisting of an amino acid sequence selected from JBT0294, JBT0295, JBT0296, JBT0297, JBT0298, JBT0299, JBT0300, JBT0301, JBT0302, JBT0303, JBT0304, JBT0305, JBT0306, JBT0307, ​​JBT0308, JBT0309, JBT0310 or JBT0311 is a substitution mutant of the amino acid sequence of JBT0293 (amino acid sequence SEQ ID NO: 1 is directly linked to a phenylalanine residue at the N-terminus and a lysine residue at the C-terminus) (see Figure 4). Amino acid substitutions include, but are not limited to, substitutions that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity, and / or (4) provide or alter other physiochemical or functional properties of the peptide. In one aspect, the substitution is a conservative substitution, in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), beta-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). However, it will be appreciated that one is not limited to making conservative substitutions, so long as the resulting peptide retains the ability to fully or partially downregulate TFPI activity. The present invention also encompasses TFPI inhibitory peptides comprising atypical, non-naturally occurring amino acids, which are well known in the art.Exemplary non-naturally occurring amino acids include ornithine, citrulline, hydroxyproline, homoserine, phenylglycine, taurine, iodotyrosine, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, γ-aminobutyric acid, 2-aminoisobutyric acid, 3-aminopropionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, Acid, fluorinated amino acids, 3-methyl amino acids, α-C-methyl amino acids, N-methyl amino acids, 2-aminoisobutyric acid, β-homoglutamic acid, β-homophenylalanine, β-homolysine, β-homoleucine, β-homoasparagine, β-homoglutamine, β-homoarginine, β-homoserine, β-homotyrosine, β-homoaspartic acid, β-homovaline, β-homoasparagine, (S)-cyclohexylalanine, (S)-citrulline, (S)-2, 4-diaminobutyric acid, (S)-2,4-diaminobutyric acid, (S)-diaminopropionic acid, (S)-2-propargylglycine, (S)-N(ω)-nitroarginine, L-homophenylalanine, (S)-homoarginine, (S)-homocitrulline, (S)-homocysteine, (S)-2-amino-5-methylhexanoic acid, (S)-homolysine, (S)-norleucine, (S)-N-methylalanine, (S)-N-methylaspartic acid, (S)- (S)-N-methylglutamate, (S)-N-methylphenylalanine, N-methylglycine, (S)-N-methyllysine, (S)-N-methylleucine, (S)-N-methylarginine, (S)-N-methylserine, (S)-N-methylvaline, (S)-N-methyltyrosine, (S)-2-aminopentanoic acid, (S)-2-pyridylalanine, (S)-ornithine, L-phenylglycine, 4-phenylbutyric acid and selenomethionine. Although all amino acids in the peptide typically adopt L stereochemistry, individual amino acids can have L or D stereochemistry where appropriate.

[0360] The present invention also includes TFPI inhibitory peptide variants, which include one or more amino acids inserted into the amino acid sequence provided herein and / or connected to the N-terminus or C-terminus. In one aspect, the peptide also includes one or more amino acids that promote the synthesis, operation, or use of the peptide, including but not limited to one or two lysines at the N-terminus and / or C-terminus to increase the solubility of the peptide. Suitable fusion proteins include but are not limited to proteins comprising a TFPI inhibitory peptide connected to one or more polypeptides, polypeptide fragments or amino acids that are generally not considered to be part of the protein sequence. In one aspect, the fusion peptide comprises the entire amino acid sequence of two or more peptides, or comprises a portion (fragment) of two or more peptides. In addition to all or part of the TFPI inhibitory peptide described herein, the fusion protein also optionally includes all or part of any appropriate peptide containing the desired biological activity / function. In fact, in some cases, the TFPI inhibitory peptide is operably linked to, for example, one or more of the following substances: a peptide with a long circulating half-life, a marker protein, a peptide that promotes the purification of the TFPI inhibitory peptide, a peptide sequence that promotes the formation of a multimeric protein, or a fragment of any of the foregoing. In one embodiment, two or more TFPI inhibitory peptides are connected by a multimerization domain or fused together by a chemical bond to produce a TFPI inhibitory peptide complex. The TFPI inhibitor peptides may be the same or different.

[0361] "Derivatives" are included in the present invention and include being chemically modified in some manner other than the addition, deletion or substitution of amino acids. In this regard, the peptides of the present invention provided herein are chemically bonded to polymers, lipids, other organic moieties and / or inorganic moieties. Examples of peptide and protein modifications are provided in Hermanson, Bioconjugate Techniques, Academic Press, (1996). In some cases, derivatives are prepared to increase solubility, stability, absorption or circulation half-life. A variety of chemical modifications eliminate or reduce any adverse side effects of the agent. In this regard, the present invention includes TFPI inhibitory peptides covalently modified to include one or more water-soluble polymer links. Useful polymers known in the art include, but are not limited to, polyethylene glycol (PEG) (e.g., PEG of about 40 kD or 1 kD in size), polyoxyethylene glycol, polypropylene glycol, monomethoxy-polyethylene glycol, dextran, cellulose, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, polyoxypropylene / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, and mixtures of any of the foregoing. In one aspect, the peptides of the present invention are PEGylated peptides. For further discussion of water-soluble polymer linkers, see U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and 4,179,337. In another instance, the peptide derivative includes a targeting moiety that is specific for a particular cell type, tissue, and / or organ. Alternatively, the peptide is linked to one or more chemical moieties that facilitate purification, detection, multimerization, and characterization of peptide activity. An exemplary chemical moiety is biotin. In addition, in one aspect, the peptide of the present invention is acylated at the N-terminal amino acid of the peptide. In another case, the peptide of the present invention is amidated at the C-terminal amino acid of the peptide. In yet another case, the peptide of the present invention is acylated at the N-terminal amino acid of the peptide and amidated at the C-terminal amino acid of the peptide.

[0362] Derivatives also include peptides containing modified or non-protein amino acids or modified linker groups (see, e.g., Grant, Synthetic Peptides: A User's Guide, Oxford University Press (1992)). Modified amino acids include, for example, amino acids in which the amino and / or carboxyl groups are replaced by another group. Non-limiting examples include modified amino acids incorporating thioamides, ureas, thioureas, acyl hydrazides, esters, olefins, sulfonamides, phosphoramides, ketones, alcohols, boric acid amides, benzodiazepines and other aromatic or non-aromatic heterocycles (see Estiarte et al., Burgers Medicinal Chemistry, 6th edition, Volume 1, Part 4, John Wiley & Sons, New York (2002)). Modified amino acids are often linked to peptides with at least one of the above functional groups instead of amide bonds. Non-protein amino acids include, but are not limited to, β-alanine (Bal), norvaline (Nva), norleucine (Nle), 4-aminobutyric acid (γ-Abu), 2-aminoisobutyric acid (Aib), 6-aminocaproic acid (ε-Ahx), ornithine (Orn), hydroxyproline (Hyp), taurine, sarcosine, citrulline (Cit), cysteic acid (Coh), cyclohexylalanine (Cha), methionine sulfoxide (Meo), methionine sulfone (Moo), homoserine methyl ester (Hsm), propargylglycine (Eag), 5-fluorotryptophan (5Fw), 6-fluorotryptophan (6Fw), 3',4' -dimethoxyphenylalanine (Ear), 3',4'-difluorophenylalanine (Dff), 4'-fluorophenylalanine (Pff), 1-naphthylalanine (1Ni), 1-methyltryptophan (1Mw), penicillamine (Pen), homoserine (Hse), tert-butylglycine, tert-butylalanine, phenylglycine (Phg), benzothienylalanine (Bta), L-homocysteine ​​(Hcy), N-methylphenylalanine (Nmf), 2-thienylalanine (Thi), 3,3-diphenylalanine (Ebw), homophenylalanine (Hfe), and S-benzyl-L-cysteine ​​(Ece). These and other non-protein amino acids may exist as either D or L isomers. Examples of modified linkers include, but are not limited to, the flexible linker 4,7,10-trioxa-1,13-tridecanediamine (Ttds), glycine, 6-aminohexanoic acid, β-alanine (Bal), and a combination of Ttds, glycine, 6-aminohexanoic acid, and Bal.

[0363] The homologues of the amino acids constituting the peptide of the present invention can be shown in Table 3.

[0364] Table 3

[0365]

[0366]

[0367] In some embodiments, the peptide (CO-NH) bonds connecting the amino acids in the peptides of the present invention are reversed, resulting in a "retro-modified" peptide, i.e., the peptide comprises amino acid residues (NH-CO bonds) assembled in the opposite direction compared to the reference peptide. The retro-modified peptide comprises the same amino acid chirality as the reference peptide. The "inverso-modified" peptide is a peptide of the present invention comprising amino acid residues assembled in the same direction as the reference peptide, but the chirality of the amino acids is reversed. Therefore, in the case where the reference peptide comprises L-amino acids, the "inverso-modified" peptide comprises D-amino acids, and vice versa. The inverso-modified peptide comprises a CO-NH peptide bond. A "retro-inverso-modified" peptide refers to a peptide comprising amino acid residues assembled in the opposite direction and having inverted chirality. The retro-inverso analog has a reversed end and a reversed peptide bond direction (i.e., NH-CO), while roughly maintaining the side chain topology seen in the reference peptide. Retro-inverse peptidomimetics are made using standard methods, including those described in Meziere et al., J. Immunol., 159, 3230-3237 (1997), which is incorporated herein by reference. Partial retro-inverse peptides are peptides in which only part of the amino acid sequence is reversed and replaced with enantiomeric amino acid residues.

[0368] The TFPI-binding peptides (e.g., TFPI-inhibiting peptides) of the present invention are prepared in a variety of ways. In one aspect, the peptide is synthesized by solid phase synthesis techniques, including those described in the following literature: Merrifield, J. Am. Chem. Soc, 85, 2149 (1963); Davis et al., Biochem. Intl., 10, 394-414 (1985); Larsen et al., J. Am. Chem. Soc, 115, 6247 (1993); Smith et al., J. Peptide Protein Res., 44, 183 (1994); O'Donnell et al., J. Am. Chem. Soc, 118, 6070 (1996); Stewart and Young, Solid Phase Peptide Synthesis, Freeman (1969); Finn et al., The Proteins, 3rd ed., vol.2 (Proteins, 3rd edition, second volume), p105-253 (1976); and Erickson et al., The Proteins, 3rd ed., vol.2 (Proteins, 3rd edition, second volume), p257-527 (1976). Alternatively, the TFPI-binding peptide (e.g., TFPI-inhibiting peptide) is recombinantly expressed by introducing a nucleic acid encoding the TFPI-binding peptide (e.g., TFPI-inhibiting peptide) into a host cell and culturing the host cell to express the peptide. Such peptides are purified from cell culture using standard protein purification techniques.

[0369] The present invention also encompasses nucleic acids comprising nucleic acid sequences encoding TFPI inhibitory peptides of the present invention. Methods for preparing DNA and / or RNA molecules are well known in the art. In one aspect, DNA / RNA molecules encoding peptides provided herein are produced using chemical synthesis techniques and / or using polymerase chain reaction (PCR). If desired, the TFPI inhibitory peptide coding sequence is incorporated into an expression vector. One of ordinary skill in the art will appreciate that any of the many expression vectors known in the art are suitable for the context of the present invention, such as, but not limited to, plasmids, plasmid-lipid complexes, and viral vectors. Any of these expression vectors can be prepared using standard recombinant DNA techniques described in the following documents: for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994). Optionally, the nucleic acid is operably linked to one or more regulatory sequences, such as a promoter, activator, enhancer, cap signal, polyadenylation signal, or other signals involved in transcriptional or translational control.

[0370] Any TFPI inhibitory peptide of the present invention or nucleic acid encoding the peptide is also provided as a composition (e.g., a pharmaceutical composition). In this regard, the peptide is formulated with a physiologically acceptable (i.e., pharmacologically acceptable) carrier, buffer, excipient, or diluent herein, which will be further described herein. Optionally, the peptide is in the form of a physiologically acceptable salt, which is encompassed by the present invention. "Physiologically acceptable salt" refers to any salt that is pharmaceutically acceptable. Some examples of suitable salts include acetates, hydrochlorides, hydrobromides, sulfates, citrates, tartrates, glycolates, and oxalates. If desired, the composition contains one or more additional effective agents.

[0371] The peptides provided herein optionally inhibit the activity of at least one tissue factor pathway inhibitor-1 (e.g., TFPI-1), such as, but not limited to, downregulating the activity of the coagulation cascade. Without being constrained by any specific mechanism of action, the proposed inhibitory mechanism may include preventing the formation of a quaternary TF-FVIIA-FXA-TFPI complex. The peptide can inhibit TFPI from binding to FXa (e.g., inhibiting the binding of TFPI's Kunitz domain 2 to coagulation factor Xa), binding to the TF / FVIIa complex (e.g., inhibiting the binding of TFPI's Kunitz domain 1 to the TF / FVIIa complex), binding to TF alone, and / or binding to FVIIa alone. As TFPI activity decreases, TF and FVIIa are free to activate FX, which then enhances the conversion of prothrombin to thrombin.

[0372] In one aspect, the peptides of the present invention show TFPI antagonistic activity in a model and / or plasma system. An exemplary model system for determining TFPI inhibitory activity is an exogenous tenase assay, which tests the ability of candidate peptides to restore exogenous complex-mediated FX activation in the presence of TFPI (which is a natural inhibitor of the FX activation reaction) (see, e.g., Lindhout et al., Thromb. Haemost., 74, 910-915 (1995)). Another model system for identifying TFPI inhibitory activity is a FXa inhibition assay, in which FXa activity is measured in the presence of TFPI (see Sprecher et al., PNAS, 91, 3353-3357 (1994)). Exogenous tenase assays and FXa inhibition assays are further described in Example 3. Optionally, the half-maximal effective concentration (EC) of the peptides of the present invention that enhances FX activation in the presence of TFPI 50 ) is less than or equal to 1x10 -4 M, less than or equal to 1x10 -5 M, less than or equal to 1x10 -6 M, or less than or equal to 1x10 -7 M.

[0373] In one aspect, TFPI antagonist activity is identified in a plasma-based assay. In the presence of a candidate peptide, thrombin formation is initiated in plasma that is substantially lacking FVIII or FIX activity (e.g., less than 1% residual coagulation factor activity). Thrombin formation can be detected using a fluorescent or chromogenic substrate as described in Example 4. Systems for measuring thrombin activity are provided by Thrombino scope BV (Maastricht, The Netherlands). Prothrombin conversion is measured using, for example, a Thrombograph TM(Thermo Scientific, Waltham, MA), and the resulting data were collected on a Thrombinoscope obtained from Thrombinoscope BV. TM Calibrated Automatic Thrombogram generated by software. In certain embodiments, the TFPI inhibitory peptide increases the peak thrombin amount generated during the analysis and / or reduces the time required to reach peak thrombin formation. For example, the peptide increases TFPI-regulated thrombin generation in the absence of FVIII (e.g., FVIII-poor plasma) to at least 1% of the TFPI-dependent thrombin generation level in normal plasma. In general, normal (non-diseased) plasma contains about 0.5U / mL to about 2U / mL coagulation factor VIII. Therefore, in some cases, the TFPI inhibitor peptide will increase thrombin formation in the absence of FVIII to at least about 1% of the level observed in the presence of 0.5U / mL to 2U / mL FVIII. In other embodiments, the peptide enhances thrombin formation in the absence of coagulation factor VIII to at least about 2%, at least about 3%, at least about 5%, at least about 7%, or at least about 10% of the thrombin formation level in normal plasma, i.e., in the presence of physiological levels of coagulation factor VIII. In various cases, the peptides are administered to animal models of thrombin deficiency or hemophilia to identify in vivo TFPI inhibitory activity. Such in vivo models are known in the art, including, for example, mice administered with anti-FVIII antibodies to induce hemophilia A (Tranholm et al., Blood, 102, 3615-3620 (2003)); coagulation factor knockout models, such as but not limited to FVIII knockout mice (Bi et al., Nat. Genet., 10 (1), 119-121 (1995)) and FIX knockout mice (Wang et al., PNAS, 94 (21), 11563-66 (1997)); rabbits induced hemophilia A (Shen et al., Blood, 42 (4), 509-521 (1973)); and Chapel Hill HA dogs (Lozier et al., PNAS, 99, 12991-12996 (2002)).

[0374] Without being bound by any particular theory or mechanism, the peptides of the invention provided herein may inhibit TFPI activity by blocking (competitively or allosterically) the binding of TFPI to FXa. Alternatively or additionally, the peptides may inhibit the binding of TFPI to the tissue factor (TF) / coagulation factor VIIa complex. Thus, in some cases, the peptides specifically bind to TFPI. Various peptides bind to TFPI from any source including, but not limited to, mice, rats, rabbits, dogs, cats, cows, horses, pigs, guinea pigs, and primates. In one embodiment, the peptide binds to human TFPI. Optionally, the TFPI inhibitory peptide binds to TFPI from more than one species (i.e., the peptide has cross-reactivity between multiple species). In some cases, the dissociation constant (K) for binding of the peptide to TFPI is D ) is less than or equal to 1x10 -4 M, less than or equal to 1x10 -5 M, less than or equal to 1x10 -6 M or less than or equal to 1x10 -7 M. Affinity can be determined using, for example and without limitation, any one, two or more of a variety of techniques, such as affinity ELISA analysis, competitive ELISA analysis, and / or surface plasmon resonance (BIAcore TM ) analysis. 50 ) When the peptide of the present invention is identified by ELISA analysis, it is preferred that the peptide of the present invention show IC 50 Less than or equal to about 50,000 nM. For example, the peptide exhibits an IC 50 Less than or equal to about 10,000 nM, such as IC 50 Less than or equal to about 5,000 nM, less than or equal to about 1,000 nM, or less than or equal to about 500 nM. In one aspect, the peptide exhibits an IC 50 Less than or equal to about 250 nM, less than or equal to about 100 nM, or less than or equal to about 50 nM. Exemplary peptides and their IC 50 Values ​​provided in Figure 32-39 In some cases, peptides were classified according to their IC 50 The values ​​are classified into groups A, B, C, D, E, F and G (see Table 4 of Example 1). In various aspects, the invention provides peptides belonging to groups A, B, C, D, E, F and / or G as defined in Table 4. Affinity can also be determined by kinetic methods or equilibrium / solution methods. Such methods are described in further detail herein or are known in the art.

[0375] As with all binding assays for binding agents, one skilled in the art recognizes that to be biologically (e.g., therapeutically) effective, a binding agent should not be detected to bind to a variety of moieties that would be exhaustive and impractical to enumerate. Thus, the term "specific binding" refers to the ability of a peptide to bind to TFPI with a higher affinity than it binds to an unrelated control protein that is not TFPI. For example, the peptide may bind to TFPI with an affinity that is at least 5, 10, 15, 25, 50, 100, 250, 500, 1000, or 10,000 times greater than the affinity it binds to the control protein. In some embodiments, the peptide binds to TFPI with a higher affinity than it binds to an "anti-target," which is a protein or other naturally occurring substance in humans to which the peptide may bind to an adverse effect. Several classes of peptides or proteins are possible "anti-targets." Because TFPI inhibitory peptides exert their activity in the bloodstream and / or at the endothelium, plasma proteins represent possible anti-targets. Proteins containing Cooney domains (KDs) are possible anti-targets because KDs of different proteins share significant similarities. Tissue factor pathway inhibitor-2 (TFPI-2) is highly similar to TFPI-1α and contains KDs like TFPI-1α (Sprecher et al., PNAS, 91, 3353-3357 (1994)). Thus, in one aspect, the peptides of the invention bind to TFPI with an affinity that is at least 5, 10, 15, 25 or 50 times higher than the affinity for the anti-target, e.g., TFPI-2.

[0376] The present invention further includes a method of inhibiting tissue factor pathway inhibitor-1 (TFPI). The method comprises contacting TFPI with a TFPI-binding peptide as described herein. Any degree of inhibition of TFPI activity is contemplated. For example, the TFPI-inhibiting peptide reduces TFPI inhibition of the exogenous pathway by at least about 5% (e.g., at least about 10%, at least about 25%, or at least about 30%). In some embodiments, the TFPI-inhibiting peptide reduces TFPI activity within the exogenous pathway by at least about 50%, at least about 75%, or at least about 90%, compared to TFPI activity in the absence of the TFPI-inhibiting peptide.

[0377] The present invention further includes methods for targeting biological structures (including but not limited to cell surfaces and endothelial linings) where TFPI is localized. The method comprises contacting the biological structure (e.g., including but not limited to cells displaying TFPI on the cell surface) with a TFPI-binding peptide described herein, optionally conjugated to a moiety that adds additional functionality to the peptide. The moiety can be a dye (e.g., a fluorescent dye), a radionuclide or a complex containing a radionuclide, a protein (e.g., an enzyme, a toxin, or an antibody), or a cytotoxic agent. For example, the peptide is linked or conjugated to an effector moiety that facilitates peptide detection and / or purification and / or comprises therapeutic properties. In one aspect, the TFPI-binding peptide or peptide conjugate is administered to a mammal to target cells that display TFPI in the mammal. Optionally, the method further comprises detecting the binding of the TFPI-binding peptide to TFPI. The method can be used to treat and diagnose diseases in which TFPI is a suitable diagnostic marker or TFPI-expressing cells are the target of a therapeutic method.

[0378] In some cases, peptide-TFPI binding is detected indirectly. In this case, the peptide is contacted with an interaction partner that binds to the peptide of the invention and does not significantly interfere with peptide-TFPI binding, and the interaction partner is detected. Exemplary interaction partners include, but are not limited to, antibodies, antigen-binding antibody fragments, anticalins and antibody mimetics, aptamers and spiegelmers. Optionally, the interaction partner comprises a detection moiety to facilitate detection of the interaction partner-peptide complex. Methods for detecting, for example, antibodies and fragments thereof are well understood in the art. Likewise, detection moieties are widely used in the art to identify biological substances and include, for example, dyes (e.g., fluorescent dyes), radionuclides and complexes containing radionuclides, and enzymes.

[0379] Thus, the present invention provides methods for diagnosing a subject having a disease or condition, or at risk of having a disease or condition, wherein the disease or condition is associated with or caused by abnormal TFPI activity. The method comprises administering a TFPI-binding peptide of the present invention to a subject and detecting a TFPI-peptide complex. In some cases, the peptide of the present invention is bound to a detectable moiety, and the method comprises detecting the detectable moiety. In other cases, the method comprises administering to a subject a TFPI-binding peptide interaction partner that binds to the TFPI-binding peptide, and detecting the interaction partner. If desired, the interaction partner comprises a detectable moiety or is bound to a detectable moiety, and the detectable moiety is detected. The presence of a detectable moiety indicates the presence of TFPI, thereby allowing diagnosis of a disease or condition associated with TFPI (e.g., the following disease or condition: (i) can be treated by inhibiting TFPI, or (ii) comprises symptoms that can be alleviated or prevented by inhibiting TFPI). If it is not desired to administer the peptide to a subject, a biological sample is obtained from the subject, the biological sample is contacted with the TFPI-binding peptide described herein, and the TFPI-peptide complex is detected.

[0380] The peptides of the invention bind to TFPI and are therefore useful for purifying TFPI or recombinant TFPI from biological samples (e.g., biological fluids such as serum), fermentation extracts, tissue preparations, culture media, and the like. The invention includes the use of TFPI binding in the industrial production of TFPI or in methods for identifying TFPI molecules. For example, the invention includes methods for purifying TFPI. The method comprises contacting a sample containing TFPI with a peptide as defined herein under conditions suitable for forming a complex between TFPI and the peptide; removing the complex from the sample; and optionally dissociating the complex to release TFPI. Exemplary conditions suitable for forming a complex between TFPI and the peptide are disclosed in the Examples, and such conditions are easily altered to dissociate the TFPI-peptide complex. In some embodiments, the peptide is immobilized on a support, such as a solid support, to facilitate the recovery of TFPI. For example, in one embodiment, the peptide is immobilized on a chromatographic stationary phase (e.g., silica, affinity chromatography beads, or chromatographic resin), a sample containing TFPI is applied to the stationary phase, a TFPI-peptide complex is formed, the remainder of the sample is removed from the stationary phase, and TFPI is eluted from the stationary phase. In this regard, in one aspect, the peptides of the invention are suitable for use in affinity chromatography techniques.

[0381] Also provided is a method for increasing thrombin formation in a subject with a coagulation factor deficiency. The method includes administering a peptide provided herein to a subject under conditions that effectively inhibit TFPI. In this regard, the TFPI inhibitory peptide is administered in an amount and condition that effectively increases the formation of thrombin in the subject. "Coagulation factor deficiency" refers to a defect in one or more blood factors, such as FVIII, FIX or FXI, required for the subject to suffer from thrombin formation. In one embodiment, the subject has a FVIII deficiency. Alternatively or additionally, the subject has a coagulation factor IX deficiency. Coagulation factor deficiency is identified by examining the amount of coagulation factors in clinical samples. Practitioners classify hemophilia according to the magnitude of coagulation factor deficiency. Coagulation factor VIII or coagulation factor IX in subjects with mild hemophilia is about 5% to 30% of the normal amount (1U / ml). Moderate hemophilia is characterized by about 1% to 5% of normal coagulation factor VIII, coagulation factor IX or coagulation factor XI levels, while subjects with severe hemophilia have less than 1% of the normal amount of coagulation factor VIII, coagulation factor IX or coagulation factor XI. Defects can be identified indirectly by the activated partial thromboplastin time (APTT) test. The APTT test measures the length of time required to form a blood clot, and patients with factor VIII deficiency (hemophilia A), factor IX deficiency (hemophilia B), and factor XI deficiency (hemophilia C) have longer times than patients with normal coagulation factor levels. Almost 100% of patients with severe and moderate coagulation factor VIII deficiency can be diagnosed with APTT. The present invention further includes increasing thrombin formation in subjects who do not suffer from coagulation factor deficiency. The method includes administering a peptide provided herein to a subject (e.g., a subject comprising a normal physiological level of a coagulation factor) under conditions that effectively increase thrombin formation.

[0382] In one aspect, TFPI inhibitory peptides are used to increase clot formation in a subject. The method of increasing clot formation includes administering the peptides described herein to a subject in an amount and under conditions effective to increase clot formation. It is to be appreciated that the method does not require complete restoration of the coagulation cascade to achieve a beneficial (e.g., therapeutic) effect. Any enhancement or increase in thrombin or clot formation that reduces the onset or severity of symptoms associated with coagulation factor deficiency is contemplated. Methods for determining the effectiveness of the method in promoting thrombin formation and coagulation are known in the art and described herein.

[0383] The present invention further includes a method for treating a coagulation disorder in a subject, the method comprising administering to the subject one or more TFPI-inhibiting peptides, such as any one or more peptides described herein, in an amount and under conditions effective to treat the coagulation disorder in the subject. In one aspect, the peptide is not a naturally occurring peptide that inhibits TFPI activity. "Coagulation disorders" include bleeding disorders caused by defects in coagulation factor activity and platelet activity. Coagulation factors include, but are not limited to, coagulation factors V (FV), FVII, FVIII, FIX, FX, FXIII, FII (causing hypoprothrombinemia) and von Willebrand factor. Coagulation factor deficiencies are caused by, for example, shortened in vivo half-life of coagulation factors, altered binding properties of coagulation factors, genetic defects of coagulation factors, and reduced plasma concentrations of coagulation factors. Coagulation disorders can be congenital or acquired. Possible genetic defects include deletions, additions and / or substitutions within the nucleotide sequence encoding the coagulation factor, and the absence, presence and / or substitution of the nucleotide sequence have a negative impact on the activity of the coagulation factor. Coagulation disorders also result from the production of inhibitors or autoimmunity (eg, antibodies) against coagulation factors. In one example, the coagulation disorder is hemophilia A. Alternatively, the coagulation disorder is hemophilia B or hemophilia C.

[0384] Platelet disorders are caused by defects in platelet function or abnormally low numbers of circulating platelets. Low platelet counts may be due to, for example, insufficient production, platelet sequestration, or unchecked overt destruction. Thrombocytopenia (not enough platelets) may have a variety of causes, including chemotherapy and other drug therapies, radiation therapy, surgery, unexpected bleeding, and other medical conditions. Exemplary disease conditions involving thrombocytopenia are: aplastic anemia; idiopathic or immune thrombocytopenia (ITP), including idiopathic thrombocytopenic purpura associated with breast cancer; HIV-associated ITP and HIV-associated thrombotic thrombocytopenic purpura; metastatic tumors causing thrombocytopenia; systemic lupus erythematosus, including neonatal lupus syndrome splenomegaly, Fanconi syndrome; vitamin B12 deficiency; folate deficiency; May-Hegglin anomaly; Wiskott-Aldrich syndrome; chronic liver disease; myelodysplastic syndrome associated with thrombocytopenia; paroxysmal nocturnal hemoglobinuria; acute severe thrombocytopenia following C7E3Fab (abciximab) therapy; alloimmune thrombocytopenia, including maternal alloimmune thrombocytopenia; thrombocytopenia associated with antiphospholipid antibodies and thrombosis; autoimmune thrombocytopenia; drug-induced immune thrombocytopenia, including These include carboplatin-induced thrombocytopenia and heparin-induced thrombocytopenia; fetal thrombocytopenia; gestational thrombocytopenia; Hughes syndrome; lupus-like thrombocytopenia; accidental and / or massive blood loss; myeloproliferative disorders; thrombocytopenia in patients with malignancy; thrombotic thrombocytopenic purpura; thrombotic microangiopathy including manifestations of thrombotic thrombocytopenic purpura / hemolytic uremic syndrome in patients with cancer; post-transfusion purpura (PTP); autoimmune hemolytic anemia; occult perforated jejunal diverticula; pure red cell aplasia; autoimmune thrombocytopenia; epidemic nephropathy; acute renal failure associated with rifampin; Paris-Trousseau thrombocytopenia; neonatal alloimmune thrombocytopenia; paroxysmal nocturnal hemoglobinuria; hematological changes in gastric cancer; hemolytic uremic syndrome (e.g., childhood uremic disease); and thrombocytopenia associated with viral infections, including hematological manifestations associated with hepatitis A virus and CMV. Platelet disorders also include, but are not limited to, Von Willebrand disease, paraneoplastic platelet dysfunction, Glanzman thrombasthenia, and Bernard-Soulier disease. Other bleeding disorders suitable for treatment with TFPI inhibitory peptides include, but are not limited to, trauma-induced hemorrhagic conditions; deficiencies in one or more contact factors, such as FXI, FXII, prekallikrein, and high molecular weight kininogen (HMWK); vitamin K deficiency; fibrinogen disorders, including afibrinogenemia, hypofibrinogenemia, and dysfibrinogenemia; and α2-antiplasmin deficiency.In one embodiment, the TFPI inhibitory peptide is used to treat massive bleeding, such as massive bleeding caused by surgery, trauma, intracerebral hemorrhage, liver disease, kidney disease, thrombocytopenia, platelet dysfunction, hematoma, internal hemorrhage, hemarthrosis, hypothermia, menstruation, pregnancy, and dengue hemorrhagic fever. All of the above are considered "coagulation disorders" within the scope of the present disclosure.

[0385] In one aspect, the TFPI inhibitory peptides of the invention are used to reverse the effects (completely or partially) of one or more anticoagulants in a subject. Numerous anticoagulants are known in the art, including, for example, heparin; coumarin derivatives, such as warfarin or dicoumarol; TFPI; AT III; lupus anticoagulant; nematode anticoagulant peptide (NAPc2); FVIIa inhibitors; active site blocked FVIIa (FVIIai); active site blocked FIXa (FIXai); FIXa inhibitors; FXa inhibitors, including fondaparinux, edaparinux, DX-9065a and lazaxaban (DPC 906); active site blocked FXa (FXai); inhibitors of FVa or FVIIIa, including activated protein C (APC) and soluble thrombomodulin; coagulation inhibitors, including hirudin, bivalirudin, argatroban and ximelagatran; and antibodies or antibody fragments that bind to coagulation factors (e.g., FV, FVII, FVIII, FIX, FX, FXIII, FII, FXI, FXII, von Willebrand factor, prekallikrein or high molecular weight kininogen (HMWK).

[0386] When used herein, "treatment" refers to any reduction in the severity and / or onset of symptoms associated with coagulation disorders. Therefore, "treatment" includes medical treatment and preventive measures. It will be appreciated by those of ordinary skill in the art that any degree of defense or relief of coagulation disorders or symptoms associated therewith are beneficial to objects, such as human patients. By reducing the severity of object symptoms and / or delaying symptom appearance to any degree, the quality of life of the patient is improved. Therefore, in one aspect, the method is performed as soon as possible after determining that the object has a risk of coagulation disorder (e.g., detection of coagulation factor (e.g., FVIII, FIX or FXI) deficiency) or after measuring coagulation disorder (e.g., hemophilia A, hemophilia B or hemophilia C). In other cases, the peptide is used to defend against excessive blood loss during injury or surgery in whole or in part.

[0387] In view of the above, the present invention provides peptides for use in methods of treating a subject, such as methods of treating a disease in which inhibition of TFPI is beneficial. In one aspect, the disease or condition is a coagulation disorder. The subject suffers from the disease or condition, or is at risk of suffering from the disease or condition (or an adverse biological event, such as excessive blood loss). The method includes administering the peptides of the present invention to the subject in an amount and under conditions effective to completely or partially treat or prevent the disease or condition. The present invention also provides peptides for the manufacture of medicaments. For example, the peptides can be used to manufacture therapeutic agents for coagulation disorders as described in detail herein.

[0388] In some embodiments, it is advantageous to administer to a subject a nucleic acid comprising a nucleic acid sequence encoding a TFPI-binding peptide of the invention (e.g., a TFPI-inhibiting peptide). In one aspect, such a nucleic acid is provided instead of or together with a TFPI-inhibiting peptide. Expression vectors, nucleic acid regulatory sequences, methods of administration, etc. are further described herein and in U.S. Patent Publication No. 20030045498.

[0389] The specific dosing regimen for a specific subject will depend in part on the TFPI inhibitory peptide of the invention used, the amount of TFPI-binding peptide (e.g., TFPI inhibitory peptide) administered, the route of administration, the specific patient being treated, considerations related to the recipient, and the cause and extent of any side effects. The amount of peptide administered to a subject (e.g., a mammal, such as a human) and the conditions of administration (e.g., time of administration, route of administration, dosage regimen) are sufficient to achieve the desired biological response within a reasonable time frame. The dosage generally depends on various factors, including the specific TFPI inhibitory peptide used, the age and weight of the subject, and the presence and severity of any disease or condition in the subject. The size of the dose will also be determined by the route, time, and frequency of administration. Therefore, the clinician can titrate the dose and change the route of administration to obtain the optimal therapeutic effect, and conventional range-finding techniques are known to ordinary professionals in the art. Purely by way of illustration, in one aspect, the method comprises administering, for example, about 0.1 μg / kg to about 100 mg / kg or more, based on the above factors. In other embodiments, the dose may be from 1 μg / kg to about 75 mg / kg; or 5 μg / kg to about 50 mg / kg; or 10 μg / kg to about 20 mg / kg. In certain embodiments, the dose comprises about 0.5 mg / kg to about 20 mg / kg (e.g., about 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.3 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg) of the peptide. Given the chronic nature of many coagulation disorders, it is conceivable that a subject will receive a TFPI inhibitory peptide over a course of weeks, months, or years, and may require one or more doses daily or weekly. In other embodiments, the TFPI inhibitory peptide is administered for a relatively short treatment period, eg, 1 to 14 days, to treat acute conditions (eg, bleeding caused by surgery or trauma, or coagulation factor inhibition / autoimmune events in subjects receiving coagulation replacement therapy).

[0390] Appropriate methods for administering physiologically acceptable compositions, such as pharmaceutical compositions comprising the peptides described herein, are well known in the art. Although more than one route may be used to administer the peptide, a specific route may provide a more direct and more effective response than other routes. Depending on the circumstances, the pharmaceutical composition is applied or instilled into a body cavity, absorbed through the skin or mucosa, taken, inhaled, and / or introduced into the circulation. In one aspect, a composition comprising a TFPI inhibitory peptide is administered intravenously, intraarterially, or intraperitoneally to introduce the peptide of the present invention into the circulation. Non-intravenous administration is also appropriate, especially in the case of low molecular weight therapeutic agents. In some cases, it is appropriate to deliver a pharmaceutical composition comprising a TFPI inhibitory peptide orally, topically, sublingually, vaginally, or rectally; by intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraportal, intralesional, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intranasal, urethral, ​​or enteral injection; by a sustained release system; or by an implanted device. If desired, the TFPI inhibitory peptide may be provided to the target area by local administration by intraarterial or intravenous administration, for example, by delivery to the leg via the femoral artery. In one embodiment, the peptide is incorporated into microparticles as described, for example, in the following U.S. Patents: 5,439,686 and 5,498,421, and U.S. Patent Publication Nos. 2003 / 0059474, 2003 / 0064033, 2004 / 0043077, 2005 / 0048127, 2005 / 0170005, 2005 / 014 2205, 2005 / 142201, 2005 / 0233945, 2005 / 0147689, 2005 / 0142206, 2006 / 0024379, 2006 / 0260777, 2007 / 0207210, 2007 / 0092452, 2007 / 0281031 and 2008 / 0026068. Alternatively, the composition is administered by implanting a membrane, sponge or other appropriate material on which the target molecule has been absorbed or wrapped. When an implant device is used, the device is implanted in any appropriate tissue on the one hand, and delivers the target molecule in multiple aspects by diffusion, time-release bolus injection, or continuous administration. In other cases, the TFPI inhibitory peptide is directly administered to exposed tissue during surgical procedures or treatment of injuries, or is administered through a transfusion procedure. Routes of delivery of therapeutic agents are well known to those of skill in the art, some of which are further described, for example, in US Pat. No. 5,399,363.

[0391] For ease of administration, in one embodiment the TFPI-binding peptide (e.g., TFPI-inhibitory peptide) is formulated in a physiologically acceptable composition comprising a carrier (i.e., a medium, adjuvant, buffer or diluent). The specific carrier used is limited only by physicochemical factors, such as solubility and lack of reactivity with the peptide and the route of administration. Physiologically acceptable carriers are well known in the art. Illustrative pharmaceutical forms suitable for injection include, but are not limited to, sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injection solutions or dispersions (see, e.g., U.S. Patent No. 5,466,468). Injectable preparations are further described in, for example, Pharmaceutics and Pharmacy Practice, JB Lippincott Co., Philadelphia. Pa., Banker and Chalmers, eds., pp. 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th edition, pp. 622-630 (1986)). The pharmaceutical composition comprising the peptide provided herein is optionally placed in a container with packaging material providing instructions for use of such pharmaceutical composition. Generally speaking, such instructions include substantial descriptions describing the concentration of reagents and the relative amounts of excipient components or diluents that may be necessary to reconstitute the pharmaceutical composition in certain embodiments.

[0392] When appropriate, the TFPI-binding peptides (e.g., TFPI-inhibitory peptides) of the present invention are combined with other substances and / or other treatment modalities to achieve additional or enhanced biological effects. Combination therapy includes, but is not limited to, plasma-derived or recombinant coagulation factors, hemophilia prophylaxis, immunosuppressants, plasma coagulation factor inhibitory antibody antagonists (i.e., anti-inhibitors), antifibrinolytics, antibiotics, hormone therapy, anti-inflammatory agents (e.g., nonsteroidal anti-inflammatory drugs (NSAIDs) or steroidal anti-inflammatory substances), procoagulants, and analgesics. In one aspect, the method is an adjunct therapy to a conventional replacement coagulation factor treatment regimen that includes administering to a subject, for example, FXIII, FXII, FXI (e.g., (Laboratoire francais du Fractionnement et des Biotechnologies, Les Ulis, France) and FXI concentrate (BioProducts Laboratory, Elstree, Hertfordshire, UK), FX, FIX (e.g. Coagulation factor IX (Wyeth, Madison, NJ); SD (Grifols, Los Angeles, CA); (CSL Behring, King of Prussia, PA); BEBULIN-VH TM (Baxter, Deerfield, IL); SD (Grifols, Los Angeles, CA); or PROPLEX T TM (Baxter, Deerfield, IL)), FVIII (e.g., ADVATE TM (Baxter, Deerfield, IL); FS (CSL Behring, King of Prussia, PA); (Wyeth, Madison, NJ), XYNTHA TM (Wyeth, Madison, NJ), and FS (Bayer, Pittsburgh, PA); (Grifols, Los Angeles, CA); HEMOPHIL M TM (Baxter, Deerfield, IL); (Talecris Biotherapeutics-USA, Research Triangle Park, NC); or MONARC-M TM (Baxter, Deerfield, IL)), FVIIa (e.g., FVIIa (NovoNordisk, Princeton, NJ) and FVII concentrate (Baxter Bioscience, Vienna, Austria, or BioProducts Laboratory, Elstree, Hertfordshire, UK), FV, Fva, FII, and / or Fill. In some cases, subjects also received FEIBA VH Immuno TM (Baxter BioScience, Vienna, Australia), which is a lyophilized sterile human plasma fraction with factor VIII inhibitor bypassing activity. FEIBA VH Immuno TMContains approximately equal units of factor VIII inhibitor bypass activity and prothrombin complex factors (coagulation factors II, VII, IX, and X, and protein C). Other exemplary combination therapies include, but are not limited to, prekallikrein, high molecular weight kininogen (HMWK), Von Willebrand factor, tissue factor, and thrombin. Alternatively or additionally, the TFPI inhibitory peptide is co-formulated with one or more different TFPI inhibitory peptides.

[0393] Thus, the present invention includes administering to a subject a combination of a TFPI-binding peptide (e.g., TFPI-inhibitory peptide) of the present invention (or multiple TFPI-inhibitory peptides) and one or more other appropriate substances, each of which is administered according to a regimen suitable for the drug. Administration strategies include simultaneous administration (i.e., administration at substantially the same time) and non-simultaneous administration (i.e., administration at different times in any order, whether or not overlapping) of a TFPI-inhibitory peptide and one or more other appropriate agents. It is to be understood that the different components are optionally administered in the same or separate compositions, and by the same or different routes of administration.

[0394] In some embodiments, the peptides of the invention are combined with moieties, such as therapeutic or diagnostic moieties such as detection moieties and the combination therapeutics described above. Alternatively or additionally, the peptides are combined with an interaction partner (e.g., an antibody, antibody fragment, anticalin, aptamer, or spiegelmer) that (a) binds to the peptide and (b) is therapeutically active and / or is linked to a moiety (e.g., a therapeutic agent, a diagnostic agent, or a detection agent) that provides additional functionality to the interaction partner. Suitable moieties include, but are not limited to, dyes, radionuclides, complexes containing radionuclides, enzymes, toxins, antibodies, antibody fragments, and cytotoxic agents, which in some cases have therapeutic activity (i.e., achieve a favorable or desired biological effect). Peptide conjugates or peptide-interaction partner pairs are suitable for use in any of the methods described herein, such as methods for treating a subject suffering from a disease or disorder or at risk of suffering from a disease or disorder.

[0395] All publications, patents and patent applications cited in this specification are hereby incorporated by reference, as if each publication or patent application is specifically and individually indicated to be cited as a reference. In addition, the entire document is intended to be associated in accordance with the integrated disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the feature combinations are not found together in the same sentence, paragraph, or chapter of this document. For example, where protein therapy is described, polynucleotide therapy (using polynucleotides / vectors encoding proteins) is specifically contemplated, and vice versa. Although the above invention has been described in more detail with the aid of illustrations and examples for the purpose of clear understanding, it should be readily recognized by ordinary professionals in the technical field that certain changes and modifications can be made according to the teachings of the present invention without departing from the connotation or scope of the claims. The present invention includes, for example, embodiments of the present invention that are narrower in scope than the variants specifically mentioned above in any way. With respect to aspects of the present invention described as generic, all individual species are individually considered to be separate aspects of the present invention. With respect to aspects of the present invention described or claimed with indicators without numbers, it is understood that this refers to "one or more", unless the context clearly requires a more limited meaning. With respect to elements described as one or more within a group, it is understood that all combinations within the group are contemplated.

[0396] Example

[0397] Example 1

[0398] The following examples describe the generation, identification and screening of peptides that bind TFPI.

[0399] Peptide candidates were obtained from commercial suppliers (e.g., Polypeptide Laboratories SAS (Strasbourg, France) and JPT Peptide Technologies GmbH (Berlin, Germany)). Methods for synthesizing candidate peptides are provided above. Candidate peptides were synthesized as trifluoroacetate (TFA) with a purity of >90% or >60%. All peptides were dissolved in DMSO to a storage concentration of 10 mM. TFPI-binding peptide sequences were identified using mRNA display libraries. mRNA display technology is superior to other library screening technologies, allowing for diversity of up to 10 within the starting pool. 14 The technique involves directly linking the mRNA to the candidate peptide it encodes via a puromycin molecule ( Figure 5). The mRNA display method is further described in International Patent Publication No. WO 2005 / 051985 and Liu et al., Methods in Enzymology, 318, 268-293 (2000). TFPI is immobilized on a solid support by biotin and exposed to a candidate peptide-RNA complex. The candidate peptide-RNA complex bound to TFPI is separated, and the RNA is reverse transcribed to obtain the encoding DNA. Using a competitive avoidance strategy, high affinity binders are obtained after six to ten rounds of selection. Many candidate peptides are 31 amino acids in length (27 randomized amino acids and 2 amino acids flanking both ends).

[0400] The selected peptides were synthesized and subjected to peptide optimization using microarray-based scanning analysis to identify peptide fragments that maintained TFPI binding affinity. For example, a microarray-based scan of JBT0047 was performed using a series of 20 amino acid fragments of the peptide whose sequences overlapped by 19 amino acids. The N- and C-terminal truncation analysis complemented the scanning analysis. The microarray scanning results demonstrated that peptide JBT0293 had the highest affinity for binding to TFPI. A series of substitution mutants based on the amino acid sequence of JBT0293 were generated and tested for TFPI binding.

[0401] The affinity of some peptides for TFPI was determined by ELISA (binding (EC 50 )ELISA) demonstrated. 96-well MaxiSorp plates (Nunc) were coated overnight with coating buffer (15mM Na2CO3, 35mM NaHCO3, pH 9.6) containing 3μg / mL TFPI. 350μl wash buffer (HNaT: 175mM NaCl, 25mM HEPES, 5mM CaCl2, 0.1% Tween 80, pH 7.35) was washed three times and then blocked with HNaT containing 200μl 2% yeast extract for 2 hours. The plates were washed three times with 350μl HNaT. DMSO stocks of biotinylated candidate peptides were diluted 1 / 200 in HNaT. If no precipitation occurred during the 1 / 200 dilution of the 10mM peptide stock, the initial peptide concentration was 50μM. If a precipitate was formed, a pre-dilution of the peptide stock in DMSO was performed. The diluted peptides were applied to the Maxisorp plates, generating serial dilutions (1 / 3), which were incubated for 1.5 hours at room temperature. The incubation was followed by three washing steps (350 μl HNaT). The bound peptides were detected by incubation with horseradish peroxidase-conjugated streptavidin (1 hour), followed by three washing steps with HNaT, and subsequent addition of TMB (3,3'5,5'-tetramethylbenzidine) for color conversion. The analysis is shown schematically in Fig. 6A middle.

[0402] In general, peptide binding to immobilized TFPI will significantly exceed the background signal. Figure 32-39 The EC of the biotinylated peptide is given 50 value. Figure 7 A binding curve of the TFPI-binding peptide JBTO132 is depicted. The EC 50 Calculated to be approximately 2.2 nM.

[0403] In addition, competition was performed using a biotinylated TFPI-binding peptide as a “tracer” (IC 50 )ELISA, competing with non-biotinylated candidate peptides for binding to TFPI. The principle of the assay is described in Figure 6B 96-well MaxiSorp plates (Nunc) were coated overnight with 3 μg / mL TFPI in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6). The plates were washed three times with 350 μl of wash buffer (HNaT: 175 mM NaCl, 25 mM HEPES, 5 mM CaCl2, 0.1% Tween 80, pH 7.35) and blocked for 2 h with 200 μl of 2% yeast extract in HNaT. The plates were washed three times with 350 μl of HNaT. The biotinylated tracer peptides were labeled with their corresponding EC values ​​determined in the binding ELISA. 90 The concentration of the competitor (median if n>2) was applied. The competitor stock solution of the peptide (10mM) was diluted 1 / 33.3 in HNaT without HSA, and a series of 1 / 3 dilutions were prepared with HNaT containing 3% DMSO. The dilution was further diluted with biotinylated tracer peptide at a ratio of 1:6 (20μl competitor dilution and 100μl tracer peptide). The mixture of competitor and tracer peptide was applied to a microtiter plate coated with TFPI and incubated for 1.5 hours. The plate was washed three times with 350μl HNaT. Streptavidin bound to HRP was applied to the microtiter plate, the mixture was incubated for one hour, the plate was washed three times with 350μl HNaT, TMB (3,3'5,5'-tetramethylbenzidine) was applied, and the subsequent color conversion of TMB by HRP was detected. Figures 8A-8D Representative IC values ​​of non-biotinylated peptides are provided. 50 Fig. ICs of JBT0303, JBT0120 and JBT0224 50 The measurement results are shown in Table 3.

[0404] Table 3

[0405]

[0406] In addition to competitive ELISA (IC 50) analysis, screening assays are used to measure a larger number of peptides in parallel. Screening ELISA is similar to competitive IC 50 ELISA, except that only three different concentrations of competitor were used (300nM, 100nM and 33.3nM for JBT0047 family, 50000nM, 16667nM and 5556nM for JBT0122 family). In some cases, the screening results are expressed as the percentage inhibition of the tracer signal relative to the competing peptide (the competing peptide for the JBT0047 family is JBT0477, and the competing peptide for the JBT0122 family is JBT1697). Figure 32-39 The competitive IC values ​​of peptides prepared and screened according to the methods described herein are provided. 50 Analyze results and filter analysis results. Figure 32-39 Average IC displayed 50 The values ​​are based on a larger number of analyses than those shown in Table 3 and therefore vary slightly. The results of the screening ELISA are expressed as a percentage of inhibition of binding of the tracer peptide JBT0131. Using IC 50 Several peptides analyzed by ELISA were selected according to their binding affinities shown in Table 4. Figure 32-39 Medium classification.

[0407] Table 4

[0408] <![CDATA[TFPI Competitive ELISA IC 50 [nM]]]> Group <50nM A 50≤x<100nM B <h2 style=";text-align:left;direction:ltr">100≤x<250nM<h2 style=";text-align:left;direction:ltr"> C 250≤x<1000nM D 1000≤x<5000nM E 5000≤x<10000nM F 10000≤x<50000nM G

[0409] Exemplary TFPI-binding peptides identified using the assays described herein are shown in Table 5. Some peptides were biotinylated, and many contained N- and C-terminal lysines to improve solubility. As described below, several peptides exhibited TFPI inhibitory activity in model and / or plasma assay systems.

[0410] Table 5

[0411]

[0412]

[0413] This example provides an exemplary method for generating and identifying TFPI inhibitory peptides. All peptides in Table 5 were found to bind to human TFPI-1α. Mutational analysis demonstrated that at least one amino acid in the TFPI binding peptides could be substituted while maintaining affinity for TFPI. The EC values ​​of the peptides in Table 5 tested for binding to TFPI-1α in the ELISA assay were 50 Less than 10μM (1x10 ～5 M) and IC 50 Less than 50μM.

[0414] Example 2

[0415] Selected TFPI-binding peptides were further characterized with respect to "anti-target" binding. This example demonstrates that TFPI-inhibitory peptides exhibit reduced affinity for non-TFPI-1 proteins.

[0416] TFPI-2 was chosen as the antitarget due to its similarity to TFPI-1. The binding kinetics of TFPI-binding peptides to human TFPI-1 (residues 29-282 fused to a 10His-tag at the C-terminus; MW 41 kDa (R&D Systems, Minneapolis, MN; Catalog No. 2974-PI)), murine TFPI-I (residues 29-289 fused to a 10His-tag at the C-terminus; MW 41 kDa (R&D Systems; Catalog No. 2975-PI)), and TFPI-2 (R&D Systems, Minneapolis, MN) were analyzed using the BIAcore 3000 TM Surface plasmon resonance analysis (GE Healthcare, Chalfont St. Giles, UK) studies. TFPI protein was immobilized on a C1 chip (GE Healthcare, order code: BR-1005-40) via amine linking chemistry with a target of 500RU. Several TFPI-binding peptides were used as analytes to interact with the immobilized TFPI protein. The flow rate adopted was 30μl / min. After 180 seconds, 180μl of six peptide solutions with different concentrations ranging from 3.84nM to 656.25nM were injected, followed by a dissociation time of 480 seconds. The chip was regenerated with 45μl 10mM NaOH. Four measurements were performed before and after each binding experiment using HBS-P buffer (10mM HEPES, pH7.4, 150mM NaCl, 0.005% P20) plus 1% DMSO and 0.8% P80. Utilizing Data were analyzed using software version 4.1 (GE Healthcare). The sensorgrams were fitted to a 1:1 Langmuir binding curve to determine k on and k off , and calculate K D .

[0417] Some of the tested peptides, such as JBT0050, JBT0121, JBT0205, and JBT0211, bound to the blank wells and no binding constants could be determined from those sensorgrams. JBT0133 showed weak binding to TFPI-1. Sensorgrams from other peptides gave reliable binding constants. Table 6 and Figure 19-21 The BIAcore analysis results of several TFPI-1 inhibitory peptides are provided. Table 6 shows the K D Less than 10 μM. The sensorgrams for both peptides are provided as Fig. 9Aand 9B .

[0418] Table 6

[0419] Peptides <![CDATA[k on (l / Ms)]]> <![CDATA[k off (l / s)]]> <![CDATA[K D (M)]]> JBT0047 <![CDATA[4.0x10 5 ]]> <![CDATA[1.9x10 -2 ]]> <![CDATA[4.7x10 -8 ]]> JBT0120 <![CDATA[1.17x10 6 ]]> <![CDATA[4.78x10 -2 ]]> <![CDATA[4.08x10 -8 ]]> JBT0131 <![CDATA[1.4x10 5 ]]> <![CDATA[6.0x10 -2 ]]> <![CDATA[4.31x10 -7 ]]> JBT0132 <![CDATA[3.55x10 4 ]]> <![CDATA[3.26x10 -2 ]]> <![CDATA[9.17xl0 -7 ]]> JBT0224 <![CDATA[6.39x10 4 ]]> <![CDATA[1.95x10 -2 ]]> <![CDATA[3.05x10 -7 ]]> JBT0293 <![CDATA[6.0x10 5 ]]> <![CDATA[5.6x10 -2 ]]> <![CDATA[9.5x10 -8 ]]> JBT0297 <![CDATA[5.0x10 5 ]]> <![CDATA[1.4x10 -2 ]]> <![CDATA[2.9x10 -8 ]]> JBT0303 <![CDATA[8.13x10 5 ]]> <![CDATA[2.75x10 -2 ]]> <![CDATA[3.4x10 -8 ]]> JBT0305 <![CDATA[7.5x10 5 ]]> <![CDATA[3.1x10 -2 ]]> <![CDATA[6.1x10 -8 ]]>

[0420] Interactions with TFPI-2 anti-target were also examined. The maximum signal generated by the interaction of candidate peptides with human TFPI-2 was much lower than that obtained with TFPI-1 as an interaction partner. Kinetic analysis of low TFPI-2 binding signals is prone to error; therefore, visual comparison of sensorgrams was used to estimate binding affinity. Fig. 10A and 10B Sensorgrams showing binding of JBT0120 to TFPI-1 and TFPI-2 are provided. JBT0120 binds TFPI-2 with 10-fold lower affinity than it binds to TFPI-1. JBT0132 was also found to have a 10-fold higher affinity for TFPI-1 than for TFPI-2.

[0421] The data provided in this example demonstrate that the TFPI-1 inhibitory peptide specifically binds to TFPI-1.

[0422] Example 3

[0423] The following example describes the use of FXa inhibition and exogenous tenase inhibition assays to identify the TFPI-1 inhibitory activity of selected peptides identified in Example 1. Both assays predict activity in plasma systems. The exogenous tenase assay is used to understand the effects of the peptides on (a) the interaction of FXa and TFPI, and (b) the interaction of the FXa-TFPI complex with the TF-FVIIa complex. The FXa inhibition assay measures only the effects of the peptides on the interaction of FXa and TFPI.

[0424] Exogenous tenase complex causes FX and FIX activation after the coagulation process begins. The exogenous complex is composed of FVIIa, tissue factor (TF) and FX substrate. In order to determine the effect of peptides on TFPI-mediated inhibition of exogenous tenase complex, an enzyme-linked assay was established. Peptides were diluted 1 / 6.25 from a 10mM stock solution (in DMSO) and further diluted by a continuous 1 / 4 dilution in DMSO to prevent unwanted precipitation. TFPI was diluted in HNaCa-HSA (25mM HEPES; 175mM NaCl; 5mM CaCl2; 0.1% HSA; pH 7.35). FVIIa, lipidated TF, phospholipid vesicles (DOPC / POPS 80 / 20) and FXa specific chromogenic substrate (S-2222 (obtained from DiaPharma, WestChester, OH)) all diluted in HNaCa-HSA were added to a 96-well plate. After the incubation period, TFPI and peptide dilutions were added to give a final concentration of 2.5% DMSO. FX activation was initiated by adding FX to the wells. FXa-mediated conversion of the chromogenic substrate was determined by observing the increase in absorbance using a microplate reader. The amount of FXa produced at certain time points was calculated from the OD readings. FXa produced 20 minutes after the start of the reaction was considered for calculation of the EC from a plot of peptide concentration versus TFPI inhibition (%). 50 .

[0425] Functional inhibition of TFPI was also examined using the FXa inhibition assay. Both the FXa-specific chromogenic substrate (S-2222) and TFPI were diluted in HNaCa-HSA and added to a 96-well plate. The peptides were diluted 1 / 6.25 from a 10 mM stock solution (in DMSO) and further diluted by serial 1 / 4 dilutions in DMSO to prevent unwanted precipitation. Peptide dilutions (2.5 μl) were added to the 96-well plate to a final concentration of 2.5% DMSO. Conversion of the chromogenic substrate was initiated by the addition of FXa, and the conversion kinetics were measured in a microplate reader. Because TFPI inhibits FXa slowly, the OD reading after 115 minutes was considered for calculating the EC from the graph of peptide concentration versus TFPI inhibition (%) 50 .

[0426] Table 7 and Figure 22-27 Results of exogenous tenase assays and FXa inhibition assays are presented.

[0427] Table 7

[0428]

[0429] Referring to Table 7, JBT0120, JBT0132 and JBT0224 restored exogenous complex-mediated FX activation in the presence of TFPI-1, EC 50The inhibitory effect of JBT0047 (EC 50 =1.4 μM), JBT0131 (EC 50 =2.2 μM) and JBT0293 (EC 50 =2.9 μM) also restored exogenous complex activity in the presence of TFPI-1. In addition, in the FXa inhibition assay, JBT0120, JBT0132, JBT0224, and JBT0303 restored FXa activity in the presence of TFPI-1, EC 50 The activity of JBT0047 (EC 50 =0.7 μM), JBT0131 (EC 50 =8.2 μM), JBT0293 (EC 50 =1.3 μM), JBT0297 (EC 50 =0.6 μM) and JBT0305 (EC 50 =2.3 μM) also restored FXa activity in the presence of TFPI-1. This example demonstrates that the peptides of the present invention are TFPI antagonists.

[0430] Example 4

[0431] In this example, the TFPI inhibitory activity of the peptides was determined using a plasma-based assay.

[0432] After slowly cleaving the thrombin-specific fluorescent substrate Z-Gly-Gly-Arg-AMC (Hemker, Pathophysiol. Haemost. Thromb., 33, 4-15 (2003)), the thrombin was detected by the Fluoroskan The effect of peptides on thrombin generation was measured in duplicate by automatically calibrated thrombus images in a reader (Thermo Labsystems, Helsinki, Finland; filters 390 nm excitation light and 460 nm emission light). Plasma (George King Bio-Medical Inc., Overland Park, KN) from patients with FVIII or FIX deficiency was obtained for testing. The residual coagulation factor activity of each plasma was less than 1%. As a model for antibody-mediated FVIII deficiency, frozen normal plasma pools (George King Bio-Medical Inc., Overland Park, KN) were incubated with high titer, heat-inactivated anti-human FVIII plasma (4490 BU / ml; Baxter BioScience, Vienna, Australia) produced in goats to 50 BU / mL. Plasma was mixed with corn trypsin inhibitor (CTI) (Hematologic Technologies, Inc., Essex Junction, VT) to inhibit coagulation factor XIIa impurities, resulting in a final concentration of 40 μg / mL.

[0433] Pre-warmed (37°C) plasma (80 μL) was added to each well of a 96-well microplate (Immulon 2HB, transparent U-bottom; ThermoElectron, Waltham, MA). In order to trigger the production of thrombin by tissue factor, 10 μL of low PPP reagents containing low amounts (12 pM) of recombinant human tissue factor and phospholipid vesicles (48 μM) (Thrombinoscope BV, Maastricht, the Netherlands) consisting of phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine were added. Peptides were diluted 1 / 7.5 with DMSO from a 10 mM stock solution and further diluted 1 / 8.33 with Aqua-Dest, resulting in a DMSO concentration of 12%, providing a 0.5% DMSO concentration in the final assay mixture. Immediately before placing the plate into the pre-warmed (37°C) reader, 5 μL of HEPES-buffered saline or Aqua-Dest containing 12% DMSO was added, followed by the addition of peptide dilutions or reference proteins (FVIII Immunate Reference Standard (Baxter BioScience, Vienna, Australia), Factor VIII Inhibitor Bypass Activity (FEIBA) Reference Standard (Baxter BioScience, Vienna, Australia), NovoSeven (Novo Nordisk, Denmark), and purified human plasma FIX (Enzyme Research Laboratories, South Bend, IL)). Thrombin generation was initiated by dispensing 20 μL of FluCa reagent (Thrombinoscope BV, Maastricht, The Netherlands) containing a fluorescent substrate and HEPES-buffered CaCl2 (100 mM) into each well. Fluorescence intensity was recorded at 37°C.

[0434] Using a Thrombinoscope TM The parameters of the resulting thrombin generation curve were calculated using the Thrombinoscope software (Thrombinoscope BV, Maastricht, The Netherlands) and the thrombin calibrator to correct for internal filter and substrate consumption effects (Hemker, Pathophysiol. Haemost. Thromb., 33, 4-15 (2003)). The peak thrombin amount (peak thrombin, nM) of each thrombin generation curve was plotted for the standard concentration and fitted by a nonlinear algorithm. Based on this correction, the equivalent activity of FVIII Immunate, FIX, FEIBA or NovoSeven was calculated. Figure 12-18 Results for various peptides are provided in Tables 28-30. Representative results are provided in Table 8. (* indicates FVIII-deficient plasma obtained from different donors.)

[0435] Table 8

[0436]

[0437] Referring to Table 8, JBT0120, JBT0132, JBT0224 and JBT0303 increased TFPI-dependent thrombin generation in FVIII-poor plasma to more than 1% of the level of thrombin generation in FVIII-containing plasma (% FVIII-equivalent activity). The tested peptides exhibited approximately 5%-40% of FVIII-equivalent activity in FVIII-poor plasma. Fig.11A and 11B As shown, JBT0120 and JBT0132 dose-dependently increased peak thrombin and peak time.

[0438] Example 5

[0439] The following examples demonstrate that the peptides of the present invention can be modified by the addition of moieties that enhance the physicochemical or pharmacokinetic properties of the peptides. As illustrated below, the addition of 40 kDa PEG to the peptides described herein significantly improved the pharmacokinetic behavior of the peptides.

[0440] Methods for conjugating chemical or biological moieties to peptides are known in the art. To add PEG (polyethylene glycol) to the peptides described herein, a functional group (AOA = aminooxyacetic acid) is added to the N-terminus of the peptide for linking aldehydes and ketones. Alternatively, a cysteine ​​is added to the C-terminal portion of the peptide for conjugation with maleimide (Hermanson, Bioconjugate Techniques, Academic Press (1996)). Peptides (JBT1586) AOA-FQSKGNVFVDGYFERL-Aib-AKL-NH2 (SEQ ID NO: 166) and (JBT1587) Ac-FQSKGNVFVDGYFERL-Aib-AKLC-NH2 (SEQ ID NO: 167) are used for N-terminal and C-terminal modification with PEG, respectively. AOA-FQSKGNVFVDGYFERL-Aib-AKL-NH2 (SEQ ID NO: 166) and Ac-FQSKGNVFVDGYFERL-Aib-AKLC-NH2 (SEQ ID NO: 167) were incubated with excess 40 kDa mPEG-propionaldehyde (SUNBRIGHT ME-400AL2, NOF, Japan) and 40 kDa mPEG-maleimide (SUNBRIGHT ME-400MA, NOF, Japan), respectively. The resulting PEGylated peptides, JBT1853 and JBT1855, showed similar affinity compared to the starting structure Ac-FQSKGNVFVDGYFERL-Aib-AKL-NH2 (JBT0740) (SEQ ID NO: 66).

[0441] The resulting PEGylated peptides exhibited significantly increased plasma stability and prolonged plasma half-life in mice. Fig.31 The results of pharmacokinetic analysis comparing the free peptide JBT0740 (Ac-FQSKGNVFVDGYFERL-Aib-AKL-NH2) (SEQ ID NO: 66) and the C-terminal PEGylated peptide JBT1855 (Ac-FQSKGNVFVDGYFERL-Aib-AKLC (PEG (40kD)) -NH2) (SEQ ID NO: 252) are shown. In contrast to the unPEGylated peptide, the PEGylated peptide was present at high concentrations in mouse plasma at 100 minutes after administration. The unPEGylated peptide was rapidly cleared from the plasma.

Claims

1. A peptide that binds TFPI, wherein the peptide consists of an amino acid sequence that is at least 80% identical to SEQ ID NO: 1040, and the amino acid sequence that is at least 80% identical to SEQ ID NO: 1040 is selected from the group consisting of SEQ ID NO: 1001-1003, 1007, 1010, 1014, 1016, 1018, 1024, 1026-1029, 1031-1033, 1035, 1037, 1038, 1040, 1042-1051, 1053, 1056, 1060, 1061, 1065, 1072, 1075, 1076, 1078, 1087, 1091, 1093, and 1097-1099.

2. The peptide according to claim 1, which consists of the amino acid sequence shown in SEQ ID NO:1040.

3. The peptide of claim 1, which consists of the amino acid sequence shown in SEQ ID NO: 1044.

4. The peptide of claim 1, wherein the peptide is conjugated to a polyethylene glycol (PEG) moiety.

5. The peptide of claim 1 or 4, for use in a method of treating a subject.

6. The peptide of claim 5, wherein the method is for treating a coagulation disorder.

7. Use of a peptide according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of coagulation disorders.

8. A pharmaceutical composition comprising the peptide according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 8, wherein the composition comprises other pharmaceutically effective agents.

10. The pharmaceutical composition of claim 8 or 9, wherein the pharmaceutical composition is used to treat a coagulation disorder.

11. Use of the peptide according to any one of claims 1 to 4 in the preparation of a medicament for targeting cells expressing TFPI.

12. The use of claim 11, wherein the cell is in a mammal.

13. The use of claim 11, wherein the peptide is conjugated to a moiety, wherein such moiety is selected from the group consisting of a dye, a radionuclide, a complex containing a radionuclide, an enzyme, a toxin, and an antibody.

14. The use of claim 13, wherein the toxin is a cytotoxic agent.

15. The use according to claim 13, wherein the dye is a fluorescent dye.

16. Use according to any one of claims 13 to 15, wherein peptide-TFPI binding is detected by detecting an interaction partner complexed with said peptide or said portion.

17. The use according to claim 16, wherein the interaction partner is selected from the group consisting of antibodies, anticalins, aptamers and spiegelmers.

18. The use of claim 16, wherein the interaction partner comprises a detection moiety.

19. The use of claim 18, wherein the detection moiety is selected from the group consisting of a dye, a radionuclide, a complex containing a radionuclide, and an enzyme.

20. The use according to claim 19, wherein the dye is a fluorescent dye.

21. Use of a peptide according to any one of claims 1 to 4 in the manufacture of a medicament for treating a subject suffering from or at risk of suffering from a coagulation disorder, wherein the peptide is conjugated to a therapeutic agent.

22. Use of a peptide according to any one of claims 1 to 4 in the manufacture of a medicament for treating a subject suffering from or at risk of suffering from a coagulation disorder, wherein an interaction partner is administered to the subject, which interaction partner: (a) binds to the peptide, and (b) is a therapeutic agent or is combined with a therapeutic agent.

23. The use according to claim 22, wherein the interaction partner is selected from the group consisting of antibodies, anticalins, aptamers and spiegelmers.

24. The use according to any one of claims 21 to 23, wherein the therapeutic agent is selected from the group consisting of a dye, a radionuclide, a complex containing a radionuclide, an enzyme, a toxin and an antibody.

25. The use of claim 24, wherein the toxin is a cytotoxic agent.

26. The use according to claim 24, wherein the dye is a fluorescent dye.

27. Use of a peptide according to any one of claims 1 to 4 in the preparation of a medicament for diagnosing a subject suffering from or at risk of suffering from a coagulation disorder, wherein the peptide is bound to a detectable portion and detection of the detectable portion indicates that the subject suffers from or is at risk of suffering from a coagulation disorder.

28. Use of a peptide according to any one of claims 1 to 4 in the preparation of a medicament for diagnosing a subject suffering from or at risk of suffering from a coagulation disorder, wherein an interaction partner that binds to the peptide is administered to the subject, the interaction partner is bound to a detectable portion, and detection of the detectable portion indicates that the subject suffers from or is at risk of suffering from a coagulation disorder.

29. The use according to claim 28, wherein the interaction partner is selected from the group consisting of antibodies, anticalins, aptamers and spiegelmers.

30. The use of any one of claims 27 to 29, wherein the detectable moiety is selected from the group consisting of a dye, a radionuclide, a complex containing a radionuclide, an enzyme, a toxin and an antibody.

31. The use of claim 30, wherein the toxin is a cytotoxic agent.

32. The use according to claim 30, wherein the dye is a fluorescent dye.

33. Use of a peptide according to any one of claims 1 to 4 in the preparation of a composition for diagnosing a subject suffering from or at risk of suffering from a TFPI-related coagulation disorder, wherein an interaction partner that binds to the peptide is administered to the subject, the interaction partner is bound to a detectable portion, and detection of the detectable portion indicates that the subject suffers from or is at risk of suffering from the coagulation disorder.

34. The use according to claim 33, wherein the interaction partner is selected from the group consisting of antibodies, anticalins, aptamers and spiegelmers.

35. The use of claim 33 or 34, wherein the detectable moiety is selected from the group consisting of a dye, a radionuclide, a complex containing a radionuclide, an enzyme, a toxin, and an antibody.

36. The use of claim 35, wherein the toxin is a cytotoxic agent.

37. The use according to claim 35, wherein the dye is a fluorescent dye.

38. A method for purifying TFPI, wherein the method comprises a) contacting a sample comprising TFPI with a peptide according to any one of claims 1 to 4 under conditions suitable for the formation of a complex between TFPI and said peptide; b) removing the complex from the sample; and optionally, c) dissociation of the complex to release TFPI.

39. The method of claim 38, wherein the peptide is immobilized on a support.

40. The method of claim 39, wherein the support comprises a solid support.

41. A method according to any one of claims 38 to 40, wherein the peptide is immobilized on a chromatographic stationary phase and step (c) comprises eluting TFPI bound to the peptide.

42. The method of claim 38, wherein the peptide of any one of claims 1 to 4 is purified by affinity chromatography.

Images