AU2014262256A1 - Tfpi inhibitors and methods of use - Google Patents

Abstract

The invention provides peptides that bind Tissue Factor Pathway Inhibitor (TFPI), including TFPI-inhibitory peptides, and compositions thereof. The peptides may be used to inhibit a TFPI, enhance thrombin formation in a clothing factor-deficient subject, increase blood clot formation in a subject, treat a blood coagulation disorder in a subject, purify TFPI, and identify a TFPI binding compound.

Description

EDITORIAL NOTE 2014262256 - There are 145 pages of Description, where the page numbers have been cut off (refer to divisional parent) Regulation 3 2 Revised 2/98 AUSTRAUiA Patents Act, 1990 ORIGINAL COMPLETE SPECIFICATION APPLICANT/S: Baxter International Inc. and Baxter Healthcare SA INVENTORS: DOCKAL, Michael HARTMANN, Rudolf FRIES, Markus SCHEIFLINGER, Friedrich EHRLUCH, Hartmu! REiNEKE, Ulrich OSTERKAMP, Frank POLAKOWSKI, Thomas ADDRESS FOR SERVICE: Peter Maxwell and Associates Level 6 60 Pitt Street SYDNEY NSW 2000 INVENTION TITLE: TFP INi-BITORS AND METHODS OF USE DETAILS OF ASSOCIATED APPLICATION NO(S): Divisional of Australia Patent Application No, 2011 227 714 filed on 11 February 2011 The following statement is a full description of this invention including the best method of performing it known to us: m:\docs\20111196\341047.doc 1 TFPI INHIBITORS AND METHODS OF USE TECHNICAL FIELD OF THE INVENTION [00011 The invention generally relates to peptides that bind Tissue Factor Pathway Inhibitor (TFP1) and uses thereof. INCORPORATION BY REFERENCE 100021 This application claims priority to U.S. Provisional Patent Application No. 61/15,758, filed March 19 20110, which is hereby incorporated by reference in its entirety. The following applications also are incorporated by reference in their entirety: U.S. Provisional Patent Application No, 61139,272, filed December 19., 2008; and U.S. Patent Application No. 12/643,818, fled Decemnber 21, 2009. BACKGROUND OF THE INVENTION [00031 Hemostasis relies on the complex coagulation cascade, wherein a series of events mediated by blood clotting factors leads to conversion of prothrombin to thrombin. Factor X (FX) activation is the central event of both the intrinsic and extrinsic pathways of the coagulation cascade. The extrinsic pathway has been proposed as the primary activator of the coagulation cascade (Mackman et at., Arterioscler. Thromb. Casc. Biol, 27, 1687-1693 (2007)). Circulating Tissue Factor (TF) and activated Factor VII (FVIIa) interact to form the "extrinsic complex," which mediates activation of FX. The coagulation cascade is amplified by the intrinsic pathway, during which successive activation of factors XII, XI, IX, and VIII results in formation of the "intrinsic" FIXa-FVIIIa complex that also mediates FX activation. Activated FX promotes thrombin formation, which is required for the body to create fibrin and effectively curb bleeding. [0004] Severe bleeding disorderssuch as hemophilia result from disruption of the blood coagulation cascade. Hemophilia A, the most common type of hemophilia, stems from a deficiency in factor VIII, while hemophilia B is associated with deficiencies in Factor IX (FIX). Hemophilia C is caused by a deficiency in Factor XI (FXI) (Cawthern et al., Blood, 91(12), 4581-4592 (1998)). There is currently no cure for hemophilia and other clotting diseases, Factor replacement therapy is the most common treatment for blood coagulation disorders. However, blood clotting factors typically are cleared from the bloodstream shortly I 1x after administration. To be effective, a patient must receive frequent intravenous infusions of plasma-derived or recombinant factor concentrates which is uncomfortable, requires clinical settings, is expensive, and is time consuming. In addition, therapeutic efficacy of factor replacement therapy can diniinish drastically upon formation of inhibitory antibodies. Approximately 30% of patients with severe hemophilia A develop inhibitory antibodies that neutralize Factor VIII (FVIl) (Peerlinck and Hermans, Haemophilia, 12, 579-590 (2006)). Few therapeutic options exist for patients with anti-Factor antibodies, [0005] Thus, there exists a need in the art for compositions and methods for treating blood coagulation disorders. The invention provides such compositions and methods, SUMMARY OF THE INVENTION [0006] The invention provides peptides that bind to Tis2sue Factor Pathway Inhibitor (TFP , including TFPI antagonistic peptides having the ability to modulate the blood coagulation cascade, For example, the invention provides a peptide comprising the amino acid sequence X 7 XsXsj 1 X 1 X ]X 1

.

16 X .

9

X

20 x (SEQ ID NO: 3109), wherein

X

7 is selected from the group consisting of L, P, K, S, W. V, N, and Q; Xs is selected from the group consisting of L. R, N, F, and I;

S

;is selected fron the group consisting of Y, P, and C;

X

10 is selected from the group consisting of F, L, and G;

X

11 is selected from the group consisting of L, W, V, A., , T, and S;

X

1 _ is selected from the group consisting of T, F, V, R, A, ), L, E, S, and Y;

X

1 is selected from the group consisting of I, M, G, Q, D, and R;

X,

4 is selected from the group consisting of 3, VA Y, L M. and I; X, is selected from the group consisting of N, P, F. H, K, and Y,

X

16 is selected from the group consising of M, D, , V. G, and K;

X

1 - is selected from the group consisting of G , I, R 8, ST, and L:

X

18 is selected from the group consisting of' M, K, L and I;

X

19 is selected from the group consisting of Y, C, R, and S;

X

20 is selected from the group consisting of A, E, S, C, and Y; and X0 is selected from the group consisting of A, V, K, and E.

[0007] 1In one aspect, the peptide comprises one or more N-terminal amino acid(s) directly linked to X, wherein the N-terminal amino acid(s) comprise the amino acid sequence selected from the group consisting of X x 5 xe,

X

3

X

4

X

5

X

6 (SEQ ID NO: 3110),

X

2

X

3

X

4 Xs2Q, (SEQ ID NO: 3111), and X sXX3XX5X0 (SEQ ID NO: 3112) wherein X, is selected from the group consisting of T and G; X 2 is selected from the group consisting of F, and V; X 3 is selected from the group consisting of V, W, Y, and F; K 4 is selected from the group consisting of D, Q, and S; Xs is selected from the group consisting of E, T, N, and S; and X 6 is selected from the group consisting of R, H, K, and A. [0008] Alternatively or in addition, the peptide comprises one or more C-terminal amino acids directly linked to X2 1 , wherein the C-terminal amino acid(s) comprise the amino acid sequence selected from the group consisting of

X

22 X23,

X

2

X

2

X.

2 -, X X(SEQ ID NO: 3113),

X

2 2

X

2 4

X

25

X

1 (SEQ IDNO: 311J4),and X2 2 X2 3

X

2 4

X

25

XX

2 2 (SEQ ID NO: 315 wherein

X

2 is selected from the group consisting of Q, I, W, Wn RA and N; X 2 is selected from the group consisting of L V, M and R; X Ais selected from the group consisting of K, L, A, and Y; X 2 5 is F; X, is 0; and X, is 1'. [0009] In one aspect, the invention provides a peptide comparing the amino acid sequence set forth in SEQ ID NOs: 1-7, such as a peptide comprising the amino acid sequence set forth in any one of JBT132, JBT0303. JBTOI193, JBTOI 78, JBTO120, and 111T0224, which inhibits TFPI activity within the blood coagulation cascade, The invention also provides a peptide that hinds TFPI comprising an amino acid sequence of at least 60% identity to the sequence Phe4Gn-Ser-Lys-Gly-Asn-Va,.Phe-Val-ApGy-TyrPhe-Glu-Arg-Len-Arg-Ala LysLenu (FQSKGNVFVDGYFERLRAKL) (SEQ ID NO: 32). [0010] In addition, the invention provides a peptide that binds TFPI, wherein the peptide comprises the structure of formula (1): X1 001 -X1 002-X 1003-NX1004-KX1005-KX1006-KX1007- X1008-X1O09-X1010-X1011-X1012-X1o3-X1o14-X115-X10l6-X1017-X1018-X11O9 X1020 (SEQ ID||NO: 3116 In formnda (li X1001 is an amino acidselected from the group consising of Bh, C , F, C I1 K, L, M, N, Nmf, Q, R, T, V, W, and Y; X 002 is an amino acid selected from the group consisting of I K, andQ; X1003 is an amino acid selected from the group consisting of A, Aib, BLs, C, D, E, F, G, H, 1, K, L, M, N, P, Q, R, S, T, V, W, and Y; X1004 is an amino acid selected from the group consisting of A, Aib Bhk, C, D, E, F, G, H, 1, K, k, L, M, N, Nmk, P, Q, R, S, T, V, W, and Y; X1005 is an amino acid selected from the group consisting of 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; X1006 is an amino acid selected from the group consisting of A, Aib, 3tq, C, D, E, F. GH , K, l N,I Q R, S F, V, W, and Y X1007 is an amino acidseleted from the group consisting of A, F, G, I, K, L, Nmv, , Q, S, V, W, and Y; X100 is an amino acid selected from the group consisting of F , K, W, and Y; X1009 is an amino acid selected from the group consisting of A A, f, I, K, and V; X10I0 is an amino acid selected from the group consisting of A, A, C, D, F G, H, 1, K, L, M, N, Nmt P, Q, R, S, T, V, W, and Y; X101 I is an amino acid selected from the group consisting of Aib, C, K, Gi, and Nmg; X1012 is Y; X1013 is an amino acid selected from the group consisting of A, Aib, C, E, F., G, H K, LM.Q, R, W,and Y; 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; X1015 is an amino acid selected from the group consisting of (omega-methyi)R, D, E, K, and R X1016 is L; X 017 is an amino acid selected from the group consisting of (omegamethylR A, Aib Bk; C, Cha, CitD. )Dab, DapE , Lag Eew, F, HL H, ar Hi HeI KN L, M N, Nie Nva, OpaOrnO Q, R, ST, V, Wand Y; Xl0i8 is an amino acid selected from the group consisting of A, Bal, C, D, E, F, G, H I K, LM, N Q, R 5, T, V, W, andY; X1019 is an anino acid selected from the group consisting of Bhk, K, R, and V; and X1020 is either present or absent, whereby, in case X1020 is present, it is an amino acid selected from the group consisting of A Bhl, C. F, , H, I, K, L, Nml, Q, R S , V, Wand Y. [0011] In one aspect, the peptide that binds TFPI comprises the structure of formula 011): X1001-Q-X1003-X1004-X1005-XI006-I/V-X1OOS-V-XI010-G-Y-C/F-X1014-R-L-X 1017 X1018--K-K/L (11) (SEQ ID NO: 3117). In formula (Il), X1001, X1003, X1 004, X1005, X 1006, X1008, Xi1O1, X1014, X1017, and X1018 are each independently selected from any amino acid. [0012] The invention further provides a TFPI-binding peptide comprising the structure of formula (V): X2001 -X2002-X2003-X2004-X2005-X2006-[X2007-X2008-X209-X2010 X2011-X2012-X2013-X2014-X2015-X2016-X2017-X2018]-X2019-X2020-X2021-X2022 X2023 (V) (SEQ ID NO: 3118). Tn formula (V), X2001, X2002, and X2023 independently are either present or absent. When present, X2001 is an amino acid selected from the group consisting of A, D, E, F, G, H, I. K, L, P, R, S, T, V, and W; and X2002 is an amino acid selected from the group consisting of A, D, E, F, 0, H, I K, L, M, P, R, S T, V, and W. Additionally, X2003 is an amino acid selected from the group consisting of A, F, K, L, R, T, 1 V W, and Y; X2004 is an amino acid selected from the group consisting of A, ), E, F, , , K, L, R, S, T, V, and W; X2005 is W; X2006 is an amino acid selected frn the group conssting o H1 K L, R V and W; X2007 is an amino acid selected from the group consisting of C, I-Iey, Dap, and K, preferably selected from the group consisting of C and Hey; X2008 is an amino acid selected f -rm the group consisting of A, G, R, and T X2009 is an amino acid selected from the group consisting of a, A, 1, K, LM, m, ne p, R, and V; X2010 is an amino acid selected from the group consisting of A, G1, K, L, P, R, S, and V; X201 I is an amino acid selected from the group consisting of D, E, 0, S, and T; X2012 isan amino acid selected from the group consisting of A, a, D, d, E, e, F, f, G, , K, k, L, 1, M, m, Nie, ne, P, p, R, r, S, s, T, t, V, v, W, and w; X2013 is an amino acid selected from the group consisting of A, D, d, E, e, F, I, K. L, R 8, s, T, V, and W; X2014 is an amino acid selected from the group consisting of A, D, E, F, G 1, K, L, MR, R, S, T, V, and W; X2015 is an amino acid selected from the group consisting of A, D, E, F, G 1, K, L, M, Me, R, S, T, V. and W; X2016 is an amino acid selected from the group consisting of A, D, E, F, , K, L, M, Me, R, S, T, V, W, and Y; X2017 is an amino acid selected from the group consisting of A, 1, , F, 0, I, K L, R, S, T, V, W, and Y; X2018 is an amino acid selected from the group consisting of C and D (preferably X2018 is C); X2019 is an amino acid selected from the group consisting of A, F, I, L, S, T, V, and X2020 is an amino acid selected from the group consisting of F and W; X2021 is an amino acid selected from te group consisting of 1, L, and V; and X2022 is an amino acid selected from the group consisting of A, D, E, F, G, 1 K, L, P, R, S, T V, and W, When X2023 is present in the peptide, X2023 is an amino acid selected from the group consisting of A, D. E, F, Gt K, 1 L, R, S,T, V, W, and Y. In one aspect, the peptide comprises a cyclic suctugeneratedba linkage between X2007 and X2018, indicated in Formula (V) by brackets.

[0013] The invention also provides a peptide that binds TFPi, where the peptide comprises at least anno acids 3-22 of the structure of formula (VI): X2001-X2002-F/Y-K W-F/H-[C-X2008-M/V-X201 0-D-X2012-X2013-G-I/T-X2016-S/T-C]-A/V-W-V-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, as well as X2001, X2002, and X2023 when present, are each independently selected from any amino acid, The peptide comprises a cyclic structure generated by a linkage between X2007 and X2018, indicated in formula (VI) by brackets. [0014] In one aspect, the invention provides a peptide that binds TFPI, wherein the peptide comprises at least amino acids 3-21 (X3003-X3021) of the structure of formula (VII): X(3001 X3002-X3003 -X3004=X3005-X3006-X3007-X3008-X3009-X 3010-X(3011-X(3012 X(3013-3X3014-X(3015-X(3016-3X3017-3X3018-X(301 9-X3020-X3021 (VIII) (SEQ ID NO: 3120). In fonnula (VIII), X3001 and X3002 are each independently present or absent. When present, X3001 is an amino acid selected from the group consisting of A, C, D, F, G, , K, L, M N, P, Q, R, S. T, W,, H, and Y; and X3002 is an amino acid selected from the group consisting of AC,) D F H, K. M, N, P R, S. T, W, Y, G, 1, and L. With respect to the remainder of formula (VIII), X3003 is an amino acid selected from the group consisting of A, C, D, E, F, G, I I, K, L,M, N P, Q R S, T, W, and Y; X3004 is an amino acid selected from the group consisting of A, CD, E, F, G HA, K,L M, N QR S T, V, W Y, and P; 3005 is an amino acid selected from the group consisting of C, D, F, 0, H, , L, M. N, P R, S, T, V, W, and Y; X3006 is an amino acid selected from the group consisting of A, W, C, K, P R, and H; X<3007 is an amino acid selected from the group consisting of Q, A, C, F, G H, 15 K, L N, R, S, T, W, and Y; X33008 is an amino acid selected fromthe group consisting of A. C, F, 0G H, K. L, M, NP Q, R S, T, V, W Y, and I X3009 is an amino acid selected from the group consisting of A, C, , G! H, I, L, M, R S, T, V, W, Y, and K: X3Oi0 is an amino acid selected from the group consisting of A, C, F, C0, , IK L, M, N, P, Q R, S, T, V, W, and i; X3011I is an amino acid selected from the group consisting of A G, I, K, L, M, N, Q, R, S T. V, WY , , ,, and H; X301 2 is an amino acid selected from the group consisting of A, C, H, I, K, L, and R; X301 3 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; X3014 is an amino acid selected from the group consisting of A, C, F , H, 1, L, M, N, Q, R, S, T, V, W, Y, and K; X3015 is an aminoacid selected from the group consisting of A, K, and R; X3016 is an amino acid selected from the group consisting of A, F K, and R; X3017 is an amino acid selected from the group consisting of A, C, F 0, I, K, L, N, Q, R, S, T, V, W, Y, H, A, and M; X301S is an amino acid selected from the group consisting of A, C, F, I, K, L, M, Q, R, V, W, and Y; X3019 is an amino acid selected from the group consisting of A, C, D E, C . H, K L, N, P, Q, R V, W. Y, and I; X3020 is an amino acid selected from the group consisting of A, C, F, 0 H, K, L, M. N, Q, R, V, V, Y, , and P; and X3021 is an amino acid selected from the group consisting of A, C, H, 1, K, L, M, N, P, Q R, T, V, W, Y, F, and G. [0015] Additionally, the 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/R-R-.X3017-X3018-X3019-X3020-X3021 (IX) (SEQ ID NO: 3121), wherein X3001, X3002, X3003, X3004, X3005, X3006, X3007, X3008, X3009, X3010, X301 1, X3013, X3014, X3017, X3018, X3019, X3020, and X3021 are each independently selected from any amino acid. In addition, the invention includes a peptide that binds TFPI wherein the peptide comprises an amino acid sequence having at least 60% identity to the sequence of formula (X): Ac-GYASFPWFVQLHVHXKRSWEMA-NH2 (SEQ ID NO: 223), [0016] The invention further provides a TFPT-binding peptide comprising the structure of formula (XI): X400I-Q-X4003-X4004-X4005-X4006-X4007-X4008-X4009-X4010-X4011 X4012-X4013 -X4014-R-X4016-X4017-X4018-.X4019-X4020 (XI). With respect to fonnula (XI), X4001 is an amino acid selected from the group consisting of F, L, M, Y, INi, Thi, Bta, and Dopa; X4003 is an amino acid selected from the group consisting of C, D, E i, Q, R, ST, Ede(O), and Cmc; X4004 isan amino acid selected from the group consistdng of Ai hFG 1, K, I_ M, P, R, W, and Y; X4005 is an amino acid selected from the group consisting of a, A, Aib, C, D, d, , G, H, K, k, l,, N Nmg, p, Q, R, NpropylG, aze, pip, tic, oc, hyp, nia, Neg, A bg, Apg, 0hz, and dtc; X4006 is an amino acid selected from the group consisting of A, C, C(NEM), D, E, G, H, K, Mi. N, Q R, S, V, Cit. C(Acm), Nie, 1, Ede(O), Cmc, Ed, Eta, Eec, Ef, Nif, and Eew; X4007 is an amino acid selected from the group consisting of , V, T Chg, Phg, and TVe; X4008 is an amino acid selected from the group consisting ofF, L Wi, 2Ni, Pmy, and Y; X4009 is an amino acid selected from the group consisting of Aib, V, Chg, Phg, Abu, Cpg, Tle, and L-2-amino-4,4,4--trifluorobutyric acid; X4010 is an amino acid selected from the group consisting of A, C, D, d, E, F, H, K, M, N, P Q, , S, T, V, V, Y, Nmd, and C(NEM); X401I is an amino acid selected from the group consisting of A, a, G, p, Sar c, and hey; X4012 is an amino acid selected from the group consisting of Y, Tym, Pty, Dopa, and Pmy; X401 3 is an amino acid selected from the group consisting of C, F, INi, Thi, and Bta: X4014 is an amino acid elected from the group consisting of A, Ail C C(NEM), D, E, L, , Q, R,

T

, , and HCy X4016 is an amino acid selected from the group consisting of L, Hey ie, and An X4017 is an amino acid selected from the group consisting of A, a, Aib, C, C, Cha, Dab, Eag, Few, H. lar, Hei. Hle, 1, K, L, M, Ne, Nva, Opa, Orn R, S, Deg, Ebe, Ea, Egz, Aic, Ape, and Egt; X4018 is an amino acid selected from the group consisting of A, Ab, Hey, hey, C, c, L, NIe, M, N, and R; X40 19 is anamino acid|selected from the group consisting of K. Rand Har; and X402( is an amino acid selected from the group consisting of K L, Hey, and An] [0017] The TFPI-binding peptide of formula (XI) does not comprise the structure formula (XII): X500I-Q-X5003-X5004-X5005-X5006-I/V-X3008-Aib/V-X5010-G-Y-X013 X5014-R-L-X5017-X5018-K-K/L (XII). In formula (XII), X5001 is an amino acid selected from the group consisting of F, L, M, and Y; X5003 is an amino acid selected from the group consisting of C, D, E, M, Q, R, S, andT X5004 is an amino acid selected from the group consisting of F, G, 1, K. L, M, P, It W, and Y; X5005 is an amino acid selected from the group consisting of a, A, Aib, C, d, E, H, K, k, M, N, Nmg, Q, R, and p; X5006 is an amino acid selected from the group consisting of A, C, D, E, G H, K, M, N, Q, R, S, and V; X5008 is an amino acid selected from the group consisting of F, H, and Y; X5010 is an amino acid selected from the group consisting of A, C, D, E, F, H, D, M, N, P, Q R, S, T, V, W, and Y; X5013 is an amino acid selected from the group consisting of Aib, C, and F: X50l4 is an amino acid selected from the group consisting of A, Atb C, D, E, K, L, MtN, QR, Trandv; X5017 is an amino acid selected from the group consisting of A, Aib, C, Cha, Dab, Eag, Eew, H, Har, Hci, Hle, I, K, L, M, Ne, Nve, Opa, Orn, R, and S; and X5018 is an amino acid selected from the group consisting of A, C, L M, N, and R [0018] The invention also includes a peptide consisting of the amino acid sequence selected from the group consisting of SEQ ID Ns: 4022, 4024, 4032, 40364047, 4049- 4078, 4086-4097, 4100-4127, 4129-4170, 4173-4195, 4200-4214,4217-4225,4228,4230, 4231, 4238, and 4239, as well as a peptide consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1294-1336, 4002, 4013, 4021 4023 40254031, 4033-4035, 4048, 4079-4085, 4098, 4099, 4128, 4171, 4172, 4196-4199, 4215, 4216, 4226, 4277, 4229, 4232, and 4233. [0019] In the context of the disclosure, any peptide encompassed by any of formulas (I) to (XI) and any TFPI-binding peptide described herein is also referred to as "the peptide of the invention" and as "a peptide as described herein. [0020] In some embodiments, the peptide of the invention binds TFPV I (e.g., TFPI- l a) and, optionally, improves TFPI-regulated thrombin generation in the absence of FVIII, FIX, and/or FXI. A composition (e.g., a pharmaceutical composition) comprising the peptide also is provided. [0021] In addition, the invention provides methods of using the peptide of the invention. For example, the invention provides a method of inhibiting a TFPI comprising contacting the TFPI with a peptide as described herein. The invention also provides a method of enhancing thrombin formation in a clotting factor-deficient subject, a method for increasing blood clot formation in a subject, and a method of treating a blood coagulation disorder in a subject. The methods are, in their entirety, also referred to herein as, e.g., "the method of the invention." The methods comprise administering to the subject a peptide as provided herein in an amount effective to enhance thrombin formation, an amount effective to enhance blood clot formation, or an amount effective to treat the blood coagulation disorder in the subject. Unless explicitly indicated to the contrary, the description provided herein with respect to one peptide of the invention or method of the invention applies to each and every peptide of the invention and method of the ivention, respectively. Further aspects of the invention include use of the pept of of the invention for the manufacture of a medicament a method for targeting a cell displaying TFPI, a method for treating or diagnosing a subjectIsuffering from a disease or at risk of suffering from a disease, a method of purifying TFPI, and a method of identifying a TFPI-binding compound. [0022] The invention also includes a method for identifying a TFPIbinding compound, the method comprising (a) contacting a peptide comprising TFPI Kunitz domain I (KDI) with a test compound, and (b) detecting binding of the test compound to a TFPI binding site defined by KD1 amino acid residues corresponding to human TFPI residues Phe28, Lys29, Ala30, Asp32, Ile46, Phe47, and He55. A method for inhibiting human TFPI, the method comprising contacting human TFPI with an inhibitor that binds human TFPI at a binding site defined by amino acid residues Phe28, Lys29, Ala30, Asp32, 1e46, Phe47, and Ie55, also is provided. The invention further provides a computer storage media having computer executable instructions that, when executed on the processor of a computer, implement a method of modeling interaction between selected three dimensional (31)) points in a TFPI Kunitz domain I (KD 1) protein and a test compound, as well as a method of comparing a test compound to selected three dimensional points in a TFPI Kunitz domain I (KDI) protein. [0023] The preceding methods are, in their entirety, also referred to herein as, e.g., "the method of the invention." [00241 The following numbered paragraphs each succinctly define one or more exemplary variations of the invention: [0025] L A peptide that binds TFPI, comprising the structure of formula (X]): X4001 Q-X4003-X4004-X4005.-X4006-X4007-X4008-X4009-X 4 010-X4011-X4012-X4013 X4014-RX4016-X4017-X4018-X4019-X4020 (XI), wherein X4001 is an amino acid selected from the group consisting of F, N, Ni, Thi, Bta, and Dopa; wherein X4003 is an amino acid selected from the group cons listing of C, D, E, M, Q, R, S, T, Ede(O) and Cmc; wherein X4004 is an amino acid selected from the group consisting of Aib, E, G,I,K, L, M, P, R, W, and Y; wherein X4005 is an amino acid selected from the group consisting of a. A, Aib, C, D, d, E, G, H, K, k, M, N, Nmg, p, Q, R, NpropylG, aze, pip, tic, oic, hyp, nia, Neg, Abg, Apg, thz, and dtc; wherein X4006 is an amino acid selected from the group consisting of A, C, C(NEM), D, E. G, H, K, M, N, Q, R, S, V, Cit, C(Acm), Nie, I, Ede(0), Cmc, Ecl, Eea, Eec, Eef, Nif, and Eew; wherein X4007 is an amino acid selected from the group consisting of I, V, T, Chg. Phg, and Tie; wherein X4008 is an amino acid selected from the group consisting of F, H, I Ni, 2Ni, Pmy, and Y; wherein X4009 is an amino acid selected from the group consisting of Aib V, Chg, Phg, Abu, Cpg, Tie, and L-2Jamino-4;4,4 trifluorobutyric acid; wherein X4010 is an amino acid selected from the group consisting of A, C, D, d, E, F. H, K, M, N. P, Q, aR S, T, V. W, Y, Nmd, and C(NEM); wherein X4011 is an amino acid selected from the group consisting of Aa, G, p Sar, c, and hey; wherein X4012 is an amino acid selected from the group consisting of Y, Tym, Pty, Dopa, and Pny; wherein X4013 is an amino acid selected from the group consisting of C, F, I Ni, Thi, and Bita; wherein X4014 is an amino acid selected from the group consisting of A, Aib, C, C(NEM), D, E, K, L, M, N, Q, R. T, V, and Hey; wherein X4016 is an amino acid selected from the group consisting of L, Hey, Hle, and Aml; wherein X4017 is an amino acid selected from the group consisting of A, a, Aib C, c, Cha, Dab, Eag, Eew, H. Har, Hei, le, 1, K, i 1 ) M, Nie, Nva, Opa, Orn, R, S, Deg, Ebe, Ec, gz Aic, Apc, and Egt; wherein X4018 is an amino acid selected from the group consisting of A, Aib Hey, hey; C, c, L, Nie, M, N, and R; wherein X401 9 is an amino acid selected from the group consisting of K R, and Har; and wherein X4020 is an amino acid selected from the group consisting of K, L, Hey, and Ar; and wherein the peptide does not comprise the following structure of formula (XI): X500 Q-X5003-X5004-X500X5006-N-X5008 Aib/ X50 0-I-Y-X501 3-X501 4-R-L-X5017 X5018&-K~KIL (XII), wherein X501 is an anino acid selected from the group consisting of F, L, M, and Y; wherein X5003 is an amino acid selected from the group consisting of C, D E, M, Q, R, S, and T; wherein X5004 is an amino acid selected from the group consisting of E, G, I, K, L, M, P, RI, W, and Y; wherein X5005 is an amino acid selected from the group consisting of a, A, Aib, C, D, d, E, G, I-H K, k, MI, N, Nmg, QR, and p; wherein X5006 is an amino acid selected from the group consisting of A, C, D, E. , H, K, M, N, Q R_ S, and V; wherein X5008 is an amino acid selected from the group consisting of F H, and Y; wherein X5010 is an amino acid selected from the group consisting of A, C, D, E, F, H, D, DM, N, P, Q, R, S, T, V, W, and Y; wherein X5013 is an amino acid selected from the group consisting of Aib, C, and F; wherein X5014 is an amino acid selected from the group consisting of A, Aib, C., D, E, K, L, M, N, Q, R, T, and V; wherein .X5017 is an amino acid selected from the group consisting of A, Aib, C, Cha, Dab, Fag, Eew, H, Har, .Hci, File, I, K, L, M, Nie, Nve, Opa, Om, R, and S; and wherein X5018 is an amino acid selected from the group consisting of A, C, L, M, N, and R. [00261 2 The peptide according to paragraph 1, wherein X4001 is an amino acid selected firm the group consisting of F, Y, 1Ni ta, and Dopa; wherein X4003 is an amino acid selected from the group consisting of D, E, and S; wherein X4094 is K; wherein X4005 is an amino acid selected from the group consisting of p. Nmg, NpropylG, aze, pip, tic, oic, and hyp; wherein X4006 is an amino acid selected from the group consisting of C, E, K, R, S, V, C(Acm), Nie, C(NEM), I, and Cit; wherein X4007 is V or Tie; wherein X4008 is an amino acid selected from the group consisting of H, 1Ni, 2Ni, and my; wherein X4009 is an amino acid selected from the group consisting of V, Abu, and Tie; wherein X4010 is an amino acid selected from the group consisting of D P, C, and T; wherein X4011 is an amino acid selected fro the group consisting of G, , c, hey, and Sar; wherein X401 2 is Y; wherein X4013 is an amino acid selected from the group consisting of F INi, and Eta; wherein X4014 is an amino acid selected from the group consisting of Aib; C, E, and Hey; wherein X4016 is an amino acid selected from the group consisting of L, AilHei, and Hey; wherein X4017 is an amino acid selected from the group consisting of A, Ab, C, c, Aic, Eca, and Deg; wherein X4018 is an amino acid selected from the group consisting of A, Aib, C, c, L, and Hey: wherein X4019 is K; and wherein X4020 is an amino acid selected from the group consisting of L, Ami, and Hey. [0027] 3. The peptide according to paragraph I or pamgraph 2 further comprising N terminal amino acids) and/or moieties linked to X4001 and selected from the group consisting of FAM-Ttds, PE, Palm, 2-phenyI acetyl, 3-phenyl propionyl, 2-(naphtha-2-y1) acetyl, hexanoyl, 2-methyl propionyl, 3-methyl butanoyl, 2-naphthylsulfonyl, and I naphthylsulfonyl. [0028] 4. The peptide according to any one of paragraphs 1-3 father comprsing X4021 linked to X4020, wherein X4021 comprises C-terninal amino acid(s) and/or moieties selected from the group consisting of C, c, C(NEM), K(Ttds-maleimidopropionyl(EtSH)), FA19205, FA1920,4 FA19203, FA03202, K(Tdts-maleiniid), K(AOA), and Cea. [0029] 5. The peptide according to any one of paragraphs 1-4, wherein the peptide comprises a cyclic structure. [0030] 6. The peptide according to paragraph 5, wherein the cyclic struture is formed between X4018 and X4021. [00311 7. The peptide according to paragraph 6, wherein (a) X4018 is C or c and (b) X4021 is Cea. [00321 8 The peptide according to paragraph 5, wherein the cyclic structure is formed between (4011 and X4014, [0033J 9. The peptide according to paragraph 8, wherein (a) X40l I is C or hey and (b) XK4014 is C or Hey, [00341 10. The peptide according to any one of paragraphs 1-9 compriing an intramolecular disulfide bond. [0035] 11. The peptide according t any one of paragraphs 1-10, wherein the IC 0 of the peptide is less than 1000 nM. [0036] 12, The peptide according to any one of paragraphs 1-- I0, wherein the ICs 0 of the peptide is less than 250 nM. [0037] 13. The peptide according to any one of paragraphs 1-10, wherein the IC 0 of the peptide is less than 50 nM.

[00381 14 The peptide according to any one of paragraphs 110, wherein the IC 0 of the pepdde is less than 10 nM. [0039] 15, A peptide consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 4022, 4024, 4032, 4036-4047, 4049-4078, 4086-4097, 4100 4127, 4129-4170, 4173-4195, 4200-4214, 4217-4225, 4228, 4230, 4231, 4238, and 4239. [0040] 16, A peptide consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1294-1336, 4002, 4013, 4021, 4023, 4025-4031, 4033-4035, 4048, 4079-4085, 4098, 4099, 4128, 4171, 4172, 4196-4199, 4215, 4216, 4226, 4277, 4229, 4232, and 4233. [041] 17. A TFPhbinding peptide comprising a homo-dimer or homo-muitimer of two or more peptides according to any one of paragraphs 1-16. [0042] 18. A TFPI-binding peptide comprising a hetero-diner or hetero-multimer of two or more peptides according to any of the paragraphs 1-16. [0043] 19. The peptide according to any one of paragraphs 1-18, wherein the peptide inhibits TFPI activity and binds to TFPI 1-alpha with a dissociation constant of less than 10 y M . [00441 20. The peptide according to any one of paragraphs 1-19, wherein the peptide is conjugated to a polyethylene glycol (PEG) moiety. [0045] 21. The peptide according to any one of paragraphs 1-20, wherein the peptide is conjugated to human serum albumin (HSA), an antibody or fragment thereof, hydroxyethy starch, a proline-alanine-serine multimer (PASylation), a C12-C18 fatty acid, or polysialic acid. [046] 22, The peptide according to any one of paragraphs 1-21, wherein the peptide is conjugated to a moiety selected from the group consisting of a photosensitizer, dye, a fluorescence dye, a radionuclide, a radionuclide-containing complex, an enzyme, a toxin, an antibody or fragment thereof, and a cytotoxic agent. [0047] 23. A peptide according to any one of paragraphs 1-22 for use in a method for the treatment of a subject. [0048] 24. The peptide according to paragraph 24 wherein the method is for the treatment of a blood coagulation disorder.

[0049] 25. Use of the peptide according to any one of paragraphs 1-22 for the manufacture of a medicament. [0050] 26 Use of the peptide according to any one of paragraphs 122 for the manufacture of a medicament for the treatment of a blood coagulation disorder. [0051] 27. A pharmaceutical composition comprising the peptide of any one of paragraphs 122 and a pharaceuically acceptable carrier. [0052] 28. The pharmaceutical composition according to paragraph 28 wherein the composition comprises a further pharmaceutically effective agent. [0053] 29. The pharmaceutical composition according to paragraph 27 or paragraph 28, whemin the pharnaceutical composition is for use in a method of treating a blood coagulation disorder. [0054] 30. A method for targeting a cell displaying TFPI, the method comprising contacting the cell with the peptide of any one of paragraphs 1=22. [0055] 31. The method of paragraph 30, wherein the cell is in a mammal, and contacting the cell comprises administering the peptide to the mammal. [0056] 32. The method according to paragraph 30 or paragraph 31 further comprising detecting peptide binding to TFPI displayed on the cell. [00571 33. The method according to paragraph 32, wherein peptide-TFPI binding is detected by detecting a moiety conjugated to the peptide and selected from the group consisting of a photosensitizer, a dye, a fluorescence dye, a radionuclide, a radionuclide containing complex, an enzyme, a toxin an antibody, and a cytotoxic agent. [0058] 34. The method according to paragraph 32 or paragraph h 33, wherein peptide-TFPI binding is detected by detecting an interaction partner complexed with the peptide or a moiety conjugated to the peptide. [0059] 35. The method according to paragraph 34, wherein the interaction partner is selected from the group consistng of an antibody or fragment thereof, an anticalin, an aptamer; streptavidin, avidi, neutravidin, and a spiegehmer. [0060] 36. The method according to paragraph 34 or paragraph 35, wherein the interaction partner comprises a detection moiety. 1 K [0061] 37. The method according to paragraph 36, wherein the detection moiety is selected from the group consisting of a dye, a florescence dye, a radionuclide, a radionuclide-containing complex, and an enzyme. [0062] 38. A method for treating a subject suffering from a disease or being at risk of suffering from a disease, the method comprising administering to the subject the peptide of any one of paragraphs 1 -22, wherein the peptide is conjugated to a therapeutic agent. [0063] 39. A method for treating a subject suffering from a disease or being at risk of suffering from a disease, the method comprising administering to the subject the peptide of any one of paragraphs 1-22, and administering to the subject an interaction partner that (a) binds the peptide and (b) is a therapeutic agent or is conjugated to a therapeutic agent. [0064] 40. The method according to paragraph 39, wherein the therapeutic agent is selected from the group consisting of a photosensitizer a radionuclide, a radionuclide containing complex, an enzyme, a toxin, an antibody or fragment thereof, and a cytotoxic agent. [0065] 41. The method according to paragraph 39 or paragraph 40, wherein the interaction partner is selected from the group consistng of an antibody or fragment thereof, an anticalin, an aptamer, streptavidin, avidin, neutravidin, and a spiegelmer. [0066] 42. A method for diagnosing a subject suffering from a disease or being at risk of suffering from a disease, comprising (a) administering to thee peptide of any one of paragraphs 1-22 conjugated to a detectable moiety and (b) detecting the detectable moiety. [0067] 43. A method for diagnosing a subject suffering from a disease or being at risk of suffering from a disease, comprising (a) administering to the subject the peptide of any one of paragraphs 1-22, (b) administering to the subject an interaction partner conjugated to a detectable moiety, and (c) detecting the detectable moiety, 10068] 44. The method according to paragraph 43, wherein the interaction partner is selected from the group consisting ofn antibody or fragment thereof, an anticalin, an aptamer, streptavidin, avidin, neutravidin, and a spiegelmer. 10069] 45. The method according to any one of paragraphs 42-44, wherein the detectable moiety is selected from the group consisting of a dye, a fluorescence dye, a radionucLide, a radionuclide-containing complex, an enzyme, and an antibody or fragment thereof. [0070] 46. The method according to any one of paragraphs 38-45, wherein the disease is a blood coagulation disorder, 1=? [9071] 47 A method for purifying TFPIwherein the method complies a) contacting a sample containing TFPI with the peptide of any one of paragraphs 1 22 under conditions appropriate to fom1 a complex between TFPfand thepeptide; b) removing the complex from the sample; and, optionally, c) dissociating the complex to release TPh [0072] ,48 The method according to paragraph 47,wherein thepeptide is immobilized to support [0073] 49. The method according to paragraph 48, wherein the peptide is immobilized to a chromatography stationary phaseand sep(c) comprises eluting TFPI bound to the immobilized peptide. [0074] 50. The method according to paragraph 48 or paragraph 49, wherein TPI is purified via affinity chromatography. [0075] 51. A method for identifying a TFPI-binding compound, the method comprising (a) contacting a peptide comprising TFPI Kunitz domain I (KD 1) with a TFPI-binding peptide of any one of paragraphs 1-22 and a test compound under conditions that allow formation of KDI-TFPI-binding peptide complexes, (b) measuring KD1-TFP-binding peptide complexes formed in step (a), and (c) comparing the number of KDI-TPI1-binding peptide complexes formed in the presence of the test compound with the number of KDI TFN-binding peptide complexes formed in the absence of the test compound, wherein a reduction in the number of KD1-TFPI-binding peptide complexes formed in the presence of the test compound compared to the number of KD1-TFPI-binding peptide complexes formed in the absence of the test compound indicates that the test compound is a TFP-binding compound. [0076] 52. The method of paragraph 51, wherein the TFP-binding peptide comprises a label that generates a signal; step (b) composes measuring signal generated by KD1-TFPI binding peptide complexes; and step (c) comprises comparing signal measured in step (b) with signal generated by KD1-FPIinding peptide complexes formed in the absence of the test compound, wherein a reduction in signal generated by IKD1-TFPbinding peptide complexes formed in the presence of the test compound compared to signal generated by KDITFPI binding peptide complexes formed in the absence of the tcst compound indicates that thje test compound is a TFFl-binding compound. [0077] 53. The method of paragraph 51 or paragraph 52, wherein step (a) comprises (al) contacting the peptide comprising KDI with the TFPIbinding peptide under conditions that allow formation of KDI-peptide complexes, and (a2) contacting KDI-TFPb-binding peptide complexes formed in step (at) with the test compound. [0078] 54. A method for identifying a TFPI-binding compound, the method comprising (a) contacting a peptide comprising TFPI Kunitz domain I (KD 1) with a test compound, and (b) detecting binding of the test compound to a TFPI binding site defined by KD amino acid residues corresponding to huinan TFPI residues Phe28, Lys29 Ala30, Asp32, e46, Phe47, and IBe55. [0079] 55. The method of paragraph 54, wherein the binding site is defined by amino acid residues corresponding to human TFPI residues Ala27, Phe28, Lys29, Ala30, A sp31, Asp32, Lys36, Re38, le46, Phe47, and Ile55. [0080] 56. The method of paragraph 54 or paragraph 55, wherein the binding site is defined by amino acid residues corresponding to human TFPI residues Ala27, Phe28, Lys29, Ala30, Asp3l, Asp32, Lys36, Ala37, Re38, Phe44, 1e46, Phe47, and Re55. 100811 57. The method of any one of paragraphs 54-56, wherein step (b) comprises determining the presence or absence of a nuclear magnetic resonance (NMR) chemical shift within the TFPI binding site, [0082] 58. The method of any one of paragraphs 54-56, wherein step (a) comprises contacting the peptide compile sing TFPI KD I with FVla in the presence of a test compound under conditions that allow binding of KDI to FVHa, and step (b) comprises comparing KDI-FVJla binding in step (a) with KDI-PVHa binding in the absence of the test compound, wherein a decrease in K1-FViIa binding in the presence of the test compound compared to KDVFV a binding in the absence of the test compound indicates that the test compound is a TFPI-binding compound. [0083] 59. The method of any one of paragraphs 54-56, wherein step (a) comprises contacting the peptide comprising TFPI KD1 with FXa in the presence of a test compound under conditions that allow binding of KDI to FXa, and step (b) comprises comparing KD1 FXa binding in step (a) with KDI-PXa binding in the absence of the test compound, wherein a decrease in KDL-FXa binding in the presence of the test compound compared to KDI-FXa binding in the absence of the test compound indicates that the test compound is a TFPI binding compound. [0084] 60. The method of any one of paragraphs 54-56, wherein the peptide comprising TFPI KD. further comprises Kunitz domain 2 (KD2), step (a) comprises contacting the peptide comprising TFPI Di and TFPI KD2 with FXa in the presence of a test compound 10 under conditions that allow binding of KD2 to FXa, and step (b) comprises comparing KD2 FXa binding in step (a) with KD2-FXa binding in the absence of the test compound, wherein a decrease in KD2-FXa binding in the presence of the test compound compared to KD2-FXa binding in the absence of the test compound indicates that the test compound is a TFPI binding compound. [0085] 61. The method of any one of paragraphs 54-56 and 58-60, wherein binding of the test compound to the TFPI binding site is detected using an enzymatic assay. [0086] 62. The method of any one of paragraphs 54-61, wherein the peptide comprising TFPI KD1 comprises amino acids 1-160 of human TFPL [0087] 63. The method of any one of paragraphs 54-61, wherein the peptide comprising TFPI KDi is full length human TFPl. [0088] 64. A composition comprising a TFPI inhibitor identified by the method of any one of paragraphs 51-63. [0089] 65 Use of a TFPI inhibitor identified by the method of any one of paragraphs 51 63 for the manufacture of a medicament. [00901 66. Use of a TFPI inhibitor identified by the method of any one of paragraphs 51 63 for the manufacture of a medicament for treating a blood coagulation disorder. [0091] 67. A method for treating a subject suffering from a disease or being at risk of suffering from a disease, the method comprising administering to the subject a TFP! inhibitor identified by the method of any one of paragraphs 51-63, [0092) 68, A method for inhibiting human TFPI, the method comprising contacting human TFPI with an inhibitor that binds human TF~fat a binding site defined by amino acid residues|Phe28, Lys29, Ala3O, Asp32 fle46 Phe47, and Re55. 10093] 69. A method for treating a subject suffering from a disease or at risk of suffering from a disease, the method comprising administering to the subject an inhibitor that binds human TFPI at a binding site defined by amino acid residues Phe28, Lys29, Ala3o, Asp32, fe46, Phe47, and Ile55. [0094] 70, The method of paragraph 68 or paragraph 69, wherein the human TFPI binding site is defined by amino acid residues A1a27, Phe28, Lys29, Ala3O, Asp3 1, Asp32, Lys36, e38, iHe46, Phe47, and Ile55 [00951 71 The method of paragraph 70,herein the human TP binding site isdefined by amino acid residues Aia27, Phe28Lys29, Aa3O, Asp3A I sp32 Lys36. Ala37 Re38, Phe44, Re46, Phe47, and Be55. [0096] 72. A method for purifying a compound that inhibits FXa activity, the method comprising (a) contacting a peptide comprising TPI Kunitz domain 1 (KD1) with a compound under conditions that allow formation of compound-KI) I complexes, (b) removing unbound compound, and (c) dissociating the eompound-KDI complexes to release the compound. [0097] 73. The method of paragraph 72, wherein step (a) comprises contacting the peptide comprising 1KD1 with a population of test compounds. [0098] 74. A computer storage media having computer executable instructions thatwhen executed on the processor of a computer, implement a method of modeling interaction between selected three dimensional (3D) points in a TFI' Kunitz domain 1 (KDI) protein and a test compound, the method comprising: obtaining a protein structure 3D model for the TFPI KDI protein; determining a 3D relationship between a selected subset of amino acids in the protein structure, wherein the selected subset of amino acids comprises Phe28, Lys29, Ala30, Asp32, Ile46, Phe47, and [le55; modeling a surface bounded by the selected subset of amino acids; obtaining a test compound 3D model of a test compound; matching the test compound 3D model to the surface bounded by the selected subset of amino acids; and identifying contact points between the selected subset of amino acids of the surface and the test compound 3D model. [0099] 75. The computer storage media of paragraph 74, wherein the selected subset of amino acids comprises Ala27, Phe28, Lys29, Ala30, Asp3l, Asp32, Lys36, 11e38, 1e46, Phe47, and Re55. [00100] 76. The computer storage media of paragraph 74, wherein the selected subset of amino acids comprises Ala27, Phe28, Lys29, Ala3O, Asp3l Asp32, Lys36, Ala37, e38, Phe44, 1e46, Phe47, and he55. 100101] 77. The computer storage media of any one of paragraphs 74-76, further comprising: determining a number of the contact points between the surface and the test compound 3D model; and recording n affinity rating for the test compound 3D model corresponding to the number of contact points, [00102] 78. The computer storage media of any one of paragraphs 74-77 wherein the test compound is a peptide. [001034 79. The computer storage media of any one of paragraphs 74-78, further comprising: determining a bond type for each contact point between the surface and the test compound 3D) model; and updating the affinity rating based on an aggregate of the bond types for each contact point between the surface and the test compound 3D model. [00104] 80S The computer storage media of any one of paragraphs 7409, further comprising: obtaining an updated test compound 3D model based on a second test compound; matching the updated test compound 3D nodel to the surface bounded by the selected subset of amino acids; and identifying the identified contact points between the selected subset of amino acids of the surface and the updated test compound 3D model on a display of the computer [00105] 81. The computer storage media of paragraph 80, further comprising: determining a number of the contact points between the surface. and the updated test compound 3D model; determining a bond type for each contact point between the surface and the updated test compound 3D model; and recording a new affinity rating based on the number of contact points and an aggregate of the bond types for each contact point between the surface and the updated test compound 3D model. [00106] 82. The computer storage media of paragraph 81, further comprising: comparing the updated affinity rating with the new affinity rating to determine whether the test compound or the second test compound has a higher affinity rating. [001074 83. The computer storage media of any one of paragraphs 8082, wherein the second test compound is a variant of the test compound. [00108] 84. The computer storage media of any one of paragraphs 74-83, further comprising displaying the contact points on a display of the computer. [00109] 85. The computer storage media of any one of paragraphs 78984 further comprising modifying the peptide to increase the number of contact points with the selected subset of amino acids or increase bond strength between amino acids of the peptide and the selected subset of amino acids. [001101 86. A method of comparing a test compound to selected three dimensional points in a TFPI Kunitz domain I (KD1) protein, the method comprising: creating a protein structure for the KD1 protein in a memory of a computer; determining a three dimensional model of a selected subset of amino acids in the KDLI protein at a processor of the computer, wherein the selected subset of amino acids comprises Phe28, Lys29, Ala30, Asp32, 1le46, Phe47, and 11e55: determining a three dimensional model of a test compound at the processor of the computer: fitting the 3D model of the test compound to the 3D model of the selected subset of amino acids at the processor of the computer; and generating an affinity of the test compound for the selected subset of amino acids at the processor of the computer, wherein the affinity is based on a number of amino acids in the subset in contact with the test compound and a bond strength at each contact point, [00111] 87. The method of paragraph 86, wherein the selected subset of amino acids comprises Ala27, Phe28 Lys29, Ala30, Asp3l, Asp32, Lys36, 1e38, [1e46 Phe47, and 1e55. [00112] 88. The method of paragraph 86, wherein the selected subset of amino acids comprises Ala27, Phe28, Lys29, Ala30. Asp31, Asp32, Lys36, Aia37, 1e38, Phe44, B1e46, Phe47, and fle55. [00113] 89. The method of any one of paragraphs 86-88, further comprising: displaying a 3D representation of the fit between the test compound and the 3D model of the selected subset of amino acids, [00114] 90. The method of any one of paragraphs 86-89, further comprising: repeating the steps of paragraph 86 for a plurality of test compounds; and saving the respective affinities for each of the plurality of test compounds. [00115] 91. A computer storage media having computer executable instructions that, when executed on the processor of a computer, implement a method of comparing a peptide to selected three dimensional points (3D) in a TFPI Kunitz domain 1 protein (KDI), the method comprising: creating a protein structure for the KDI protein; determining a three dimensional model of a selected subset of amino acids in the KDI protein, wherein the subset of amino acids comprises Phe28, Lys29, Ala30, Asp32, Ie46, Phe47 and Ie55; determining a three dimensional model of a peptide; fitting the 3D model of the peptide to the 3D model of the selected subset of amino acids; and generating an affinity of the peptide for the selected subset of amino acids, wherein the affinity is based on a number of amino acids in the subset in contact with the peptide and a bond strength at each contact point. [00116] 92. The computer storage media of paragraph 91, wherein the selected subset of amino acids comprises Ala27, Phe28, Lys29, Ala3O, Asp3l, Asp32, Lys36, 11e38, Ie46, Phe47, and Ile55.

[00117] 93. The computer storage media of paragraph 9 . wherein the selected subset of amino acids comprises Ala27, Phe2S, Lys29, Ala3, Asp31, Asp32, Lys36, Ala37, lie38, Phe44, Ile46, Phe47, and 1e55. DESCRIPTION OF THE FIGURES [00118] figure 1 is an ilustration of the blood coagulation cascade, [001191 Figure 2 is an illustration of the secondary structure of Tissue Factor Pathway !nhibitor- . [001203 Figure 3 is an ilustration of the formation of a quaternary complex comprising Tissue Factor, Factor Xa (FXa), Factor Vila (FVIa), and TFPL [00121] Figure 4 is a listing of amino acid sequences of various TFPI-inhibitory peptides denoting amino acid substitutions (bolded and underlined) in reference to peptide JBT0293. [00122] Figure 5 is an illustration of mRNA display selection of TFPI-inhibitor peptides, [00123] Figure 6A is an illustration of the EC 5 binding ELJSA and Figure 6B is an illustration of the K50 EISA described in Example 1. [001241 Figure 7 is a binding ELISA curve comparing % OD (yaxis) and concentration [nM] (x-axis) for biotinylated peptide13T0132. [00125] Figures 8A8D are competition EUSA curves comparing % OD (y-axis) and concentration [nM] (x-axis) for exemplary peptides of the invention, [00126] Figures 9A and 9B are sensorgrams plotting RU (y-axis) against time in seconds (x-axis) for peptides JBT0120 and JBT0132. [00127] Figures 10A and 10B are sensorgrams plotting RU (y-axis) against time in seconds (x-axis) for peptide IBT0120 interaction with Tissue Factor Pathway Inhibitor-1 and Tissue Factor Pathway Inhibitor-2. [00128] Figures 11A and 33 113 are graphs comparing amount of thrombin generated (nM) (y-axis) and tirne in minutes (x-axis) for peptide JBTO120 and peptide JBT0132 in a plasma based assay. [00129] Figures 12-18 are tables listing the amino acid sequences of various TFPI inhibitory peptides; ECS and percent inhibition of TFPT observed in the FXa inhibition assay;

EC

50 and percent inhibition of TFPI observed in the extrinsic tense inhibition assay; and I A FEIBA, Factor VIII (FVIll) Immunate, or Factor IX (FIX) equivalent activities (mU/mL) in plasma-based assays, "*" denotes negative controls, [00130] Figure 121 are tables listing the results from BlAcore analysis of several TFPI binding peptides. "*" denotes negative control. [00131] Figures 22-30 are tables listing the amino acid sequences of various TFPI-binding peptides; ECs 0 and percent inhibition of TFPI observed in the FXa inhibition assay; EQ5 0 and percent inhibition of TFPI observed in the extrinsic tenase inhibition assay; and FElBA, FVIII Inimunate, or FIX equivalent activities (mU/mL) in plasma-based assays. "*" denotes negative controls. [00132] Figure 31 is a graph comparing a pharmacokinetic characteristic (concentration of peptide (y-axis) versus time after administration (x-axis)) of a PECylated TFPI-binding peptide to the pharmacokinetic characteristic of same peptide lacking PEG. The peptides were administered intravenously to C57B16 mice at a dose of 10 mg/kg. Three biological samples were analyzed for the presence of peptide at each time point. [00133] Figures 32-39 are tables listing the amino acid sequences and IC 50 or EC 0 values of various peptides of the invention, "*" denotes negative controls. [00134] Figure 40 is a graph illustrating a pharmacokinetic characteristic (concentration of peptide (nM) (y-axis) versus time after administration (minutes) (x-axis)) of a PEGylated TFPI-binding peptide following subcutaneous administration to mice at a dose of 10 mg/kg. [00135] Figure 41 is a graph correlating the amount of thrombin generated (nM) (y-axis) with time (minutes) (x-axis) for peptide JBT1855 in a plasma-based assay of hemophilia A patient plasma. [00136] Figure 42 is a graph illustrating the amount of blood loss ( 1; y-axis) observed following a nail-clip in mice treated with JBT-1855 (intravenous or subcutaneous administration), anti-TFPI antibody (intravenous administration), or vehicle (intravenous administration) (x-axis). 100137] Figure 43 is a graph plotting TFPI160 amino acid residue (X-axis) against the chemical shift differences of HSQC signals for free TFPI160 and TFPI160 bound to JBT0303 (y-axis). [00138] Figure 44 is a ribbon model of the secondary structure of TFPI illustrating regions of chemical shift changes of HSQC signals when TFPI160 is complexed to JBT0303 compared to uncomplexed (free) TFP1160, [00139] Figure 45 is a graph plotting TFPII60 amino acid residue (x-axis) against the chemical shift differences of HSQC signals for free TFPII60 and TFPII60 bound to JBT0122 (y-axis). [00140] Figure 46 is a ribbon model of the secondary stmucture of TFPI protein illustrating regions of chemical shift changes of HSQC signals when TFPI160 is complexed to JBT0122 compared to uncomplexed (free) TFPI1.60 [00141] Figure 47 is a table listing assignments for the carbonyl carbon (C), the alpha carbon (CA), the beta carbon (CB), the aide proton (H), and the amide nitrogen (N) of JBT0788 based on HSQC, HNCACB, HNCA, HNCO and HNN spectra. [09142] Figure 48 is a ribbon model of the secondary structure of free JBT0788. [001431 Figure 49 is a tabie listing assignments for the carbon carbon (C), the alpha carbon (CA), the beta carbon (CB), the amide proton (1-), and the aide nitrogen (N) of JBT0788 complexed with TFP1160 based on HSQC, HNCACB, HNCA, HCCOCA, and HNCO spectra. [00144] Figure 50 is a bbon model of the secondary structure of JBT 88 when comnplexed with TFPII6O. [00145] Figure 51 is a table listing assignments for the carbonyl carbon (C), the alpha carbon (CA), the beta carbon (CB), the amide proton (H), and the aide nitrogen (N) of JBT0616 based on HSQC, HNCACB, and HNN spectra. [00146] Figure 52 is a ribbon model of the secondary structure of free JBT0616. [00147] Figure 53 is a table listing assignments for the carbonyl carbon (C), the alpha carbon (CA), the beta carbon (CB), the anmide proton (H), and the amide nitrogen (N) of JBT0616 complexed with TFPI based on HSQC HNCO, HNCA, and HNCOCA spectra. [00148] Figure 54 is a ribbon modelf the secondarystructure of JBTO61I6when complexed with TFPI160. [00149] Figure 55 is a ribbon structure of the energetically minimized best model of KDI (residues 22-79) in complex with JBTO3O3 with residues proposed to drive the protein protein interaction displayed as sticks. Italicized and underlined residues belong to JBT3O3; the remaining residues belong to KDI of TFPL [00150] Figure 56 is a rotational thromboelastogram correlating sample elasticity (mm) with time in seconds (5) for JBT23 17 [00151) Figure 57 is a rotational thromboelastogram correlating sample elasticity (mm) with time in seconds (s) for JBT2329. [00152] Figure 58 is an illustration of a computing device. [00153] Figure 59 is an illustration of a three dimensional (3D) model of a KDl protein. [00154] Figure 60 is an illustration of a 3D model of a TFPbinding peptide. [00155] Figure 61 is an illustration of a method of modeling protein and peptide interaction, [00156] Figure 62 is a table listing the amino acid sequences and IC 50 or EQ 0 o values of various peptides of the invention. Designation "n-a" i-s "not analyzed." Progression curve data were obtained using the FXa inhibition assay described in Example 3 with recombinant human fulil length TFPI Assayconcentration ofprogression curve assay was 0,0025% (0.1% Tween80 used in peptide dilution buffer), [00157] Figure 63 is a graph correlating concentration of peptides JBT2325-JBT2329 (nM) (y-axis) with time following intravenous administration (hours) (x-axis). Peptides comprising higher weight PEG moieties exhibited a prolonged in vivo half life in mice. Each time point is represented by the mean of three independent samples quantified by ELISA. [00158] Figure 64A-64C are graphs correlating concentration of peptides JBT2401, JBT2404 and JBT2410 (nM) (y-axis) with time following intravenous administration (hours) (x-axis). Each time point is represented by the mean. of three independent samples quantified by ELISA. Solid circles symbolize intravenous data, sold triangles symbolize subcutaneous data, [00159] Figure 65 is a table listing the amino acid sequences of various peptides of the invention. DETAILED DESCRIPTION OF THE INVENTION [00160] The invention provides peptides that bind Tissue Factor Pathway Inhibitor-1 and, in some instances, block the inhibitory activity of Tissue Factor Pathway Inhibitor-1 (herein referred to as TPPI) within the blood coagulation cascade, Upon vascular injury, Tissue Factor (TF) conplexes with Factor Vita to form the "extrinsic complex" or "extrinsic tenase complex," which activates Factors IX and X (Figure it TFPI is the main natural regulator of TF/FVILa extrinsic complex activity and by extension, plays a role in controlling thrombin generation (Panteieev et at, Eur. J. Bioche,, 249, 2016-2031 (2002)). TFPI is a 43 kDa serine protease inhibitor comprising three Kunitz-type inhibitory domains (Figure 2). Kunitz domain I of TFPI binds FVIa and Kunitz domain 2 binds FXa, enabling the inhibitor to form a quaternary FXa-TFPI-FVIa-TF complex that blocks activity of the TF/FVIIa extrinsic complex (Figure 3). TFPI binding of 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 invention provides, e.g., TFPI-jnhiitory peptides that block TFPI's inhibitory action on the blood coagulation cascade, thereby enhancing thrombin formation. [00161] 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 set forth in Table 1. TABLE 1 3-letter I-letter Amino acids 3-letter 1-letter Amino acids codes code codes code Ala A Alanine Met N Methionine Cys C Cysteine Asn N Asparagine Asp D Aspartic acid Pro P Proline Gli H Glutanic acid Gin Q Glutamine Phe F Phenylalanine Arg R Arginine Gly G Glycine Ser S Serine His H Histidine Thr T Threonine ie I Isoleucine Val V Valine Lys K Lysi ne Trp W Tryptophan Leu L Leucine T1yr Y Tyrosine [001621 Examples of non-conventional amino acids and additional peptide building blocks are identified according to a three-letter code (with the exception of Ttds and Dopa, which are common four-letter abbreviations) found in Table 2. Additional building blocks designated by three-, four- or seven-number/letter designations or abbreviations also are listed in Table 2. The stmctures of some building blocks are depicted with an exemplary reagent for introducing the building block into a peptide (e., the structure provided for 2-naphthyl sulfonyl comprises a chloride). TABLE 2 Name .......... Abbrev ation Structure Phenyl acetyl OH 2-Naphthyl sulfonyl 972 Sc cc I S'2 972 1i-Naphtbyl sulfonyl 973 973 3-Phenyl propionyi 1281 _ _ _ _ _ _ _ _ _ _ OH "1281 -- _________ _ Hcxa-novl 1525 1525 3-Methyl butanoyl 3067 OH 307 lehl Nm____ _A bbrcviation tructure ....... 2-ehy rpiony1 46J5 .0.. ))"OH >-(Napht m2 yI) acetyl 5963 OH 5963 N}(4-'minobutyl)glycine Abg

H

0 H~O

H

2 N H 2 4 O H 2 nIAmio-indan&0'icacbid. Aib H2N AkA Ale Name Abbreviation __ _Structure, pxperidiny carboxylicc acid OH ~N-(4-ainop opyl} -glycile

H

2 N H N

H

2 IY-Ae ine-~"aioxvic cid aze OH 0NH P-Homogjutamatic acid Phe a I J-HornophenyialaWin Bh Bbl OH f$4-lrnoiucin ...... .............. - ------------------- - - ------------ --------- -------- - ------------ ... ........ .OH....... ,, I Name EA: :____________ 4bbreviatzij S.........tructr PAionloasparaglIleBi I HO 2 N -H m g itw n ------ .. .- ... ---- NH2 H~ j- lomf1o arguuln eMi ri BhH HO W,-Uomn.oaspatc acid Blid ' ______ _____ _____ ____ O N L L H _ _ _ HOr 2 c . ~~~~ ~ ~ H ' ...... -----.....

O-----H ......

Nam ______Abbreviation Structuire _____ L - .- lenzothi eiiyilaa inel Bta 0 OH Bta P 3 i.Aceramino- C(Acm) methbylsulfanyi7ado 2 ~K prophonic acid OH, C(Acm) N Aniino ahvlthiol Cea HAN (SY-cyciollxylalanine SHa L-Cycilohexylgl yin e ~ Chg I0 HO K HO 0 NH 2 . ~ ~ ~ ~ ~ ~ _ _ ..... --------------- 2 ..... -------- Name _____Abbreviationx Structure Carboxyrne hiylen cyste'n COne 0 HOH H0 N-ethyhuialefido cysteine C(NEM'H -OH 0 L-Cy clopentygiycine CpgLN N (S>j2Diaminoopnni aid Dapb 2 NO OH

H

2 N OH 545 D methy Dehialidn--______ 'arboxvlic acid H 0 H -2 A Name Abbreviation Structure 4-DihydroxyphenyPaanine Dopa

H

2 N OH OH Dopa OH (S)-2-Propargylglycine Eag OH 1Amino-cyclopropane- - Ebc carboxvlic acid H2N carboxylic acid OH CKs(3-propionic acid amide) Ecl 0 OH NS Sulfoxid of Carboxyecthyleystein Ede(O) OH O OH ........ -------------- ........ --------------- ......... 0..

Name__________ Structure Cys( 5-.methyl~en-2- oxazoiidinoni) Eea H 2 N N Eec \'

H

2 N -O E efN

-P

ha~pa-DiatYginin 4e ~FAN

HH

Name ___ Abbe! 'iaion ~jStructure L-onphen yilani e HWe H N

HO

HN N HN OH rn~o~cyNineHHN HSS H-S OH

NH

2 D- Horno-cysitei-nf fhc' S OH hey SH (S)>2A Anno-5- 1elhv Iexanoic H acid NHH (S)-Hkrooysine HlyH 2 2-Anino-6-(2-aminooxy K(AOA) acetylamino)-hexanoic acid C OH 0 acid-NH

HN

Name Abbreviation _______ Structure I-Nrpbthyiahiinc IMi -1OH 2Ng N U..y........ .y.......N.g.. 'O Nif NO, HO H0 ---- et -~pfi acid---- j..

.OH...

Name______ j______ ST sructure (s>,NMethi -phe-.nylalanirie Ninf HNH N-mn - - e Nin (S 1 TNet hyIedfI earNiniis ----------- ~~ .- O . ..... -- -- ......

Name jAbbrfwiant Srctr ...... Structurersin N _....... L__ _ __ _ _ __ _ _ _ - --- --__- - ----------- .. ...... Nn10 HN (S)-2Anino-pentanoic acid Niva (S)>2-P vridyvbalaninc op *oN N\//" -AC ------ H Hcid0H Ci Name Abbreviat} Structure OH

NH

2 Palmitoyl Pahn O Palmm 0 [m~rdOH Pal L-Phenylglycinl Phg Polyethlene glycol PEG DPipecohic acid pip N OH L-Tyrosi(-Met yl)-OH P O

H

2 N OH Pmy LPhosphotrosinePty 0 H OH P S H------------------------------------------------------ AlI ----- ------- --- -------------- ; ----- ~ r n t ..... ......-------..... o~ -?Y~etAhVagexicarc Wi Sun, ~~OH 1 - ------- ...... ........ tid al~ucrnmi aidTts "'>" )"~'~ rN~rNN0 _ _ _ ~ ~ ~ ~ ~ ~ ~ ~ ~ , N__ _ _ 1 _ _ _ _ _ _ _ _ _ _ -- - -- - -- -- -- . ... - -- - . .... .. ... .. .. .

Name Abbreviation Structure Ttds Maleimidopropionyl(RtSH)) H H 0 o Ttds-Maleimidopropiony(EtSH) 3-NtroLtyrosine Tvm{ 0 OH NO2 OH CaroxyhioescinFAM NO HO0 [2-(2-Ainno-ethoxy)-ethoxy]- FA03202 acetic acid H 2 N O H 3-. {2- [2-(-Amino-ethoxy)- FA 19203 ethoxy]-ethoxy}-propionic acid H 2 N O OH 3-(2-{2~[2~(2-Amino--ethoxy)- FA 19204 H 2 0 ethoxy]-ethoxy}-etboxy) N OH propionic acid O 3-[2-(2 2[2-(2-Amino- FA19205 ethoxy)-ethoxy]-ethoxy -OH ethoxy)-*ethoxy]-propionicacid [00163] The amino acid sequences of the peptides provided herein are depicted in typical peptide sequence format, as would be understood by the ordinary skilled artisan, For example, the three-letter code or one-letter code of a conventional amino acid, or the three-, four--, or seven-number/etter code additional building blocks, indicates the presence of the A'2 amino acid or building block in a specified position within the peptide sequence. The code for each non-conventional amino acid or building block is connected to the code for the next and/or previous amino acid or building block in the sequence by a hyphen. Adjacent amino acids are connected by a chemical bond (typically a anmide bond). The formation of the chemical bond removes a hydroxyl group from the I -carboxyl group of the amino acid when it is located to the left of the adjacent amino acid (eg., HIe-adjacent amino acid), and removes a hydrogen from the amino group of the amino acid when it is located on the right of the adjacent amino acid (eg. adjacent amino acid-Hie). It is understood that both modifications can apply to the same amino acid and apply to adjacent conventional amino acids present in amino acid sequences without hyphens explicitly illustrated. Where an amino acid contains more than one amino and/or carboxy group in the amino acid side chain, the 2- or 3-amino group and/or the 1-earboxy group generally are used for the formation of peptide bonds. For non-conventional amino acids, a 3-letter code was used where the first letter indicates the stereochemistry of the C-a-atom. For example, a capital first letter indicates that the L-form of the amino acid is present in the peptide sequence, while a lower case first letter indicating that the D-form of the correspondent amino acid is present in the peptide sequence. When one-letter code is used, a lower case letter represents a D-amino acid, while an upper case letter represents an L-amino acid. Unless indicated to the contrary, the amino acid sequences are presented herein in N- to C-terminus direction. [00164] The C-terrini of several TFPI-binding peptide sequences described herein are explicitly illustrated by inclusion of an OH, NH 2 , or an abbreviation for a specific terminating amine linked to the C-terminal amino acid code via a hyphen. The N-termini of several peptides described herein ae explicitly illustrated by inclusion of a hydrogen (for a free N terminus), or an abbreviation for a specific terminating carboxylic acid or other chemical grouplinked to the N-terminal amino acid ode via a hyphen. [10165] The invention provides a peptide comprising the amino acid sequence

XX

9

X

1

X

1

X

1 2 XXXXXXXX (SEQ ID NO: 3109), wherein (using single letter codes for amino acids)

X

7 is selected from the group consisting of L; P, K, S, WV, N, and Q; XA is selected from the group consisting of L, R. N, F, and Q; X is selected from the group consisting of Y, V, P, and C;

X

1 w is selected from the group consisting of F, L, and G;

X,

1 is selected from the group consisting of L, W, V, A, M, T, and 3;

X

12 isselected from the group consisting of T, F, V, R, A, D, L, E, , and Y;

AA

X

1 is selected from the group consisting of I M, G, Q, D, and R; X is selected from the group consisting of G, W, Y L, M, and H;

X

1 5 is selected from the group consisting of N, P, F, H, K, and Y;

XI

1 is selected from the group consisting of M, D, E, V, and K:

X

17 is selected from the group consisting of G, 1, R, S, T, and L; Xis is selected from the group consistig of M, K, L, and I;

X

9 is selected from the group consisting of Y, G, R, and S;

X

20 is selected from the group consisting of A, E, S, C, and Y; and

X

2 1 is selected from the group consisting of A, V, K, and E. [00166] In, addition to the core streture set forth above, X , other structures that are specifically contemplated are those in which one or more additional amino acids are attached to the core stmuture (eg., linked to the N-terminus or the C-terminus of the amino acid sequence X-X 21 ). Thus, the invention includes peptides comprising the core streture and further comprising one or more N-terminal amino acids) comprising an amino acid sequence selected from the group consisting of:

X

6 ,

X

4

XX

6 ,

X

5

X

1

X

5

X

6 (SEQ ID NO: 31O)

X

2

X

3

X

4 XsX 6 dSEQ ID NO: 3111), and

X

1

X

2

X

3

X

4

X

5

X

6 (SEQ I) NO" 3 I1); wherein X 6 is directly linked to X 7 of the core structure amino acid sequence, and Xj; is selected from the group consisting of T and G;

X

2 is selected from the group consisting of F and V;

X

3 is selected from the group consisting of V, W, Y, and F:

X

4 is selected from the group consisting of D, Qand S; Xs is selected from the group consisting of E, T, N, and Q; and

X

6 is selected from the group consisting of R, H, K, and A. The peptide of the invention in one aspect comprises or consists of the amino acid sequence QSKKNVFVFGYFERLRAK (SEQ ID NO: J). [00167] In another embodiment, the peptide of the invention comprising the core structure comprises one or more C-terminal amino acid(s) compri.sing an amino acid sequence selected from the group consisting of:

X

22

X

23 ,

X

2 2

X

2 3

X

2 4 ,

X

22

X

2

X

2 X' (SEQ ID NO: 313)

X

2 2

X

2 3

X

2 4

X

25

X

26 (SEQ ID NO: 3114), and

X

22

X

23

X

2 4 X2X2 6 X27(SEQ ID NO: 3115), wherein X 2 is directly linked to X of the core structure amino acid sequence, and

X

2 is selected from the group consisting of Q, E, W. R, L, and N;

X

3 is selected from the group consisting of L, N and R.;

X

4 is selected from the group consisting of K, L, A, and Y;

X

2 4 s F; X2y is G; and

X

7 is T' [00168] In one aspect, the peptide of the invention comprises or consists of the amino acid sequence VIVFTFRHNKLJGYERRY (SEQ ID NO: 4). It is also contemplated that the peptide of the invention comprises additional amino acids at both the N-terminus and the C-> terminus of the core structure, In this aspect, the peptide comprises or consists of the amino acid sequence TFVDERLLYFLTIGNMGMYAAQLKF (SEQ ID NO: 3), GVWQTHIPRYFWTMWPDIKGEvWyLFGT (SEQ [ID NO: 5), KW5FCGMRDMKTMSCVWVKF (SEQ ID NO: 6), or ASFPLAVQLHVSKRSKEMA (SEQ lID NO: 7). 100169] The invention further includes peptides comprising the anino acid sequence X3XAsX -- X?~NVF-XI!XIG Y-Xl 5 XwRLR AK-X) (SEQ IID NO: 2), wherein X 3 is Y or F; X4 is Q or S; X5 is N or S;X7is K, N, or Q; X is V, A, S, or T;X 1 2 is F, A, D, L, Q, S, or Y; Xis is F, K, or Y; X 16 is E or D; and X 22 is L or N, [00170] In addition, the invention provides a peptide that binds TFPI, wherein the peptide comprises the structure of formula (I): X1001-X 002-X1003-X1(04-XI1005-XI006-X1007 XI008-XI 009-X1010-X101 I -X1012-X1013-X1O14-X1015-X1I16-X1o17-X1018-X1019 X1020 (SEQ ID NO: 3116). In formula (I), X1001 is an amino acid selected from the group consisting of Bhf, C, DFG, H, I, K, L, M, N, Nmf Q, R, T, V W, and Y; X1002 is an amino acid selected from the group consisting of G K, and Q; X1003 is an amino acid selected from the group consisting of A, Aih, Bhs, C, D E. F, G, H, L K, L M N, P, Q, R S, T, V, V, and Y; X1004 is an amino acid selected from the group consisting of, A, Aib, Bhk, C, D, EU F, (3, H, , K k, L, M, N, Nmk; P, Q R, S, T, V, W, and Y; X1005 is an amino acid selected from the group consisting of a, A, Aib, Bal, C, D, d, E, F, 0 I, K, k, L, M, N, Ning, p, Q, R,S, T, V, W, and Y; X1006 is an amino acid selected from the grup consisting of Aib, Btq, C, D, E, F, G, H, I, K, L, RI, N, Q. RS T, V, W, and Y; X1007 is an amino acid selected from the group consisting of A, F, G, 1, K, L Nmv, P Q, S, V, W, and Y; XI008 is an amino acid selected from the group consulting of F, H, K, Vt and Y: X1009 is an amino acid selected front the group consisting of A, Aib, 1 K, S5T and V; X1010 is an amino acid selected from the group consisting of A, Aib, C, D, E, F, G, H, 1, K, L, M, N, Nmf, P, Q, R, S, T, V, W, and Y; X 011 is an amino acid selected from the group consisting of Aib, C, K, G, and Nmg:; X1012 is Y; X1013 is an amino acid selected from the group consisting of A, Aib, C, E. F, G, H, K, LN I. Q, RI W and Y; X014 is an amino acid selected from the group consisting of A, Ailb, Bhe, C, D, D , F, 0, H, LI, L, M, N, P, Q, R, S, T, V, W, and Y; X1015 is an amino acid selected from the group consisting of (omega-methyi)-R, D, E, K. and R; X1016 is L; X1017 is an amino acid selected from the group consisting of (omega-methyI--R, A, AiT, Br, C, Cha, Cit D, Dah, Dap, E, Eag, Eew, F, G. H, Iar, hei I ie, I, K, L, M, N, NOe Nva, Opa, On, Q, R, S , , V and Y; X1OlS is an amino acid selected from the group consisting of A, Ba C, D , F, G, H, , K L, M, N, Q R S, T, V. W, and Y; and X1019 is an amino acid selected from the group consisting of Bhk, K, R, and V.

X1020 is either present or absent in formula (1) (i.e, in some instances, the peptide of the invention comprises the structure X 001-X1002-X1003-X1004-XOO-X1 006 X1007 X1 008 X1010-X1011-X1012-X101 3-X10144X101 5-X1016-X1017eXl01 8-XI019 (SEQ ID NO: 3116))4 When X1020 is present, it is an amino acid selected from the group consisting of Aib, Bh, C, F, G, H, 1, K, L, Nmbl, R, S, T, V, W, and Y. [00171] For example, the peptide of the invention comprises the structure of formula (I) wherein X1001. is an amino acid selected from the group consisting of C, F, , K, L, Nmf, V, M, W. and Y; X1002 is Q; X1003 is an amino acid selected from the group consisting of A, C, D, E, H, K, M, I, N, Q, R, S, T, and V; X1004 is an amino acid selected from the group consisting of A, Aib. C, D, E, G, -1, F, I, K, k, L, M, N, Nmk, P, Q, R., , X, W, and Y; X1005 is an amino acid selected from the group consisting of 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 the group consisting of A, Btq, C, D, G, I, K, 11, L, M, N, Q, R, S. V, and Y; X1007 is an amino acid selected from the group consisting of 1, K. L, Q, V, and Y; X1008 is an amino acid selected from the group consisting of F, 11, and Y; X1009 is an amino acid selected from the group consisting of f, 1, and V; X1010 is an amino acid selected from the group consisting of A, D, E, F, G H, K, L, M, N, P, Q, R, S, T, V, V, and Y; X1011 is an amino acid selected from the group consisting of G and Nmg; X1012 is Y; X1013 is an amino acid selected from the group consisting of Aib, C, F, -1, L, W, and Y; X1014 is an amino acid selected from the group consisting of A, Aib, Bhe, C, D, D , B, , K, L, M, N, Q, R, S, T, V, W, and Y; X1015 is an amino acid selected from the group consisting of E and R; X1016 is L; X1017 is an amino acid selected from the group consisting of (omega-methyl)-R, A, Aib, Bhr, C, Cha, Cit, Dab, Dap, Eag, Eew, F, 1, Har. Hei, Hle, I, K, L, M, N, Nie, Nva, Opa. Orn, R, S. T. V, and Y; 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 X 1020 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). [001721 In one aspect the peptide of the invention comprises the structure of formula (I) wherein X1001 is an amino acid selected from the group consisting of F, L, Y, and M; X1002 is Q; X1003 is an amino acid seated from the group consisting of N, Q, R, S, T, and C; X1 004 is an amino acid selected from the group consisting of Aib, K, L, P, R, E, G, , Y, M, and W; X 1005 is an amino acid selected from the group consisting of 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 the group consisting of A, C, ). (, HK, N, Q, R, S, and M; X1007 is an amino acid selected from the group A Q consisting of I and V; XI00S is anl ammo acid selected from the group consisting of F, H, and Y; X1009 is V; XI 00 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; X0is G; is 0; X101s Y; Xl013 is C or F; Xi14 is an amino acid selected from the group consisting of A, C D, EI K, L, M, N. Q R. T V, and Ait X1015is R XR016 is L; Xl017 is an amino acid selected from the group consisting of A, Aib, C, Cha, Dab, Dap, Eag, Few H, Har I- Fle , K, Me, Nva, Opa, Grn R, I L8, and M; Xl0IS is an amino acid selected from the group consisting of A, L N, M, and R; X1019 is K; and XlO20 isK or L. [00173] When amino acid X1020 is absent from formula (1), the peptide of the inention in one aspect further comprises amino acid X1000 at the N-terminus of formula (1), such that the peptide comprises or consists of the structure of formla (II): X1000-X1001-X1002- X1003-Xl1004X005-X1006X1007X1O00-Xl0O9X1010-X1011Xl -X102X01 3-X1O 14 XI15-X1016-Xl0l7-XI 8-X1019 (Hi) (SEQ ID NO: 3122 When X1000 is present in the peptide, X1000 is an amino acid selected from the group consisting of A, E, and P. vhile the amino acids of X10l-X1019 areas defined above. [00174] In an additional aspect, the TFPI-binding peptide of the invention comprises the structure of formula (11): XiOOI-Q-X1003-X1004-XlOO5-X 1006-I/V-X10O8-V-X1010-G Y~C/F-XO14-R-L-X1017-X1018-KIK/L (11) (SEQ TID NO: 3117). As used herein, amino acid designations separated by "/" refer to alternative amino acid residues at the indicated position. For example, with respect to formula (IH), the amino acid residue at position 7 is isoleucine orvaline. XIOOI. X1003, X1004,X1005, X1006, XI008, X1OIO, X1Oi4, X1017 and X1 018 in formula (il) are each independently selected from any amino acid. For example, in formula (IIH), X1001 is optionally an amino acid selected from the group consisting of Bhf, C, D. F, G H, T, K, L. M, N, Nmf, Q, R<T, V, W, and Y, such as an amino acid selected front the group consisting of C, F, , K, L, Nmf, V, M W, and Y (eg, an amino acid selected from the groupconsisting of F, L, Y and a); X1003 is optionally an amino acid selected from the group consisting of A, Aib, Bhis, C, D, E, F, G, 1-1, 1, K, L, M, N, P, Q, R, S, T, V, W, and Y, such as an amino acid selected from the group consisting of A, C, , , -, K, M, I, N, Q, R, S, T, and V (e. the amino acid is M, Q, R, S, T or C); X1004 is optionally an amino acid selected from the group consisting of, A, Aib, Bhk, C, D, F, F, 6, H, I, K, k. L JM, N,Nmk, i, Q, R, S, T, V, V and Y, suh as an amino acid

AA

selected from the group consisting of A, AlH, C, D, F, . .H, F. I, K, k, L, M, N, Nnk, P, Q, R, S, V, W, and Y (eig., an amino acid selected from the group consisting of Aib, K, L. P, R; E GI, Y, M, and W); X1005 is optionally an amino acid selected from the group consisting of a, A, Aib, Bal, C, P, d, E, F, 0, H, K. k, L, M, N, Nmg, p, Q, R, S, T, V, W, and Y such as an amino acid selected from the group consisting of a, A, Aib, Bal, C, d, E, D, F, G, H, K, k, L, M, N, Nmg,. p, Q, R, S, T, and Y (eg., the amino acid is a, Aib, D, d, G H K, k, N, Nmg, p, Q, R, A, E, C, orM); X1006 is optionally an amino acid selected from the group consisting of A, Aib, Btq, C, DE, F, R G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y, such as an amino acid selected from the group consisting of A, Btq, C, D, C, t K, H, L, M, N, Q, R., S, V, and Y (e.g. an amino acid selected from the group consisting of A, C, D, G, H, K. N, Q, R, S, and M); XI008 is optionally an amino acid selected from the group Consisting of F, H, K, W, and Y, such as an amino acid selected from the group consisting of Ft H, and Y; XIOlO is optionally an amino acid selected from the group consisting of A, Aib, C, D, E, F, G, HI, I, K, L, M, N, Nmf, P, Q, R, S, T, V, W, and Y, such as an amino acid selected from the group consisting of 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 the group consisting of A, D, E, K, M, N, Q, R, F, H, P, S, V, W and Y); X1014 is optionally 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, T, V, W, and Y, such as an amino acid selected from the group consist ig of A, Aib Bhe, C, D, F, H, 1, K, L, M, N, Q, R, S, T, V, W, and Y (eng., A, C, D E K, L, MI, N, Q, R, T, V, or Ab); X 017 is optionally an amino acid selected from the group consisting of (ornega methyi)=R, A, Aib, Bhr, C, haCit, Pt Dab, Dap F, Eag, Few, F, 0, GH, Har, 1i, Hile, I, K, B, M, N, Nie, Nva, Opa, Orn Q, R, S, T, V, W, and Y, such an amino acid selected from the group consisting of (omega-methyl)-R, A,Aib, Bhr, C, Cha, Cit, Dab, Dap, Eag, Eew. F, H, Har, Hci, Hit, I, K, L M, N, Nie, Nva, Opa, Om, R, S, T, V, and Y (eg., an amino acid selected from the group consisting of A, Aib, C, Cha, Dab, Dap, Fag, Few, H, iH-a Heci, Hie, K, Nie Nva, Opa, On, R, I, L, S, and M); and/or X108 is optionally an amino acid selected from the group consisting of A BalC D, F, F Gi H, I, K, L, M, N, Q, R S T, V, W, andi such as an amino acid selected from the lc f group consisting of A, C D , F, , K, L, L, N, Q, R, V, and W (eg,. an amino acid selected from the group consisting of A, L, N, M, and R), [00517] In sone embodimentsithe peptide of the invention comprises one or more additional amino acid residues attached to the N- or C-terninus of the amino acid sequence. For example, the peptide comprising the structure of any one of formulas (l)-(III), in some embodiments, further comprises one or more N-terminal amino acid(s) directly linked to X1001, wherein the N-terminal amino acid(s) comprise the amino acid sequence selected from the group consisting of X1000, X999-X1000, X998-X999-X1000, X99X998X99- X000 SEQ D NO: 3123). X996-X997- X998--X999-X1000 (SEQ ID NO: 3124), X995-X996-X997-X998--X999-X1000 (SEQ ID NO: 3125), X994-X995-X996-X997-X998-X999-X1000 (SEQ ID NO: 3126), X993-X994-X995-X996-X997--X998 -X999-X1000 (SEQ ID NO: 3127), X992-X993-X994-X995-X996-X997-X998-X999-X1 000 (SEQ ID NO: 3128), X991-X992-X993 -X994-X995-X996-X997-X998-X999-X1000 SEQ ID NO: 3 129), and X990-X991 -~X992-X993-X994-X995-X996-X997-X998-X999-X1000 (SEQ TD NO: 3130). When the peptide0omprisesone or more N-terminal amino acids Xl000 i A or K; X999 is V or; K; XoK; X997 is L or K; X996 is R or r X95 i G or K: X994 is V orK; X993||is G||or K; X992 is S or K; X991 s K; and X990 is K. [00176J in addition to the corc strctures set foth in formulas|1k11) other stretures that are specifically contemplated are those in which one or more additional amino acids are attached to the C-tenninus of the core structure directly linked to X1020. For example, the C-terminal addition optionally comprises an amino acid sequence selected from the group consisting of X1021, X1021-X1022, X1021-X1022-X1023, and X1021-X1022-X1023 X1024 (SEQ D NO: 3131), wherein X1021 is T or K; X1022 is S or K; and X1023 and X 2104 are K, [00177] The invention further includes a TFPT-binding peptide comprising or consisting of an amino acid sequence having at least 60% identity (eg, 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-NVFVDGYERL-Aib-AKL-NH2 (formula v) (SEQ ID NO: 164). In some instances, the peptide comprises or consists of the amino acid sequence of any one of formulas (I)-(IIl), as described herein. The invention also includes a peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-978 (e g, a peptide comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 8-741 and 962-972 (such as SEQ ID NOs: 8-741, 962-968, 971, or 972) and/or selected from the group consisting of 742961 (such as SEQ ID NOs: 744-961) and/or selected from the group consisting of SEQ ID NOs: 973-978). [00178] The invention includes peptides that comprise a cyclic structure. In this regard, the invention includes peptides comprising cyclic structures within the peptide (e.g., one or mor loops fonned by linkage between amino acids other than the N- and C-terminal amino acids), peptides comprising a cyclic structure formed by the interaction of a terminal amino acid with an amino acid within the peptide sequence, and peptides cyclized head to tail. The peptide may also be part of a larger cyclic struture formed by surrounding additional amino acids or chemical substituents. The peptides of the invention, in some instances, comprise intramolecular disulfide bonds. In some embodiments, the intramolecular disulfide bonds are formed by cysteine residues. Peptides comprising cyclic structures formed by non-cysteine residues, or a non-cysteine residue and a cysteine residue, also are provided. For example, in one embodiment, the inventive peptide comprises at least one non-conventional amino acid or chemical moiety that mediates cyclization. Suitable non-conventional amino acids or chemical moieties include, but are not limited to, FA19205, FM9204, FA19203, FA03202, Hey, hey, Cea, and c, The amino acids or moieties responsible for cyclization are sufficiently spaced apart to allow formation of a loop structure, eg., the amino acids or moieties are separated by two, three, four, five, six, seven, eight, or more residues, [00179] In one aspect, the peptide comprising the structure of formulas (i)-(U) contains at least two cysteine residues (e g., the peptide contains two cysteine residues) that are spaced apart by at least three amino acid residues such that the cysteines fonn an intramolecular disulfide bond. In some instances, the cysteines are spaced apart by more than three amino acid residues. For example, in the peptide comprising the structure of formulas (1), (II or (III) any two of X1000, X1001, X1003, X1004, X1005, X1006, X1010, X1011, X1013, ?1) X1014, X1017, X1018, X1020 and X1021 are optionally cysteines capable of forming a disulfide bridge. Accordingly, in some aspects, the peptide contains two cysteine residues: one of X1000, X1005. X1010 and X1014 is cysteine, and one of X1006, X1010, X1017 and X1021 is a cysteine. The invention contemplates all of the possible combinations of cysteine pairs, e.g., X1000 and X1006 are C; X1000 and X1010 are C; X1000 and X1017 are C; X1005 and XI017 are C; X1010 and X1017 are C; X1010 and X1021 are C; or X1014 and X1021 are C. Other exemplary cyclic peptides of the invention include, e.g., JBT2441, JBT2450, JBT2466-JBT2469, JBT2489- JBT2495, JBT2497-JBT2499, and J:BT2513 JBT2518 (SEQ ID NOs: 4159, 4167, 4181-4184, 4204-4210. 4-212-4214, and 4228-4233, respectively.). [00180] The invention further provides a peptide that binds TFPI, the peptide comprising the structure of formula (V): X200 I-X2002- X2003-X2004-X2005-X2006-[X2007-X2008 X2009-X2010-X2011-X2012-X2013-X2014-X2015--X20 16-X2017-X2018]-X20 19-X2020 X2021-X2022-X2023 ('V) (SEQ 11) NO: 3118), wherein the peptide forms a cyclic structure generated by a linkage, e.g., a disulfide bond, between X2007 and X2018 (denoted as brackets within formula (V)). In formula (V), X2001, X2002, and X2023 are independently either present or absent. When present, X2001 is an amino acid selected from the group consisting of A, D, E, F, G, -1, I, K, L, P, R, S, T, V, and W; X2002 an amino acid selected from the group consisting of A, D, E, F, G, II, IKL M P R, S, T, V and XV; and X2023 is an amino acid selected from the group consisting of A, D, F, G1, K. L, R, S T, V, W, and Y. In addition, X2003 is an minor acid selected from the group consisting of F, I K, L, R, S, T, V, W, and Y; X2004 is an amino acid selected from the group consisting of A, D, E, F, G. 1, K, L, R, S, T, VandW; X2005 is W X2006 is an amino acid selected fromthe group consisting of F, H, 4K, L R V, and X2007 is an amino acid selected from the group consisting of C, Hcy, Dap and K (e.g, C or Hey); X2008 is an amino acid selected from the group consisting of A, 0, R, S. and T; X2009 is an amino acid selected from the group consisting of a, A, T. K, L M, mm Nie, p, R, Sem, and V; X2010 is an amino acid selected from the group consisting of A,G. LK L, P, R, S,T and V; X2011 is an amino acid selected from the group consisting of D, E G, S, and T; X201 2 is an amino acid selected from the group consisting of A, a, D, d, E, e, F, f, G, L K, k, L, 1, M, n, le, nie, P, p, R, r, S, s, Sem, T, T V, v, , and v; X2013 is an amino acid selected from the group consisting of A, D, d, E, e, F, i, L, K, L, R, S, s, T V, and W; X2014 is an amino acid selected from the group consisting of A, D E, F G, G K, L M, R, S, YV and W X2015 is an amino acid selected from the group consisting of A, D, E, F, G, , K, L, M, Ne, R, S,T, V, and W; X2016 is an amino acid selected from the group consisting of A, D, E, F, , K L, M, Nle, R, S, Sem, T, V, W, and Y; X20W 7 s anamino and selected from the group consding of A, E E, F, G, IK, L, R S T, V, W, and Y; X2018 is an amino acid selected from the group consisting of C and 1) (e.g., X2018 is C); X2019 is an amino acid selected from the group consisting of A, F, I, L, S, T, V, and W; X2020 is an amino acid selected from the group consisting of F and XV X2021 is an amino acid selected from the group consisting of L L, and V; and X2022 is an amino acid selected from the group consisting of A, D, E, F, GC I, K, L P, R, S, T, V, and W. 1001811 In some instances, in the peptide of the invention comprising the structure of formula (V), X2001 is optionaly an amino acid selected from the groupconsisting of A D, F, G, Hi K, L, P, and St such as an amino acid selected from the group consisting of A, 0, F, , H, K, L, and S (when X2001 is present).;

CA

X2002 is optionaly an amino acid selected from the group consisting of A, D, F, G, H, K, L, P, R, and , such as an amino acid selected from the group consisting of A, F, HI, K, L, M, R, and S (eg., H, F, M or R) (when X2002 is present); X2003 is optionally an amino acid selected from the group consisting of A, F, K, L, S and Y, such as an amino acid selected from the group consisting of F, S, and Y (e.g, F or Y); X2004 is optionaly an amino acid selected from the group consisting of A, D' F, G3, K, L, and S (eg, K); X2005 is optionaly W; X2006 is optionally an amino acidelected from the group consisting of F, -1, K, and L (e.g, F or H); X2007 is optionally an amino acid selected from the group consisting of C and HeY (e.g, X2007 is C); X2008 is optionaly an amino acid selected from the group consisting of A; G, and S; X2009 is optionally an amino acid selected from the group consisting of a, A, K, L, V, M, m, Nie, Sm, and p, such as an amino acid selected from the group consisting of M, Nle, p, and V (e g., M, Sem, or V); X2010 is optionally an amino acid selected from the group consisting of A. G, K, L, P. R and S, such as an amino acid selected from the group consisting of A, K, L, P, R and S (eg, K, P or R); X201 is optionaly an amino acid selected from the group insisting of D C, and S (e.g., D or S); X2012 is optionally an amino acid selected from the group consisting of A, a, D, d, F, f, G, K k L, 1, Mi n Nic, P, S, and s, such as an amino acid selected from the group consisting of D, d, F, f G, K, k, L, 1, M Ne, P, S. and SemI (e g. an amino acid selected from the group consisting of F, L, 1, Sem, and M); X2013 is optionaly an amino acid selected from the group consisting of A, DI d, F, G, K, L, S, and s, such as an amino acid selected from the group consisting of A D, F G, K, U and S (eDg, D, C, K., or S); X2014 is optionaly an amino acid selected from the group consisting of D, FE Q K, L, and S (e.g., D or 0); X2015 is optionally an amino acid selected from the group consisting of A, D, F, G, L K, L, M NLe, 5, and T (eg, I or T); X201 6 is optionally an amino acid selected from the group consisting of D, F, K, L, M, Me, S and Y, such as an amino acid selected from the group consisting of D, F, K, L, M, Nie, S Sent and Y (eg, D, F, M, Sem, or Y); X2017 is optionally an amino acid selected from The group consisting of A, D, F, G-i, K L, S T and Y (eg -S or T); X201 is optionally C; X2019 is optionally an amino acid selected from the group consisting of A, F, L, S, and V (e g, A or V); X2020 is optionally an amino acid selected from the group consisting of F and W (e.g., W); X2021 is optionally an amino acid selected from the group consisting of L and V (e.g., V); X2022 is optionally an amino acid selected from the group consisting of A, D, F, G, K, L, P, R, S, and W, such as an amino acid selected from the group consisting of A, F, G, K, L, P, R. S, and W(e.g.an amino acid selected from the group consisting of F, L, K, R, P. and W); and X2023 is optionally an amino acid selected from the group consisting of A, D, F, G, K. L, NI, S, and Y, such as an amino acid selected from the group consisting of A, D, F, G, L M, S, and Y (eg, an amino acid sequence selected from the group consisting of A, D, F, M, S and Y) (when X2023 is present). [001821 The invention further includes a peptide That binds TFPI, wherein the peptide comprises the structure of formula (VI): X2001-X2002-F/Y-.K-W-F/H[C-X2008-M/V X2010 -D-X201220I 3-0-IT-~X2Ol 6-S-C}AN-W-V-X2O22-X2O23 (VI) (SEQ ID NO: 3119). In the peptide comprising the structure of formula iVD. X200I, X2002 and X2023 are each independently present or abset. If X2001, X2002, and/or X2023 are present any of X2001, X2002 and X2023 is independently selected from any amino acid, In addition, X200S, X20 10, X2012, X2013, X2016, and X2022 are each independendy selected from any amino acid. [00183] In some aspects, in the peptide of formula (VI), X2001 is optionally an amino acid selected from the group consisting of A,, D, , F, G, H, I, K, L, P, R, S, T, V, and W, such as an amino acid selected from the group consisting of A, D F, G1- K L, |P and Seg, an amino acid selected from the group consisting of A, D, FG (3, H, K, L, and S) (when X2001 is present); X2002 is optionally an amino acid selected from the group consisting of A, D E, F, G H, I, K, L, M, P, R, S, T, V, and W, such as an amino acid selected from the group consisting of A, D, F, G, H, K, L, M, P, R, and S (eig., an amino acid selected from the group consisting of A, F, H, K, L, M, Rand S, such as H', F, M, or R) (when X2002 is present); X2008 is optionally an amino acid selected from the group consisting of A, GR, S, and T, such as an amino acid selected frm the group consisting of A, 3, and S; X201 0 is optionally an amino acid seected from the group consisting of A, GI, K, L, P, R, S, T, and V, such as an amino acid selected from the group consisting of A, G, K, L, P, R, and S (e.g., an arnino acid selected from the group consisting of A, K, L, P, R, and S, such as K, P or R); X2012 is optionally an. amino acid selected from the group consisting of A, a, D, d, E, e, F, f, G, I, I, K, k, L, 1, M, m, Nle, nie, P. p, R, r, S, s, Sem, T, t, V, v, W, and w, such as an amino acid selected from the group consisting of A, a, D, d, F, f, G, K, k, L, 1, M, m, Nle, P, S, s, and Sem (e.g., an amino acid selected from the group consisting of D, d, F, f G, K, k, L. 1, M, Nle, P, S, and Sem, such as F, L, 1, Sem, or M); X2013 is optional an amino acid selected from the group consisting of A, Dd dB e F., I, KL, R,8 Ss TV, and WX, such as an amino acid selected from the group consisting of A, D, d, F, G .K, L, S, and s (e g. an amino acid selected from the group consisting of A, D, F, G, K, L, and S, such as D, G, K, or S); X2016 is optionally an amino acid selected from the group con si sting of A, D, E, F, I, K, L, M, Nle, R, S, Sem, T, V, W, and Y, such as an amino acid selected from the group consisting of D, F, K, L, M, Ne, S, Sem, and Y (e.g., an amino acid selected from the group consisting of D, F, K, L, M, Nle, S, and Sem, such as F, Sem, or M); X2022 is opdonally an amino acid selected from the group consisting of A, D, E F, G, 1, K, L, P, R. S, T, V. and W, such as an amino acid selected front the group consisting of A, D, F, G, K, L, P, R, S, and W (e g., an amino acid selected from the group consisting of A, F,k, KL P R, S and W-such as F, L, K, P, or W); and/or X2023 is optionally an amino acid selected from the group consisting of A, D, E, F, G, 1, K, L, R, M, S, T, V, W, and Y, such as an amino acid selected from the group consistmg of A, D, F, G, K, L, I, S, and Y (e g. an amino acid selected from the group consisting of A, D, F, G, L M, S. and Y, such as A, D, F, M, S, or Y) (when X2023 is present). [00184] The TFM-binding peptide of the invention, in one aspect, comprises an amino acid sequence having at least 60% identity (eg., 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% identity) to the sequence of formula VII: Ac-FYYKWH[CGMRDMKGTMSC]AWVKF-NHl2 (VII) (SEQ ID NO: 1040). Optionally, the peptide comprises or consists of the amino acid sequence of formula (V)-(VII) as defined herein. The invention also includes a peptide comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1001-1293 (e.g., a peptide comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1001-1212 and 1290-1291 (such as SEQ ID NOs: 1001-120, 1290, or 1291) and/or selected from the group consisting of SEQ ID NOs: 1213-1289 and/or selected from the group consisting of 1292 and 1293). [001851 The invention further provides a TFPI-binding peptide comprising at least amino acids 3-21 (X3003-X3021I) of the structur of formula (VIII): X3001-X3002-X3003-X3004 X3005-X3006-X3007-X3008-X3009-X301I 0-X30 1 -X30 2301 -X301 4-X3015-X3016 X3017,X3018 X3019-X3020-X3021 (VIII) (SEQ ID NO: 3120). In formula (VUII), X3001 and X3002 are independently either present or absent in the peptide. If present, X3001 is an amino acid selected from the group consisting of A, C, ), F, G 1, K, L, M, N1 P, Q, R, S, T, W, E, H, and Y; and X3002 is an amino acid selected from the group consisting of A, C, D, F, H, K, M, N, P, R, S, T, W, Y, 0, I, and L. In addition, X3003 is an amino acid selected from the group consisting of A, C, D, E, F, G, B I, K, 1, M, N, P, Q, R, S, T, W, and Y; X3004 is an amino acid selected from the group consisting of A, C, ), E, F, G, H, 1, K, L, MN, Q, R, S, T, V, W, Y, and P; X3005 is an amino acid selected from the group consisting of C, D, F, G, H, 1, K, L, M, N1P,R,S, T, V, andY; X3006 is an amino acid selected from the group consisting of A, W, C, K., P, R, and H; X3007 is an amino acid selected from the group consisting of Q, A, C, F, G, H, L K, L, N, R, S, T, W, and Y; X3008 is an amino acid selected from the group consisting of A, C, F, C. H, K, L, M, N, P, Q, R, S, T, V, W, Y, and I; X3009 is an amino acid selected from the group consisting of A, C, F, G, L, L, M R S, , V, W,) and K; X3010 is an amino acid selected from the group consisting of A, CF G7 HLK,L, M, N, P. Q, R, S, T, V, W, and Y; X3011 is an amino acid selected from the group consisting of A, G, 1, K, L, M, N Q, R, 8, T, V, W, Y, C, F, and H; X3012 is an amino acid selected from the group consisting of A, CH, - I, K, Land R; X3013 is an amnino acid selected from the group consisting of A, C, F, G H, K, L, M, R, S, V, W, Y, and , X3014 is an amino acid selected from the group consisting of A, C, F, G, H, 1, L, M, N, Q, R, S, T, V, W, Y, and K; X301 5 is an amino acid selected from the group consisting of A, K, and R; X3016 isan an amino acid selected from the group consisting of A, F, KI and R X301 7 is an amino acid selected from the group consisting of A, C, F, . , K.K, N, Q. R, S, T, V, W, Y, H, A, and M; X3018S is an amino acid selected from the group consisting of A, C, F, 1L K, L, M, Q, R, V, W, and Y; 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 ; 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, , and P; and X3021 is an amino acid selected from the group consisting of A, C, H, , K, L, M, N, P, Q, , T. V, W, Y, F, and 0. [00186] In some aspects of the invention, the peptide comprises the sequence of formula (VI), wherein X3001 is optionally an amino acid selected from the group consisting of A, C, D. C, 1, K, L, M, N, P, Q, R, S, T, W, F, H., and Y, such as an amino acid selected from the group consisting of A, C, D, G, K, L, M N, P, R, S, T, & H, and Y (when X3001 is present); X3002 is optionay an amino selected from the group consisting of C F, H K, R, S, W, Y, , 1, and L, such as an amino acid selected from the group consisting of C, K, R, W, Y, C, d Lwhen X3002 is present); X3003 is optionally an amino acid selected from the group consisting of A, C, D, F G, H. 1 K. L, M, N, P, Q, R, S, T, and W, such as an amino acid selected from the group coising of A C, G, GH, I, K. L. M, R, S, T, and W; X3004 is optionally an amino acid selected from the group consisting of A, C, D, G, H, 19 K, L, M, N, R, S, T, V, and P, such as an amino acidfselected from the group consisting of A, C, (3, H, L K, L, M, N, R, S, T, and P; X3005 is optionally an amino acid selected from the group consisting of C, F, H, 1, K, M, R, T. W, and Y, such as an amino acidselected from the group consisting of C, IK, R, and W; X3006 is optionally an amino acid selected from the group consisting of P, H, and A; X3007 is optionally an amino acid selected from the group consisting of C, G. R, W, A, and L, such as an amino acid selected from the group consisting of L, C, R, and W; X3008 is optionally an amino acid selected from the group consisting of A, C, F, G, H, K, L, M, N Q, R, T, V, W, Y, and L such as an amino acid selected from the group consisting of A, C, F, H, K, R, V, W, Y, and I; X3009 is optionally an amino acid selected from the group consisting of C. I, R, V, and K, such as an amino acid selected from the group consisting of C, R, V, and K; X-3010 is optionally an amino acid selected from the group consisting of A, C, H, I, K, L, M, Q. R, S. and T, such as an amino acid elected from the group consisting of A, C, K, L, Q, R, and S; X301ioptionaly an amino acid selected from the group consisting of A, L K, L, M, R, S, V W, C, F, and H, such as an amino acid selected from the group consisting ot K, L.M RV, W,|C F,and H; X3012 is optionally an amino acid selected from the group consisting of H and R (e.g., H); X3013 is optionally an amino acid selected from the group consisting of C, F, K, L, M, R, V and I, such asan amino acid selected from the group consisting of C, K R V, and I; X3014 is ptionally an amino acid selected from the group consisting of A M C, F, H, I, L, N, R, S, V, W, and K, such as an amino acid selected from the group consisting of A S, C H F H I, R and K; X3015 is optionally K or R; X3016 is optionally K or R; X3017 is optionally an amino acid selected from the group consisting of A, C, F, G, I, K, L, N, Q, R, S, T, V, W, I-, A, and M, such as an amino acid selected front the group consisting of C, G. 1, K. L, N, Q, R, S, T, V, H, A, and M; X3018 is optionally an amino acid selected from the group consisting of A, KC, L L, R, and W (e, K, C, I R, or W); X3019 is optionally an amino acid selected from the group consisting of A, C, E, H, K, N, Q, R, and I, such as an amino acid selected from the group consisting of C, F, H, K, R, and I; X3020 is optionally an amino acid selected fromn the group consisting of C, H, L, M, R, V, , and P (e.g, C, M, I, or P) and X3021 is optionaly an amino acid selected from the group consisting of A, C, H, K, L, M, N, Q R, V, W, Y, F, and 0, such as an amino acid selected from the group consisting of A, C, H, 1K, L, M, N, Q R, V, W, F, and G. [00197] The invent tion further provides a peptide that binds TFPI and comprises at least amino acids 3-21 (X3003-X3021) of the structure of formula (IX): X3001 -X3002-X3003 X3004-X3005-X3006-X3007-X3008-X3009-X3010-X301 1-H-X3013-X301I 4-K/R-R X3017-X3018-X3019-X3020-X3021 (TX) (SEQ ID NO: 3121). In formula (LX), X3001 and X3002 are independently either 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 the group consisting of A, C, D, F, G, 1, K, L, M, N, P, Q, R, S, T, W, E, H, and Y, such as an amino acid selected from the group consisting of A, C. D, G, I, K, L, M, N, P, Q, R, S, T, W, E, H, and Y (eag, an amino acid selected from the. group consisting of 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 the group insisting of C, D. F, K, M, N, R, S T, W, Y, G,1, and L, such as an amino acid 6.1 selected from the group consisting of C, F, H, K, R, S. W, Y, G, 1, and L (eg., an amio acid selected from the group consisting of C K, R, W, Y, G 1, and L). Also with respect to fonnmda (LX) X3003 is optionally an amino acid selected from the group consisting of A, C D, F, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, and Y, such as an amino acid selected from the group consistin of A, C , F, G, H, 1, K, L M, N, P, Q, R, 8, T, and W (e.g., an amino acid selected from the group consisting of A, C, G, H, I, K, L, M, R, S, T, and WV); X3004 is optionally an amino acid selected from the group consisting of A, C, D, E, F. G, H, L K, L, M, N Q, S, T, V, W, Y, and P, such as an amino acid selected from the group consisting of A, C, D, G, H, I, K, L, M, N, R, S, T, V, and P (e.g., an amino acid selected from the group consisting of A, C, G, H, , K, L, M, N., R, T, and P); X3005 is optionally an amino acid selected from the group consisting of C, D, F, G, H, L K, L, M, N, P, R, S, T, V, W. and Y. such as an amino acid selected from the group consisting of C, F, H 1, K, M, R, T, W, and Y (e~g., an amino acid selected from the group consisting of C, F, H, K, R, and W); X3006 is optionally an amino acid selected. from the group consisting of A, W, C, K, P, R and H, such as an amino acid selected from the group consisting of P, I-L and A; X3007 is optionallyan amino acid selected from the group consisting of Q, A, C, F, GH, I- K. L, N, R, S, T, W and Y such as an amino acid selected from the group consisting of C, 0 R W, A, and L (eg, L, C, R, or W); X3008 is optionally 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, such as an amino acid selected from the group consisting of A, C, F, G, H, K, L, M, N, Q, R, T, V, W, Y, and I (e g.,an an mino acid selected from the group consisting of A, C, F, H, K, R, V, W, Y, and I; X3009 is optionally an amino acid selected from the group consisting of A, C, F, 0 H, , L, M, R, 8, T, V, W, Y, and K, such as an amino acid selected from the group consisting of C, I R, V, and K (e . , R, V. or K); X3010 is optionaly an amino acid selected from the group consisting of A, C, F, G, H, I K, L, M, N, P, Q I 8, T, V, W, and Y, such as an amino acid selected from the group consisting of A, C, G, H, I, K, L, M. Q, R. S, and T (eg, an amino acid selected from the group consisting of A, C, K, L, Q, R, and S); X301! is optionally an amino acid selected from the group consisting of A, G,1, K, L, M, N, Q, R, S. T, V, W, Y, C, F, and 1, such as an amino acid selected from the group consisting of A. 1, K, L, M, R, S, V, W, C, F, and H (e.g., an amino acid selected from the group consisting of , K, L, M, R, V, W, C, F, and H); X3013 is optionally an amino acid selected from the group consisting of A, C, F, G, H, K, L M, R., S, V, W, Y, and , such as an amino acid selected from the group consisting of C, F, K, L, M, R, V, and I (e.g., C, K, R, V, or I); X3014 is optionally an amino acid selected from the group consisting of A, C, F, G H, L, , N, Q, R, s, 1, V, W. Y, and K, such as an amino acid selected from the group consisting of A, M, C, F, II, [,L, N, R, S V, W, and K (e.g., an amino acid selected from the group consisting of A, S, C, F H, I, R, and K): X301 7 is optionally an amino acid selected from the group consisting of A, C, F, G, 1, K, L. N, Q, R, T, V, W, Y, H, A, and MV, such as an amino acid selected from the group consisting of A, C, F, G, 1, K, L, N, Q, R, S, T, V, W, H, A, and M (eg., an amino acid selected from. the group consisting of C, G, 1 K, L, N, Q, R, S, T, V, H, A, and M); X301 8 is optionally an amino acid selected from the group consisting of A, C, F 1l, L, M, Q, R, V, W, and Y, such as an amino acid selected from the group consisting of A, K, C, I, L, R, arid W (e.g., K, C, L, R, or W); X30 is optionallyn anaino acid selected fromthe group consisting of A, C, D, E, F, G, H, K. L, N, P. Q, R, V, W, Y, and , such as an amino acid selected from the group consisting of A, C, E, H, K, N, Q, R, and I (e.g., C, E, H, K, R, or I); X3020 is optionally an amino acid selected from the group consisting of A, C, F, G, H, K, L, M, N, QR, V, W, Y, L and P, such as an amino acid selected from the group consisting of C, H, L M, R, V, I, and P (eg, C, M, , or P);and/or X3021 is optionally an amino acid selected from the group consisting of A, C, H, , K, L, M, N, P, Q, R, T, V, W, Y, F, and G, such as an amino acid selected from the group consisting of A, C, H, L K, L, M, N, Q, R, V, W, Y, F, and G (e.g, an amino acid selected from the group consisting of A, C, H, 1, K, L, M, N, Q, R, V, W, F, and G). [001881 The TFPbinding peptide of the invention comprises, in some aspects, an amino acid sequence having at least 60% identity (e.g.at least 65%at t least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95% or 100% identity) to the sequence of formula (X): Ac-GY ASFPWFVQ LHVHKRSWEMKANH2 (X) (SEQ ID NO: 223).

Optionally, the peptide comprises or consists of the amino acid sequence of formula (VUL> (IX) as defined herein. As used herein, "at least 60% identity" and similar terms encompass any integer from, e.g., 60%, to 100%, such as 60%, 61%, 62%, and the like. Also, the term "at least [percentage] identity" encompasses any percentage that is greater than or equal to the number of identical amino acids divided by the total number of amino acids of the peptide of the invention ([at least percentage identity] [number of identical amino acids] / [total number of amino acids of the peptide of the invention]). [00189] The invention also includes a peptide comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 2001-2498 (eg, a peptide comprising or consisting of the amino acid sequence selected rom the group consisting of SEQ [D NOs; 2001-2296 and 2498 (such as SEQ ID NOs: 2001-2126, 2128-2296, or 2498) and/or selected from the group consisting of SEQ I) NOs: 2297-2497 (such as SEQ ID NOs: 2298-2497)). The invention further provides a peptide comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 3001-3108 (e ga peptide comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs; 3001-3064 (such as SEQ ID NOs: 3001-3048, 3051-3053, 3055, or 3057-3064) and/or selected from the group consisting of SEQ ID NOs: 3065-3084 (such as SEQ ID NOs: 30663084) and/or selected from the group consisting of SEQ ID NOs: 3085-3108). [00190] The peptide of SEQ ID NOs: 1-7 also, in some aspects comprises one or more amino acids attached at the N- or C-tenminus of SEQ ID NOs: 1-7. For example, the invention includes a peptide comprising or consisting of the amino acid sequence of JBT0047, JBT00051, JBT055, J.BT131, JBTO0132, JBT0133, JBTOI55, JT0158, JBT0162, JBTOI63, JB3T0164, JB3TOI66, 31BT0169, JBTO17O, JBT0i7I, 1JBT0174, 1BT0175, or JBT0293, all of which comprise the amino acid sequence of SEQ ID NO: 1. Exemplary peptides comprising the amino acid sequence of SEQ ID NO: 2 include peptides comprising or consisting of the amino acid sequence of JBT0294, JBT70295, JBT0296, JBT0297, JBT0298, JBT0299, JBT0300, JBT0301, JBTO302, JBT0303, JBT0304, JBT0305, JBT0306, JBT0307, JBTO3OS, JBT0309, JBT0310, or JBT031.. Exemplary peptides comprising the amino acid sequence of SEQ ID NO: 3 comprise or consist of the amino acid sequence of JBT0049, JBT0053, JBT0057, JBT0190, JBT0193, or JBT0197. The invention further includes a peptide comprising or consisting of the amino acid sequence of JBT0050, JBT0054, JBT0058, JBT30129, JBT0130, JBT0205, JBT0208, JBTO21I1, JBT0212, JBT02 17, JBT0218, or JBT0219, all of which include the amino acid sequence of SEQ ID NO: 4. Exemplary peptides comprising SEQ ID NO: 5 include those comprising or consisting of the

J--A

amino acid sequence of JBT011BT0052. JBTOIO3, JBT0178, or JBTOI82, The invention additionally includes a peptide comprising or consisting of the amino acid sequence of JBT0120, JBT0124, JBT0247, JBT0248, JBT0251, or JBT0252, each of which include the amino acid sequence of SEQ ID NO: 6. A peptide including the arrino acid sequence of SEQ f) NO: 7, e.g., a peptide comprising or consisting of the amino acid sequence of JBT0122, JBT0126. JBT0221, JBT0224, JBT0225, JBT0226, JBT0228, JBT0232, or JBT0233, also provided by the invention. The peptides described herein are set forth in Table 5 of Example I and in Figures 12-8. [00191) The invention further includes a TFPI-binding peptide comprising the structure of formula (Xl): X(4001 Q. X4003-X4004-X4005 -X4006-X(40017-X4008-X4009-X4010-X40 11 X401 2-X413-X4014-R-X4016-X4017-X4018-X4019-X4020 (XI), With respect to formula (XI), X4001 is an amino acd selected from the group consisting of F, L, M, Y, Ni Thi, Bta, and Dopa (e.g., F, Y, 1Ni, ita, or Dopa); X4003 is an amino acid selected from the group consisting of C, ), E, M, Q, R, S, T, Ede(O) and Cme (e-g. D, E, or S) X4004 is an amino acid selected from the group consisting of Ab, E , 1, K, L, Mi P, R, W, and Y (e g , K); X4005 is an amino acid selected from the group consisting of a, A, Aib, C, D, d, F, 0, H-, K, k, M. N, Nmg p, Q. R, NpropylG, aze, pip, tic, oi, hyp, nma, Ncg, Abg, Apg, thz. and dtc (esg, p, Nmg, NpropylG, aze, pip, tic, oie, or hyp); X4006 is an amino acid selected from the group consisting of A, CC(NEl), D, E, G, H, K, M, N, Q, R, S, V. Cit, C(Acm), Me, 1, Ede(O), Cmc, Eel, Eea, Eec, Eef, Nif, and Eew (e.g., C, E, K, R, S, V, C(Acm), Ne, C(NEM), I, or Cit); X4007 is an amino acid selected from the group consisting of I, V, T, Chg, Phg, and Tle (e.g., V or Tle); 2(4008 is an amino acid selected front the groupconsisting of F, HINi 2Ni, Pmy, and Y (e g HNi2Ni, or Iny; X4009 is an amino acid selected from the group consisting of Aib, V, Chg, Phg, Abu, Cpg, Tie, and L-2-amino-4,4,4-trifluorobutyric acid (e.g., V, Abu, or Tle); X4010 is an amino acid selected from the group consisting of A, C, D, d, E, F, H, K, M, N, P, Q R, S, T, V. W, Y, Nmd, and C(NEM) (e.g. D, P, C or Tl; X401 I is an amino acid selected from the group consisting of A, a, G, p, Sar, c, and hcy (e.g., G, a, c, hey, or Sar); X4012 is an amino acid selected from the group consisting of Y, Tym, Pty, .Dopa, and Pmy (e.g., Y); X4013 is an amino acid selected from the group consisting of C, F, 1Ni, Thi, and Bla (e.g,, F, 1Ni, or Bta); X4014 is an amino acid selected from the group consisting of A, Aib, C, C(NEM), ), E, K, L, M, N, Q, R, T, V, and Hey (e.g., Aib, C, E, or Hey); X4016 is an amino acid selected from the group consisting of L, Hey, We, and Aml; X4017 is an amino acid selected from the group consisting of A, a, Aib, C, c. Cha, Dab, Eag, Eew, H, Har, , Hit, I, K, L, M , Nme Nya, Opa, Orn, R, S, Deg, Ebe, Eca, Egz, Aic, Ape, and £gt (e.g., A, Aib, C, c, Aic; Eca, or Deg); X4018 is an amino acid selected from the group consisting of A, Aib, Hey, hey, C, e, L, Ne, M, N, and R (e.g, A, Aib, C, c. L, or Hey); X4019 is an amino acid selected from the group consisting of K, ,and Har (eg K); and X4020) is an amio acid selected from the group consisting of K, L, -Icy, and Ani (e.g., L, Aml, and Hey). [001921 The TFP-binding peptide of formula (XI) does not comprise the structure of formula (X ig: X5001 -Q-X5003-X5004-X5005-X5006-I/V-X5008-Aib/V -X5O10- - X5013X5014-R-L-X5017X508 -K/L XII) In formula (XII) X5001 is an amino acid selected from the group consisting of F, L, M, and Y; X5003 is an amino acid selected from the group consisting of C, D, E, M, Q, R, S, and T; X5004 is an amino acid selected from the group consisting of E, G , K. L, M, P, R, W, and Y; X5005 is an amino acid selected fronthe group consisting of a A, Aib, C, D- d, E, G, H.K k M,N NmgQ R and ; X5006isanamino acid selected from the group consisting of A, C, i E H, K, M, N, Q R, S., and V; X50O8 is an amino acid selected from the group consisting of F, H and Y; X50I0 is an amino acid selected from te group consisting of A, ,B F, H, D, M, N, P Q, R, S, V, W, and Y; X5013 is an amino acidselected from the group consisting of AibCand F; X504is an amino acid selected fom the group consisting of A, A:,, C F D K., 111 M, N, Q, R, T, and V; X5017 is an amino acid selected from the group consisting of A, Aib, C, Cha, Dab, Eag, Eew, H, Har, Hei, Hle, I, K, L, M, Ne, Nve, Opa, Orn, R, and S; and X5018 is an amino acid selected from the group consisting of A, C, L, M, N, and R. [00193] In one aspect, the TFPI-binding peptide of formula (XI) further comprises N terminal amino acid(s) and/or moieties linked to X4001, The N-terminal amino acid(s) and/or moieties are optionally selected from the group consisting of FAM-Ttds, a proline glutamate tag ("PE"), Palm, 2-phenyl acetyl, 3-phenyl propionyl, 2-(naphtha-2-yl) acetyl, hexanoy1, 2-methyl propionyl, 3-methyl butanoy, 2Tnaphthylsulfonyl, and I naphthylsulfonylh Alternatively or in addition, the TFPI-binding peptide of formula (Xl) further comprises one or more amino acids) and/or moieties linked to X4020. The C terinal amino acids) and/or moieties are designated herein as X4021 and are optionally selected from the group consisting of C, c, C(NEM), K(Ttds-maleimidopropionyh(EtSH)), FA19205, FA19204, FA19203, FA03202, K(Tdts-maleimid), K(AOA), and Cea. [00194] In one embodiment, the peptide comprises a cyclic structure formed between X4018 and X402L In this regard, X4018 is optionally C ore, and X4021 is optionally Cea. In another embodiment, the peptide comprises a cyclic structure formed between X4011 and X4014, In this regard, X4011 is optionaly e or hey, and X4014 is optionally C or Hey. [00195] The invention also includes a peptide consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 4022, 4024, 4032, 4036-4047, 4049 4078, 4086-4097,4100-4127, 4129-4170 41.734195, 4200-4214, 42174225, 4228, 4230, 4231, 4238, and 4239, as well as a peptide consisting of the amino acid sequence selected from the group consisting of SEQ I) NOs: 1294-1336, 4002, 4013, 4021, 4023, 4025-4031, 4033-4035, 4048, 4079-4085, 4098, 4099, 4128, 4171, 4172, 4196-4199, 4215, 4216, 4226, 4277, 4229, 4232, and 4233. [00196] In certain embodiments, the peptide of the invention comprises or consists of the amino acid sequence of JBT0047, JBT0049, JBTO1OI, JBT0120, or JBTI0122 or any of the inventive peptides described herein (e,g- a peptide comprising or consisting of the amino acid sequence of any one of SEQ TD NOs: 1-3108, such as 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, 121 3-1289, 1290-1293 2001-2126, 2 128-2296, 2298-2498, 300 -3048, 3051 -3053, 3055, 3057-3064, and 3067-3108; a peptide comprising or consisting of the arnino acid sequence of any one of SEQ ID NOs: 4022,4024, 4032, 4036-4047, 4049-4078,4086-4097,4100-4127, 4129-4170. 4173-4195, 4200-4214, 4217-4225. 4228, 4230, 4231 4238 and 4239; or a peptide comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1294-1336, 4002, 4013. 4021, 4023, 4025-4031, 4033-4035, 4048, 4079-4085, 4098, 4099, 4128, 4171, 4172, 4196-4199, 4215, 4216, 4226, 4277, 4229, 4232, and 4233), or a variant of any of the foregoing. By "variant" is meant a peptide comprising one or more amino acid substitutions, amino acid deletions, or amino acid additions 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 of the amino acid sequences provided herein while retaining the ability to bind TFPI and/or inhibit TFPJ activity. In one embodiment, the peptide comprises or consists of the amino acid sequence of JBT0132, JBT0303, JBT0193, JBT0178, JBT0120. or JBT0224. [00197] In one aspect, the peptide of the invention consists of 40 amino acids or less, such as 35 amino acids or less. Optional, the peptide of the invention consists of 25 amino acids or less. or 10 amino acids or [ess, In various embodiments, the peptide comprises 15-3|5 amino acid residues (esg., 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 a peptide described herein comprising one or more deletions is suitable in the context of the invention so long as the peptide binds TFPI and, optionally, blocks TFPT inhibition of the coagulation cascade. In some aspects, amino acids are removed from within the amino acid sequence, at the N terminus, and/or at the C-terminus. Such peptide fragments can comprise 3-14 amino acid residues (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acid residues). [00198] Optionally, the peptide of the invention comprises one or more amino acid substitutions (with reference to any of the amino acid sequences provided herein) that do not destroy the ability of the peptide to bind and/or inhibit TFPL For instance, peptides comprising or consisting of the amino acid sequence selected from the group consisting of 1BT0294, JBT0295; JBTO296, JBT0297, JBTO298, JBT0299, JBT0300, J13T301, JBT0302, JBT0303, JBT0304, JBT0305, JBT0306, JBT0307, JBT0308, JBT0309, JBT0310, or JBTQ3 11 are substitutional mutants of the amino acid sequence of JBT0293 (the amino acid sequence of SEQ ID NO: 1 directly linked to a phenylalanine residue at the N-terminus and a lysine reside at the C-terminus) (see Figure 4). [00199] Amino acid substitutions include, but are not limited to, those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinities, and/or (4) confer or modify other physiochemical or functional properties on a peptide. In one aspect, the substitution is a conservative substitution, wherein an amino acid residue is replaced with an amino acid residue having a similar side chain, Families of amino acid residues having similar side chains have been defined within 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), beta-branched side chains (eg, threonine, valine, and isoleucine) and aromatic side chains (eig., tyrosine; phenylalanine, tryptophan, and histidine) t will be appreciated however, that a practitioner is not limited to creating conservative substiuions so long as the resulting peptide retains the ability to downregulate, in whole or in part, TFPI activity. The invention also embraces TFPI-inhibitory peptides comprising atypical, non-naturally occurring amino acids, which are well known in the art. exemplary no-naturally occurring amino acids include ornithine, citrulline, h ydroxyproline. homoserine, phenylglycine, taurine, iodotyrosine, 2,4 diaminobutyric acid, -amino isobutyric acid, 4-aminobutyric acid, 2-amino butyric acid, y amino butyric acid, 2-amino isobutyric acid, 3-amino propionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, P-alanine, a fluoro-amino acid, a 3-methyl amino acid, a-C-methyl amino acid, a N-methyl amino acid, 2-amino-isobutyric acid, p--homoglutamatic acid, p homophenylalanine, fi-homolysine, $-homoleucine; $-hoimoasparagine, Dhomoglutamine, $i homoarginine., Jhomoserine, phomotyrosine, ihomoaspartic acid, $Shomovaline, pV homoasparagin, (S) -cyclohexylalanine, (S)-citrullin, (S)-2,4diaminobutyric acid, (S)-2,4 diaminobutyric acid, (S)-diaminopropionic acid, (S)-%propargyiglycine, (S)-N(omega)-nitro argmnine, L-homophenylalanine, S)-homo-arginine, (SI-homo-citrulline, (5)-homo-cysteine, (S)-2-amino-5-methyl-hexanoic acid, (Shomo-lysine, (S)-norleucine, (S)-N-methylalanine, (S)-N-methyl-aspartic acid, (S)ONmethyl-glutamic acid, (S)-N-methyi-phenylalanine, N methyl-glycine, (S)-N-methyl-lysine, (S)-N-methyl-leucine, (S)-N-methyl-arginine, (S)-Nmethbylserine, (S)N-methyl-valine, (S)-Nmnethyl-tyrosine, (S)-2-amino-pentanoac acid, (S)- 2-pyridyl-alanine, (S)-ornithine. L-phenylgiycin, 4-phenyl- butyric acid and selenomethionine. The individual amino acids may have either L or D stereochemistry when appropriate, although the L stereochemistry is typically employed for all of the amino acids in the peptide. [00200] The invention further includes TFPI-inhibitory peptide variants comprising one or nore amino acids inserted within an amino acid sequence provided herein and/or attached to the N-terminus or C-terminus. In one aspect, the peptide further comprises one or more amino acids tat facilitate synthesis, handling, or use of the peptide, including, but not limited to, one or two lysines at the N-terminus andar C-terminus to increase solubility of the peptide. Suitable fusion proteins include, but are not limited to, proteins comprising a TFPI inhibitory peptide linked to one or more polypeptides, polypeptide fragments, or amino acids not generally recognized to be part of the protein sequence In one aspect, a fusion peptide comprises the entire amino acid sequences of two or more peptides or, alternatively, comprises portions (fragments) of two or more peptides. In addition to all or part of the TFPI-inhibhory peptides described herein, a fusion protein optionally includes all or part of any suitable peptide comprising a desired biological activity/function. Indeed, in some aspects, a TFPJ-inhibitory peptide is operably linked to for instance, one or more of the following: a peptide with long circulating half life, a marker protein, a peptide that facilitates purification of the TFP-inhibitory peptude, a peptide sequence that promotes formation of multimeric proteins, or a fragment of any of the foregoing, Suitable fusion partners include, but are not limited to, a His tag, a FLAG tag, a strep tag, and a myc tag. Optionally, the TFPIinhibitor peptide is fused to one or more entities that enhance the half life of the peptide. Half life can be increased by, e.g., increasing the molecular weight of the TFPI binding peptide to avoid renal clearance and/or incorporating a ligand for the nFec receptor mediated recycling pathway. In one embodiment, the TFPI-binding peptide is fused to or chemically conjugated to (as described further below) an albumin polypeptide or a fragment thereof (e.g., human serum albumin (HSA) or bovine serum albumin (BSA)). The albumin fragment comprises 10%, 25%, 50%, or 75% of the full length albumin protein. Alternatively or in addition, the TFPI-binding peptide comprises an albumin binding domain or fatty acid that binds albumin when administered in vivo, Other suitable fusion partners include, but are not limited to, a proline-alanine-serine multimer (PASylation) and an antibody or fragment thereof (e.g., an Fe portion of an antibody).

[002011 In one embodiment, two or more TFPI-inihibitory peptides are fused together, linked by a multimerization domain, or attached via chemical linkage to generate a TFPI inhibitory peptide complex. The TFPI-inhibitor peptides may be the same or different. Thus, the invention provides a homo-dimer (i.e., a dimer comprising two identical TFP-binding peptides), a homo-multimer (i.e. a complex comprising three or more identical TFPI-binding peptides), a hetero-dimer (i.e., a dimer comprising two different TFPI-binding peptides), and heteromultimer (i.e, a complex comprising three or more TFPI-binding peptides, wherein at least two of the TFPI-binding peptides are different) comprising or consisting of any of the peptides described herein, optionally attached by one or more liners. An exemplary TFPI binding peptide dimer is JBT2496 ($EQ 1D NO: 4211) [002021 "Derivatives" are included in the invention and include TEIN-inhibitory peptides that have been chemically modified in some manner distinct from addition, deletion, or substitution of amino acids. In this regard, a peptide of the invention provided herein is chemically bonded with polymers, lipids, other organic moieties, and/or inorganic moieties. Examples of peptide and protein modifications are given in Hernanson, Bioconjugate Techniques, Academic Press, (1996). The TFPI-binding peptides described herein optionally comprise a functional group that facilitates conjugation to another moiety (e.g., a peptide moiety). Exemplary functional groups include, but are riot limited to, isothiocyanate, isocyanate, acyl azide. NHS ester, sulfonyl chloride, aldehyde, epoxide, oxirane, carbonate, arylating agent, imidoester, carbodiimide, anhydride, alkyl halide derivatives (e.g., haloacetyl derivatives), maleimide, aziridine, acryloyl derivatives, arylating agents, thiol-disulfide exchange reagents (e.g., pyridyl disulfides or TNB thiol), diazoalkane, carboyldiimadazole, N,N'-Disuccinyl carbonate, N-Hydroxysuccinimidyl chlorofornmate, and hydrazine derivatives. Maleimide is useful, for example, for generating a TFPI-binding peptide that binds with albumin in vivo. [00203] Derivatives are prepared in some situations to increase solubility, stability, absorption, or circulating half life. Various chemical modifications eliminate or attenuate any undesirable side effect of the agent i one aspect, the invention includes TFPI-binding peptides covalently modified to include one or more water soluble polymer attachments. A water soluble polymer (or other chemical moiety) is attached to any amino acid residue although attachment to the N- or C-terminus is preferred in some embodiments. Optionally, a polymer is attached to the peptide via one or more amino acids or building blocks that offer functional groups that facilitate polymer attachment. For example, JBT2315 comprises a C terminal cysteine (position X4021 with respect to formula (XI)), which facilitates the addition -71 of e g. a maleimide polyethylene glycol (PEG). Useful polymers include, but are not limited to, PEG (e.g , PEG apprxiniately 40 UkD 30 kD, 20 kDI 10, kID 5 k D, or I kD in size), polyoxyethylene glycol, polypropylene glycol, monomethoxy-polyethylene glycol, dextran, hydroxyethyl starch, cellulose, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polysialic acid (PSA), polyoxyethylated polyols (e.g, glycerol) and polyvinyI alcohol, as well as mixtures of any of the foregoing. hi one aspect, the peptide of the invention is a PEGylated peptide. PEG moieties are available in different shapes, e.g., linear or branched. For further discussion of water soluble polymer attachments, see U.S. Patent Nos, 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and 4,179,337. Other moieties useful for improving peptide half life or stability are described herein and include, for instance, albumin (optionally modified to allow conjugation to the inventive peptide), fatty acid chains (e.g., C12-C18 fatty acid, such as a C14 fatty acid), an antibody or fragment thereof (e.g., an Fc portion of an antibody), and proline-alanine-scrine multimers. [00204] In another aspect, a peptide derivative includes a targeting moiety specific for a particular cel type, tissue, andlor organ. Alternatively, the peptide is linked to one or more chemical moieties that facilitate purification, detection, multimerization, binding with an interaction partner, and characterization of peptide activity, An exemplary chemical moiety is biotin. Other moieties suitable for conjugation to the TFP1hinding peptide of the invention include, but are not limited to, a photosensitizer, a dye, a fluorescence dye, a radionuclide, a radionuelide-containing complex, an enzyme, a toxin, and a cytotoxic agent. Photosensitizers include, e.g Photofrin, Visudyne Levulan, Foscan Metvix, Hexvix, Cysview

TM

, Laserphy'rin, Antrin, Photochior, Photosens, Photrex, Lumnacan, Cevira, Visonac, BF-200 ALA, and Amphinex If desired, a His tag a FLAG tag a strep tag, or a myc tag is conjugated to the peptide. [00205] In addition, in one aspect, the peptides of the invention are acylated at the N terminal amino acid of the peptide. in another aspect, the peptides of the invention are amidated at he C-terminal amino acid of the peptide. in a still further aspect, the peptides of the invention are acylated at the N-terminal amino acid of the peptide and are amidated at the C-terminal amino acid of the peptide. [00206] Derivatives also include peptides comprising modified or non-proteinogenic amino acids or a modified linker group (see- e.g., Grant, Syntheic Peptides: A User's Guide, Oxford University Press (1992)). Modified amino acids include, for example, amino acids wherein the amino and/or carboxyl group is replaced by another group. Non-imiting examples include modified amino acids incorporating thioamides, ureas, thioureas, acyihydrazides, esters, olefines, sulfonamides, phosphoric acid aides, ketones. alcohols, boronic acid aides, benzodiazepines and other aromatic or non-aromatic heterocycles (see Estiarte a al, Burgers Medicinal Chemistry, 6th edition, Volume 1, Part 4, John Wiley & Sons, New York (2002)). Modified amino acids are often connected to the peptide with at least one of the above mentioned functional groups instead of an amide bond, Non proteinogenic amino acids include, but are not liMited, to p-alanine (Bal), norvaline (Nva), norleucine (Nie), 4-aminobutyric acid (yAbu), 2-aminoisobutyric acid (Aib), 6 aminohexanoic acid (:-Ahx), ornithine (Orn), hydroxyproline (Hyp), taurine, sarcosine, citrulline (Ci), cysteic acid (Coh), cyclohexylalanine (Cha), methioninesulfxide (Meo), methioninesuifone (Moo), homoserinemethylester (Hsm), propargyiglycine (Bag), 5 fluorotryptophan (5 Fw), 6-fluorotryptophan (6Fw), 3,4'-dimethoxyphenyi-alanine (Ear), 3',4'-difluorophenylalanine (Dffl 4='fluorophenyl-alanine (Pff, 1 -naphthyl-alanine (Ni), I.

mnethiyitryptophan (1Mw), penicilamine (Pen). homoserine (Hse), t-butylglycine, t butylalanine; phenylglycine (Phg), benzothienylalanine (Bta), Lhomo-cysteine (Hey), N methyl-phenylalanine (Nuf, 2-thienylalanine (Thi), 33-diphenyiaianine (Ebw), homophenylalanine (Hife) and S-benzyl-L-cysteine (Ece). The structures of many of the non proteinogenic amino acids are provided in Table 2. These and other non-proteinogenic amino acids may exist as D- or L- isomers. Examples of modified linkers include, but are not limited to, the flexile e linker 4,7,10-trioxa- , 13tridecanediamine (Ttds) glycine, 6 aminohexanoic acid, beta-alanine (Bal), pentynoic acid (Pyn), and combinations of Ttds, glycine,6-aminohexanoic acid and al. [00207] Homologs of the amino acids constituting the peptides of the invention may be as set fothh in Table 3. In any embodiment, one or more amino acids of the TFPL-binding peptide are substituted with a homolog. TABLE3 Amio Acid Expemplary homologs A A'b, BaEagE Nna .bu , . Nva.. Ne CS, A, Hey, Ml L- L V Nc-Cysteine'. D EHmguai acid, Tyrx-ltmcaiTabx gltai aid mxd $-sat Nai, ,Cseai E--- --- -- --- -- --- -- --- -- - - a c-d--y d r oc a c- -C- glutamic acid, ac-Aminoadipic acid, Nme, f-glutamxic acid, Q, N. Cysteic acid F W e, Nmf, p-Phenylalanine, Phg, Bhf, Thienylalanine, Benzothienylalanine, Bromophenylalanine, Iodophenylalanioe, Chlorophenylalanine, Methylphenylalanine, Nitrophenylalanine, Y, W, Naphtylaanine, Tic G A, N Ng H Nibh 1-Methyihis idin 3c - ethy histidine, Thieny-alanine i L, V, Hie Nva, Nie, $-lsoleucine. Nmil 14 Nmi K Nnk, Nnu $VLysine, Dab. Dap 51-Piperazinylalanine 26 Diamino-4-hexynoic acid, delta-Hydroxy-lysine, Har, omega Hydroxy-norarginine, omega-Amino-arginine, omega-Methyl arginine, P-(2-Pyridyl)-alanine, $-(3-Pyridyl)-alanine, 3-Amino tyrosine, 4-Amino-phenylalanine, Hei, Cit L IV, le Ne, Na, p-Isoleucine, Nm, M S: V He, Nva, R, Har, Nimn, Methioninesulfone N Nmn, -Asparagine, Q, Nmq, S-Glutamine, Cys(3-popionic acid arnide)-OH, Cys(02-3-propionic acid amide)-OH P Azetidine-2-carboxylic acid, Hyp, a-Methyl-mnethionine, 4 Hydroxy-piperidine-2-carboxylic acid, Pip, x-Methyl-Pro Q N, Nmn, Nm nq V-ltamin, Cys(3apropionic acid amnidei-OHE Cys(O2-3-propionic acid adi e--O R. Nmk, K, Nmar, .rysine, Dab, Dap.0, m3-O(Piperazinyl)-aihmine. 2 6 Diamnino-4hxc.yno'e a id, delta*Hydroxy-1ysie, Mar, omega Hydroxy--norargintine, omega- Amino-arginine, omnega-Methyl arginine. $3(2-Pyidyl)-alanine, p-(3-Pyrdyl >alanine, 3-Amino~ yrosine 4-Amnino-phen ylalanine. Hei Cit, H e, L, Nie, M S I Hse $A-erine, C, $-Cyano-alainie, allo-hreonine Ts oehen S Thenn aloTrenn --- -- -- -- --- -- -- -- -- --- -- -- -- --- -- -- -- -- -- - -- - - - - --- - - - -- - - - -- - - - - - -- - -- - -- - - - - - -- - - - - -- - - -- - -- - - - - --- - - - - -- - - -- - -- - - - - --- - - - - -- - - S, ~ )~s~~.5t2QU2 iOitcnr ---- --- ---- --- --- ---- --- --- ---- --- ---- --- --- -- ---- --- ---- --- ---- --- --- ---- --- -- L, I le, Nva, Nle, -Valine, Nmv, M, Nmi, Nml W Nmw, j-Tryptophan, F, Hfe, Nmf, p-Phenylalanine, Phg, Bhf, Thienvialanine, Benzothienylalanine, Bromophenylalanine, lodophenylalanine, Chlorophenylalanine, Methylphenylalanine, Nitrophenylalanine, Y, Naphtylalanine, Tic Y Nmy, j-Tyrosine, , F, Hfe, Nmf, [S-Phenylalanine, Phg, Bhf, Thienylalanine, Benzothienylalanine, Bronophenylalanine, Iodophenylalanine, Chlorophenylalanine, Methylphenylalanine, Nitrophenylalanine, W, Naphtylalanine, Tic [00208] Derivatives also include peptides comprising amino acids having modified substituents, such as amino acids modified by halogenation with, e.g., fluorine, chlorine, iodine, or bromine. In some embodiments, the TFP-binding peptide comprises a halogenated aromatic amino acid, such as phenylalanine. [00209] In some embodiments, the peptide (CO-NH) linkages joining amino acids within the peptide of the invention are reversed to create a "retro-modified" peptide, i.e., a peptide comprising amino acid residues assembled in the opposite direction (NH-CO bonds) compared to the reference peptide. The retro-modified peptide comprises the same amino acid chirality as the reference peptide. An "inverso-modified" peptide is a peptide of the invention comprising amino acid residues assembled in the same direction as a reference peptide, but the chirality of the amino acids is inverted. Thus, where the reference peptide comprises L-amino acids, the "inverso-modified" peptide comprises D-amino acids, and vice versa. Inverso-modified peptides comprise CO-NI-I peptide bonds. A "retro-inverso modified" peptide refers to a peptide comprising amino acid residues assembled in the opposite direction and which have inverted chirality A retro-inverso analogue has reversed termini and reversed direction of pepide bonds (i.e., NH-CO), while approximately maintaining the side chain topology found in thereference peptide Retro-inverso peptidomirnetics are made using standard methods, including the methods described in Meziere et al, J. Immune, 159, 3230-3237 (1997), incorporated herein by reference, Partial retro-inverso peptides are peptides in which only part of the amino acid sequence is reversed and replaced with enantiomeric amino acid residues. [00210] TFPIbinding peptides of the invention (including TFPI inhibitor peptides) are made in a variety of ways. In one aspect, the peptides are synthesized by solid phase synthesis techniques inchding those described in Menrifield, /. Am. Chemn Soc., 85, 2149 (1963); Davis et al.Biochem. Int., 10, 394-414 (1985); Larsen et al, . Am. Chem. Soc., 115, 6247 (1993); Smith et at., . Peptide Protein Res,, 44, 183 (1994); O'Donnell et al, J. Am. Chemt. Soc, 118, 6070 (1996); Stewart and Young, Solid Phase Peptide Synthesis, Freeman (1969); Finn et al, The Proteins, 3rd ed., vol, 2, pp. 105-253 (1976); and Erickson et aL, The Proteins, 3rd ed., vol. 2, pp. 257-527 (1976). Alternatively, the TFPI-binding peptide (e.g, the TFPIinhibitory peptide) is expressed recombinantly by introducing a nucleic acid encoding a TFP-binding peptide (eg. a TFPf-inhibitory peptide) into host cells, which are cultured to express the peptide. Such peptides are purified from the cell culture using standard protein purification techniques. [00211] The invention also encompasses a nucleic acid comprising a nucleic acid sequence encoding a TFPJ-hinhibitory peptide of the invention. Methods of preparing DNA and/or RNA molecules are well known in the art. In one aspect, a DNAIRNA molecule encoding a peptide provided herein is generated using chemical synthesis techniques and/or using polymerase chain reaction (PCR), If desired, a TFPhhibitory peptide coding sequence is incorporated into an expression vector. One of ordinary skill in the art will appreciate that any of a number of expression vectors known in the art are suitable in the context of the invention, such as, but not limited to, plasmids, plasmid-liposome complexes, and viral vectors, Any of these expression vectors are prepared using standard recombinant DNA techniques described in, e.g, Sambrook et al, Molecular Cloning) a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and Ausubel et al, Current Protocolin Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994). Optionally, the nucleic acidl is operably linked to one or more regulatory sequences, such as a promoter, activator, enhancer, cap signal, polyadenylation signal, or other signal involved with the control of transcription or translation, [00212] Any of the TFPI-inhibitory peptides of the invention or nucleic acids encoding the peptides also is provided in a composition (eg., a pharmaceutical composition). In this regard, the peptide is formulated with a physiclogically-acceptable (i.e, pharmacologically acceptable) carrier, buffer, excipient, or diluent, as described further herein, Optionally, the peptide is in the form of a physiologically acceptable salt, which is encompassed by the invention, "Physiologically acceptable salts" means any salts that are pharmaceutically acceptable. Some examples of appropriate salts include acetate, hydrochloride, hydrobromide, sulfate, citrate, tartrate, glycolate, and oxalate. If desired, the composition comprises one or more additional pharmaceutically-effective agents.

[002131 The peptide provided herein optionally inhibits at least one TFPI- (e.g. T-FPl 1 or TFPI-1p) activity such as, but not limited to, an activity that downregulates the blood coagulation cascade. Without being bound by any specific mechanism of action, a proposed mechanism of inhibition may involve preventing formation of the quaternary TF-FVIIA FXA-TFPI complex. The peptide may inhibit binding (competitively or allosterically) of TFPL to FXa (e.g. inhibit binding of TFPT Kunitz domain 2 to Factor Xa or interrupt binding of TFPT Kunitz domain 1 to an exosite of Factor Xa), the TF/FVIIa complex (e.g, inhibit binding of TFPI Kunitz domain I to the TF/FVIIa complex), TF alone, and/or FVIIa alone, With TFPI activity diminished, TF and FVI1a are free to activate FX which, in turn, enhances conversion of prothrombin to thrombin. Surprisingly, in one embodiment, the peptide of the invention that binds Kunitz domain I interferes with TFPI-mediated inhibition of FXa, Thus, the invention provides a method of, e~g., inhibiting TUPI-mediated downregulation of the extrinsic and/or common pathway of the coagulation cascade and/or enhancing FXa-mediated conversion of prothrombin to thrombin, by administering to a subject a peptide described herein that binds Kunitz domain 1 [00214] In one aspect, the peptide of the invention exhibits TFPI antagonistic activity in model and/or plasmatic systems. An exemplary model system for determining TFPI inhibitory activity is the extrinsic tenase assay, which tests the ability of candidate peptides to restore extrinsic 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. Haernost., 74, 910 915 (1995)). Another model system for characterizing TFPI-inhibitory activity is the FXa inhibition assay, wherein PXa activity is measured in the presence of TFP! (see Sprecher et al., PA4S, 91, 3353--3357 (1994)). The extrinsic tenase assay and the FXa inhibition assay are further described in Example 3. Optionally, the peptide of the invention enhances IFX activation in the presence of TFPI with a half maximal effective concentration (ECo) of less than or equal to 1 x Mless than equal to 1 x 10M less than or equaltoI x 10 MI, or less than or equal to I x 10 4 M. 100215] In one aspect, TFPI-antagonist activity is characterized in a plasma-based assay. Thrombin formation is triggered in plasma substantially lacking FVIiI or FIX activity (e.g., the residual coagulation factor activity is lower than 1%) in the presence of a candidate peptide. Thrombin formation can be detected using a fluorogenic or chromogenic substrate, as described in Example 4. A system for measuring thrombin activity is provided by Thrombinoscope BV (Maastricht, The Netherlands). Prothrombin conversion is measured using, e.g. a Thrombograph T M (Thermo Scientific, Waltham, MA), and the resuming data is compiled into a Calibrated Autonatic Thrombogram generaed by ThrombinoscopeTM software available from Thrombinoscope BV. In certain embodiments, the TFPI-inhibitory peptide increases the amount of peak thrombin generated during the assay and/or decreases the time required to achieve peak thrombin formation. For example, the peptide improves TFPI-regulated thrombin generation in the absence of FVIII (e.g., in FVIII-depleted plasma) to at least I % of the level of TFPI-dependent thrombin generation in normal plasma. Generally, normal (unafflicted) plasma contains about 0.5 U/mL to about 2 U/mL Factor V11, Accordingly, in some instances, a TFPI-inhibitor peptide will enhance thrombin formation in the absence of FVII to at least about 1 % of that observed in the presence of 0.5 U/mL to 2 U/rnL FVIL. In further embodiments, the peptide enhances thrombin formation in the absence of 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 level of thrombin formation in normal plasma, i.e., in the presence of physiological levels of Factor VIII. In various aspects, the peptide is administered to an animal model of thrombin deficiency or hemophilia to characterize TFPI inhibitory activity in vivo. Such in vivo models are known in the art and include for example, mice administered anti-FVIII antibodies to induce hemophilia A (Tranholm et al, Blood, 102, 3615-3620 (2003)); coagulation factor knock-out models such as, but not limited to, FVIII knock-out mice (Bi et al, Nat. Genet, 10(1), 119-121 (1995)) and FIX knock-out mice (Wang et aL PNAS., 94(21), 11563-66 (1997)); induced hemophilia-A in rabbits (Shen et at, Blood, 42(4), 509-521 (1973)); and Chapel Hill HA dogs (Lozier et al., PN.AS, 99, 12991 12996 (2002)). [00216] Various peptides bind TFPI from any source including, but not limited to, mouse, rat, rabbit, dog, cat, cow, horse, pig, guinea pig, and primate. In one embodiment, the peptide binds human TFle Optionally, the TEninhibitory peptide binds TFP from more than one species (i.e., the peptide is crosreactive among multiple species). In certain aspects, the peptide binds TFPI with a dissociation constant (K 0 ) of less than or equal to 1 x 104 M, less than or equal to 1 x 10t than or ehan or equal to I x 106M, or less than or equal to 1 x 10 M. Affinity may be determined using, for example and without limitation, any one, two, or more of a variety of techniques, such as affinity ELISA assay, a competitive ELISA assay, and/or surface plasmon resonance (BlAcoreTM) assay. When characterized using a competitive (IC4 0 ) ELISA assay, the peptide of the invention optionally demonstrates an IC 0 o of less than or equal to about 50,000 nbA For example the peptide demonstrates an IC 0 o of less than or equal to about 10,000 nM, such as an IC.( of 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 1170 aspect, the peptide demonstrates an 1Cso of less than or equal to about 250 niM, less than or equal to about 100 nM, less than or equal to about 50 nM, or less than or equal to about 10 nM. Exemplary peptides and their ICo values are provided inFigures 32-39; in some instances, the peptide are classified into Groups A, B C, D , F, and G(see Table 4 in Example 1) based on their ICso values. In various aspects, the invention provides peptides falling within Groups A, B, C, D, E, F, and/or G as defined in Table 4. Affinity may also be determined by a kinetic method or an equilibrium/solution method. Such methods are described in further detail herein or known in the art. [00217] Another suitable assay for charaterizing the inventive peptides is a k 1 assay, which examines a peptide's release from TFP. The ka assay result is not the dissociation rate constant, but a percentage of competitor peptide blocked from TPI binding by a test peptide after an incubation period with TFPL An exemplary k; assay includes the following steps: 1) incubation of a TFPV-coated microtiter plate with an amount of test peptide resulting in approxinately 90% TFP! occupation; 2) removal of unbound test peptide; 3) addition of a biotinylated tracer (ie, competitor) peptide that competes with the test peptide for binding to TFPP, 4) incubation for a period of time during which binding sites released by the test peptide is occupied by the tracer; 5) removal of unbound tracer and test peptide;, and 6) detection of bound tracer by a chromogenic reaction using strptavidin-horseradish peroxidase conjugate. The rsulting signal is indicative of binding sites freed by the test peptide A test peptide that does not dissociate from TFPI during the incubation period yields a weaker signal compared to an analyte that dissociates completely. [00218] As with all binding agents and binding assays, one of skill in the art recognizes that the various moieties to which a binding agent should not detectable bind in order to be biologically (e.g., therapeutically) effective would be exhaustive and impractical to list. The_-rr the term "specifically binds" refers to the ability of a pepide to bind TFPI with greater affinity than it binds to an unrelated control protein that is not TFPL For example, the peptide may bind to TFPI with an affinity that is at least, 5, 0, 15, 25, 50, 100. 250, 500, 1000, or 1.000 times greater than the affinity for a control protein., in some embodiments, the peptide binds TFI with greater affinity than it binds to an "anti-target," a protein or other naturally occurring substance in humans to which binding of the peptide might lead to adverse effects. Several classes of peptides or proteins are potential anti-targets. Because TFPhinhibitory peptidesoexert their activity in the blood stream and/or at the endothelium, plasma proteins represent potential anti-targets. Proteins containing Kunitz domains (KDs) are potential anti-targets because KDs of different proteins share a significant similarity.

Tissue Factor Pathway Inhibitor-2 (TFPI-2) is highly similar to TFPLIa and, like TFPI-] a, contains KDs (Sprecher et aL, PAS, 9], 3353-3357 (1994)). Thus, in one aspect the peptide of the invention binds to TFPI with an affinity that is at least 5 10, 15, 25, or 50 times greater than the affinity for an anti-targetsuch as TFPE2. [002191 Optionally, the TFPI-binding peptide demonstrates one or more desired characteristics described herein, and the amino acid sequence of a peptide can be modified to optimize binding, stability and/or activity, if desired. An exemplary TFPN-binding peptide binds TPPI with a K of less than or equal to 20 nM and/or exhibits a binding affinity for TFPJ that is at least 100 tines greater than the binding affinity for an anti-target. Alternatively or in addition, the TFP-binding peptide enhances FX activation in the presence of TFPI with an ECO (as measured using any suitable assay, such as the assays described here) of less than or equal to 50 nM and/or enhances Lhrombin formation in the absence of Factor VII to at least about 20% (eig,40%) of the level of thrombin formationin plasma containing physiological levels of Factor VIRI Alternatively or in addition, the TFPbinding peptide achieves a desired level of plasma stability (e.g., 50% or more of a dose remains in plasma after 12 hours) and/or demonstrates a desired half life in vivo (e.g., at least two, three, four, five, six, seven, eight, nine, or ten hours), Alternatively or in addition, the TFPL-binding peptide exhibits a desired level of bioavailability, such as a desired level of bioavailability following subcutaneous administration (e.g., greater than or equal to 5%, 10%, 15%, 20%, 25%, 30%, or 50%) and/or demonstrates a desired level of TFPIinhibitory activity at a given dose in vivo. [00220] The invention further includes a method of inhibiting TFP- L The method comprises contacting TFP[ with a TFPIbinding peptide as described herein, Any degree of TFPi-activity inhibition is contemplated. For example, a TFPI-inhibitory peptide reduces TFPIinhibition of the extrinsic pathway at least about 5% (e g, at least about 10%, at least about 25%, or at least about 30%),. In some embodliments, the TFPL-inhibitory peptide reduces TFPI activity within the extrinsic pathway at least about 50%, at least about 75%, or at least about 90% compared to TFPI activity in the absence of the peptide. [0022t] In one aspect of the invention, TFPI-binding peptides arc used to detect and/or quantify TFPI in vivo or in vitro. An exemplary method of detecting and/or quartifying TFPI in a sample comprises (a) contacting a sample with a TFP -binding peptide of the invention, and (b) detecting binding of the TFPbinding peptide to TFPL on [002223 The invention further includes a method for targeting biological structures (including, but not limited to, cell surfaces and endothelial lining) where TFPI is located. The method comprises contacting the biologicalstreture (e g. including without limitation, a cell displaying TFPI on the cell surface ith a TFP-binding peptide described herein, optionally conjugated to a moiety that adds additional functionality to the peptide. The moiety can be a dye (such a a fluorescence dyea radionuclide or a radionuclide-containing complex, 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 a TFPI displaying cell within the mammal Optionallythe method further comprises detecing binding of dhe TFPIbinding peptide to TFP. The method is useful for therapy and diagnosis of disease where TFPIis a suitable diagnostic marker or TFP-expressing cells are a target for a therapeutic approach. [00223] Peptide-TIFPI complexes are directly or indirectly detected, Detection moieties are widely used in the art to identify biological substances and include, for example, dye (e.g., fluorescent dye), radionuclides and radionuclide-containing complexes, and enzymes. In some aspects, peptide-TFPI binding is detected indirectly. In this regard, the peptide is optionally contacted with an interaction partner that binds the peptide of invention without significantly interfering 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 nimetics, aptamers, streptavidin, avidin, neutravidin, and spiegelmers. Optionally, the interaction partner comprises a detection moiety to facilitate detection of an interaction partner-peptide complex. The TFPlbinding peptide is, in some embodiments, modified to facilitate binding of an interaction partner. For example, in one aspect, the TFPI-binding peptide is conjugated to biotin, which is bound by an interaction partner comprising streptavidin. An exemplary interaction partner comprises strepavidin fused to horseradish peroxidase, which is detected in, e.g., an ELISA-like assay. Alternatively, the TFPI-binding peptide is modified to include an antibody epitope, and binding of the corresponding antibody to the peptide-TFPI complex is detected. Methods of detecting, e.g, antibodies and fragments thereof, are well understood in the art. [002241 Peptide-TFPI complexes and interaction partner-peptide complexes are identified using any of a number of methods, such as, but not limited to, biochemical assays (e.g., enzymatic asays)pectroscopy(g, detection based on optical densityfluorescence FRET, BRET, TR-FRET, fluorescence polarization, electrochemohuminescence, or NMR), positron emission tomography (PET), and single Photon Emission Computed Tomography (SPECT). Detectable moieties that facilitate fluorescence detection of peptide-TEPI complexes or interaction partner-peptide complexes include, but are not limited to, fluorescein, Alexa Fluor@ 350, Marina BlueTM, Cascade YellowTM, Alexa Fluor@ 405, Pacific BlueTM, Pacific OrangeTM, Alexa Fluor@ 430, Alexa luor® 488, Oregon Green® 488, Alexa Fluor@ 500, Oregon Green@ 514, Alexa Fluor@ 514, Alexa Fluor@ 532, Alexa Fluor@ 555, Tetramethylrhodamine, Alexa Fluor@ 546, Rhodamine B, Rhodamine RedM-X, Alexa Fluor@ 568, Alexa Fluor® 594, Texas Red@, Texas Red@-X, Alexa Fluor@ 610, Alexa Fluor@ 633, Alexa Fluor® 635, Alexa Fluor@ 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor@ 700, Alexa Fluor@ 750, B-Phycoerythrin, R-Phycoerythrin, Allophycocyanin, BODIPY@, Cy3, Cy5, TAMRA, and fluorescent proteins (GFP and derivatives thereof). An example of a TFPI-binding peptide comprising a fluorescent detection moiety is JBT2454 (FAM-Ttds- FQSKpNVHVDGYFERL-Aib-AKL--NH2 (SEQ ID NO: 4171)), which is labeled with 5,6-cartoxyfluoresceine. [00225] Radioactive labels also are used to detect biological materials (e g., TFPI, TFP binding peptides, or TFPbinding peptide-TFPI complexes), and, in some instances are attached to peptides or interaction partners using a chelator, such as (but not limited to) EDTA (ethylene diamine tetra-acetic acid), DTA (diethylene triamine pentaacetic acid), CDTA (cyclohexyl 1,2-diamine tetra-acetic acid), EGTA (ethyleneglycol-O,Ob'-is(2 aninoethyl)-N,N,N'N'-tetra-acetic), HBED (N,N-bis(hydroxybenzyl)-ethylenediamine- N,Nt diacetic acid), TTIA (triethylene tetramine hexa-acetic acid), DOTA (1,4,7,10 tetraazacyclododecane-N,N',N",N'"-tetra-acetic acid), HEDTA (hydroxyethyldiamine triacetic acid), or T[ETA (l,4,8-tetra-azacycdotetradecaneN.N'N",N"' -tetra-acetic acid). Examples of radioactive labels include 99 1e, 2 Pb, tGa, 6 Ga, bGa As, 11 In, 1 3 "In JJ,~ '' 6', 6 1 52m qc,186R '5 77 90~ 67 1, "In, Ru, "Cu, Cu, e, smn 5 1 CissRe. Re As Y. Cu, Etr, "rSn iSn, 'TTe, NIPr, Pr, 'IAu Au Th, 1 Tb, 'Pd, 'y, "Pm, Pm "Sm ad, Ho 2 ' 'Y L ' 0 sRh and " 1 Ag. Paramagnetic metals also are detectable moieties that are suitable for attachment to TFPI-inding peptides or interaction partners, optionally via chelator complex Examples of paramagnetic metals include, for example, Cr, Mn, Fe, Co, Ni, Cu, Pr, Nd, Srn, Yb, Gd, Tb, Dy, Ho, and Er. [00226] TEP-binding peptides, themselves, are, in some aspects, modified to include one or more amino acids with detectable substituents or nuclides. In this regard, in one embodiment, the TFPI-binding peptide comprises at least one amino acid comprising a 0', detectable isotope (e.g., 13C, 14C 35S, 3H 180 or 15N), and/or an amino acid that is halogenated with, e.g1, m Br,

T

Br, r or ,Br. Amino acids suitable for halogenation include, but are not limited to, tyrosine and tryptophan. [00227] The invention also provides a method for diagnosing a subject suffering from a disease or disorder, or at risk of suffering from a disease or disorder, wherein the disease or disorder is associated with or caused by aberrant TFP1 activity. The method comprises administering to the subject the TFPI--binding peptide and detecting the TFPI-peptide complex. In some instances, the peptide is conjugated to a detectable moiety, and the method comprises detecting the detectable moiety. Exemplary detectable moieties are described herein. In other instances, the method comprises administering to the subject a TEN-binding peptide interaction partner that binds the TFPI-binding peptide, and detecting the interaction partner. If desired, the interaction partner comprises or is conjugated to a detectable moiety, and the detectable moiety is detected. The presence of the detectable moiety indicates the presence of TFP, thereby allowing diagnosis of a disease or disorder associated with TFPI (e g., a disease or disorder which (i) can be treated by inhibiting TEPI or (ii) comprises symptoms which can be ameliorated or prevented by inhibiting TFPI) If administration of the peptide to the subject is not desired, a biological sample is obtained from the subject, contacted with the TFPI-binding peptide as described herein, and TFPI-peptide complexes are detected, [00228] The peptides of the invention bind TFPI and, therefore, are useful for purifying TEIN or recoinbinant TFPI from a biological sample (e.g, a biological fluid, such as serum), fermentation extract, tissue preparations, culture medium, and the like, The invention includes methods of using the TEIN-binding peptide in the commercial production of TFP! or in a method of characterizing TFPI molecules. For example, the invention includes a method of purifying TFPL The method comprises contacting a sample containing TFPI with a peptide as defined herein under conditions appropriate to form a complex between TFPI. and the peptide; removing the complex from the sample; and, optionally, dissociating the complex to release TFP. Exemplary conditions appropriate to form a complex between TFPI and the peptide are disclosed in the Examples, and such conditions can be easily modified to dissociate the TFPI-peptide complex. In some embodiments, the peptide is immobilized to a support, e.g., a solid support, to facilitate recovery of IT[Pl. For example, in one embodiment, the peptide is immobilized to chromatography stationary phase (e.g., silica, affinity chromatography beads, or chromatography resins), a sample comprising TEE! is applied to the stationary phase such that TFPI-peptide complexes are formed, the remainder of the sample is removed from the stationary phase, and TFPI is ehuted from the stationary phase. In this regard, the peptides of the invention are, in one aspect, stable for use in affinity chromatography techniques. [002291 A method of enhancing thronibin formation in a clotting factor-deficient subject also is provided. The method comprises administering to the subject a peptide provided herein under conditions effective to inhibit TFPL In this regard, the TFPI-inhibitory peptide is administered in an amount and under conditions effective to enhance thrombin formation in the subject. By "clotting factor-deficient" is meant that the subject suffers from a deficiency in one or more blood factors required for thrombin formation, such as FVIII, FIX, or FXI Indeed, in one embodiment, the subject is deficient in FVM. Alternatively or in addition, the subject is deficient in Factor IX, Clotting factor deficiencies are identified by examining the amount of factor in a clinical sample. Practitioners classify hemophilia according to the magnitude of clotting factor deficiency. Subjects suffering from mild hemophilia have approximately 5% to 30% of the normal amount (1 U/ml) of Factor VIII or Factor X. Moderate hemophilia is characterized by approximately 1% to 5% of normal Factor VIN, Factor IX, or Factor XI levels, while subjects suffering forn severe hemophilia have less than I% of the normal amount of Factor VIT Factor IX, or Factor XI Deficiencies can be identified indirectly by activated partial thrombopiastin time (APTT) testing APTT testing measures the length of time required for a blood clot to form, which is longer for patients with Factor VII Deficiency (hemophilia A), Factor IX Deficiency (hemophilia B), and Factor XI Deficiency (hemophilia C) compared to patients with nonnal clotting factor levels, Almost 100% of patients with severe and moderate Factor VI deficiency can be diagnosed with an APTT. The invention further includes enhancing thrombin fonnation in a subject that does not suffer from a clotting factor deficiency. The method comprises administering to a subject (e.g., a subject comprising normal, physiological levels of clotting factor) a peptide provided herein under conditions effective to enhance thrombin formation. [00230] In one aspect, the TFPI-inhibitory peptide is used for increasing blood clot formation in a subject, The method of increasing blood clot formation comprises administering to the subject a peptide described herein in an amount and under conditions effective to increase blood clot formation. It will be appreciated that the method need not completely restore the coagulation cascade to achieve a beneficial (e.g., therapeutic) effect. Any enhancement or increase in thrombin or blood clot formation that reduces the onset or severity of symptoms associated with clotting factor deficiencies is contemplated. Methods 0 ,4 of determining the efficacy of the method in promoting thrombin formation and blood clotting are known in the art and described herein. [00231] The invention further includes a method of treating a blood coagulation disorder in a subject, the method comprising administering to the subject one or more TFPI-inhibitorv peptides, such as any one or more of the peptides described herein, in an amount and under conditions effective to treat the blood coagulation disorder in the subject, In one aspect, the peptide is a recombinant or synthetic peptide that inhibits TFP activity. "Coagulation disorders" include bleeding disorders caused by deficient blood coagulation factor activity and deficient platelet activity. Blood coagulation factors include, but are not limited to, Factor V (FV) FY11, FINl, FDX FX, FXL FXHI, FH (responsible for hypoprothrombinemia), and von Willebrand's factor. Factor deficiencies are caused by, for instance, a shortened in vivohalf life of the factor, altered binding properties of the factor, genetic defects of the factor, and a reduced plasma concentration of the factor. Coagulation disorders can be congenital or acquired. Potential genetic defects include deletions, additions and/or substitution within a nucleotide sequence encoding a clotting factor whose absence, presence, and/or substitution, respectively, has a negative impact on the clotting factor's activity. Coagulation disorders also stem from development of inhibitors or autoimmunity (eg., antibodies) against clotting factors. In one example, the coagulation disorder is hemophilia A ltematively, the coagulation disorder is hemophilia B or hemophilia C. [00232] Platelet disorders are caused by deficient platelet function or abnormally low platelet number in circulation, Low platelet count may be due to, for instance, underproduction, platelet sequestration, or uncontrolled patent destruction. Thrombocytopenia (plateict deficiencies) may be present for various reasons, including chemotherapy and other drug therapy, radiation therapy, surgery, accidental blood loss, and other disease conditions. Exemplary disease conditions that involve thrombocytopenia are: aplastic anemia; idiopathic or immune thrombocytopenia (TP), including idiopathic thrombocytopenic purpura associated with breast cancer; HIV-associated UP and TV related thrombotic thrombocytopenic purpura; metastatic tumors which result in thrombocytopenia; systemic lupus erythematosus, including neonatal lupus syndrome splenomegaly; Fanconi's syndrome; vitamin B 12 deficiency; folic acid deficiency; May Regglin anomaly; Wiskott-Aldrich syndrome; chronic liver disease; myelodyspiastic syndrome associated with thronbocytopenia; paroxysmal noctural hemoglobinuria; acute profound thrombocytopenia following C7E3 ab (Abeiximab) therapy; alloimmune thrombocytopenia, including maternal aloimmune thrombocytopenia; thrombocytopenia associated with andtphospholipid antibodies and thrombosis; autoimmnune thrombocytopenia; drug-induced immune thrombocytopenia, including carboplatin-induced thrombocytopenia and heparin-induced thrombocytopenia; fetal thrombocytopenia; gestational thrombocytopenia; Hughes' syndrome; lupoid thrombocytopenia; accidental and/or massive blood loss; myeloproliferative disorders; thrombocytopenia in patients with malignancies; thrombotic thrombocytopenia purpura, including thrombotic microangiopathy manifesting as thrombotic thrombocytopenic purpura/hemolytic uremic syndrome in cancer patients; post transfusion purpura (PTP); autoimmune hemolytic anemia; occult jejunal diverticulum perforation; pure red cell aplasia; autoimmune thrombocytopenia; nephropathia epidemica; rifampicin-associated acute renal failure; Paris-Trousseau thrombocytopenia; neonatal alloimmune thrornbocytopenia; paroxysmal nocturnal hemoglobinuria; hematologic changes in stomach cancer; hemolytic uremic syndromes (e.g., uremic conditions in childhood); and hematologic manifestations related to viral infection including hepatitis A virus and CMV associated thrombocytopenia. Platelet disorders also include, but are not limited to, Von Willebrand Disease, paraneoplastic platelet dysfunction, Glanzman's thrombasthenia, and B~eitard 2 Soier disease. Additional bleeding disorders amenable to treatment with a TFPI inhibitory peptide include, but are not limited to, hemorrhagic conditions induced by trauma; a deficiency in one or i ore contact factors, such as FX, FX1L, prekallikrein, and high molecular weight kininogen (IMWK); vitamin K deficiency; a fibrinogen disorder, including afibrinogenemia, hypofibrinogenemia, and dysfibrinogenemia; and alpha2-antiplasmin deficiency. In one embodiment, the TFPI-inbibitory peptide is used to treat excessive bleedingsuch as excessive bleeding caused by surgery, trauma, intracerbral hemorrhage, liver disease, renal disease, thrombocytopenia, platelet dysfunction, hematomas, internal hemorrhage, hemarthroses, hypothennia, menstruation, pregnancy, and Dengue hemorrhagic fever. All of the above are considered "blood coagulation disorders" in the context of the disclosure. [002331 In one aspect, the TFPI-inhibitory peptide of the invention is used to reverse the effects (in whole or in part) of one or more anticoagulants in a subject. Numerous anticoagulants are known in the art and include, for instance, heparin; coumarin derivatives, such as warfarin or dicumarol; TFPI; AT III; lupus anticoagulant; nematode anticoagulant peptide (NAPc2); FVIIa inhibitors; active-site blocked FVIla (FVHai); active-site blocked FIXa (FIXai); FIXa inhibitors; FXa inhibitors, including fondaparinux, idrapainux, DX 9065a, and razaxaban (DPC906); active-site blocked FXa (FXai); inhibitors of FVa or FVIa, including activated protein C (APC) and soluble thrombomodulin; thrombin inhibitorsincluding hirudin bivalirdin, argatrobanand ximelagatran; and antibodies or antibody fragments that bind a clotting faor (egV FIX, FX, FXil FT, FXI, FXI, von Willebrand factor prekaiiikreinohigh molecular weight kininogen (HMW.K)). [002341 As used herein, "treating" and "treatment" refers to any reduction in the severity and/or onset of symptoms associated with a blood coagulation disorder. Accordingly, "treating" and "treatment" includes therapeutic and prophylactic measures. One of ordinary skill in the art will appreciate that any degree of protection from, or amelioration of, a blood coagulation disorder or symptom associated therewith is beneficial to a subject, such as a human patient. The quality of life of a patient is improved by reducing to any degree the severity of symptoms in a subject and/or delaying the appearance of symptoms. Accordingly, the method in one aspect is performed as soon as possible after it has been determined that a subject is at risk for developing a blood coagulation disorder (e.g., a deficiency in a clotting factor (e.g., FVII, FIX, or FXI) is detected) or as soon as possible after a blood coagulation disorder (e.g., hemophilia A, hemophilia B, or hemophilia C) is detected. In an additional aspect, the peptide is administered to protect, in whole or in part, against excessive blood loss during injury or surgery. [00235] In view of the above, the invention provides a peptide for use in a method for the tmatment of a subject, such as a method for the treatment of a disease where the inhibition of TFPI is beneficia. In one aspect, the disease or disorder is a blood coagulation disorder The subject is suffering from a disease or disorder or is at risk from suffering from a disease or disorder (or adverse biological event, such as excessive blood loss). The method comprises administering to the subject the peptide of the invention in an amount and under conditions effective to treat or prevent, in whole or in pail, the disease or disorder. The invention further provides a peptide for use in the manufacture of a medicament. For example, the peptide can be used in the manufacture of a medicament for the treatment of a blood coagulation disorder, as described in detail herein. [002361 In some embodiments, it is advantageous to administer to a subject a nucleic acid comprising a nucleic acid sequence encoding a TFPI-binding peptide (e.g., TFPI-inhibitory peptide) of the invention, Such a nucleic acid, in one aspect, is provided instead of, or in addition to, a TFPI-inhibitory peptide. Expression vectors, nucleic acid regulatory sequences, administration methods, and the like, are further described herein and in U.S. Patent Publication No. 20030045498. 0o7 [00237j A particular administration regimen for a particular subject will depend, in part, upon 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 particular ailment being treated, considerations relevant 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, timing of administration, route of administration, dosage regimen) are sufficient to affect the desired biological response over a reasonable time frame. Dosage typically depends upon a variety of factors, including the particular TFPL-inhibitory peptide employed, the age and body weight of the subject, as well as the existence and severity of any disease or disorder in the subject. The size of the dose also will be determined by the route, timing, and frequency of administration. Accordingly, the clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect, and con ventonal range-finding techniques are known to those of ordinary skill in the art. Purely by way of illustration, in one aspect, the method comprises administering, e.g., from about 0.1 gg/kg to about 100 mg/kg or more, depending on the factors mentioned above. In other embodiments, the dosage may range from I pg/kg up to about 75 mg/g; or 5 pg/kg up to about 50 mg/g; or 10 pgg up to about 20 mg/g. In certain embodiments, the dose comprises about 0.5 mg/kg to about 20 mg/kg (eg., about I 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 mgkg 4.5 mg/g, 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 peptide. Given the chronic nature of many blood coagulation disorders, it is envisioned that a subject will receive the TFPI-inhibitory peptide over a treatment course lasting weeks, months, or years, and may require one or more doses daily or weekly, In other embodiments, the TFPI-inhibitory peptide is administered to treat an acute condition (eg., bleeding caused by surgery or trauma, or factor inhibitor/autoimmune episodes in subjects receiving coagulation replacement therapy) for a relatively short treatment period, e.g. one to 14 days. [00238] Suitable methods of administering a physologically-acceptable composition, such as a pharmaceutical composition comprising a peptide described herein, are well known in the art. Although more than one route can be used to administer a peptide, a particular route can provide a more immediate and more effective reaction than another route. Depending on the circumstances, a pharmaceutical composition is applied or instiled into body cavities, absorbed through the skin or mucous membranes, ingested, inhaled, and/or introduced into circulation. In one aspect, a composition comprising a TFPI-inhibitory peptide is administered intravenously, intraarterially, or intraperitoneally to introduce the peptide of the

PQ

invention into cireuladio Non-intravenous administration also is appropriate, particularly with respect to low molecular weight therapeutics. In certain circumstances, it is desirable to deliver a pharmaceutical composition comprising the TFPI-inhibitory peptide orally, topically, sublingually, vaginally, rectally, pulmonary; through injection by intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraportal, intralesional, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intranasal, urethral, or enteral means: by sustained release systems; or by implantation devices. If desired, the TFPI-inhibitory peptide is administered regionally via intraarterial or intravenous administration feeding a region of interest, e.g., via the femoral artery for delivery to the leg In one embodiment, dhe pepride is incorpornted into a microparticle as described in, for example, U.S. Patents 5,439,686 and 5,498,421, and U.S. Patent Publications 2003/0059474, 2003/0064033 2004/0043077, 2005/0048127, 2005/0170005 2005/042205 2005142201, 2005/0233945, 2005/0147689. 2005/0142206 2006/0024379, 2006/0260777, 2007/0207210, 2007/0092452, 2007/028 031, and 2008/0026068. Alternatively, the composition is administered via implantation of a membrane, sponge, or another appropriate material on to which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device in one aspect is implanted into any suitable tissue, and delivery of the desired molecule is in various aspects via diffusion, timed-release bolus, or continuous administration, i other aspects; the TFPI inhibitory peptide is administered directly to exposed tissue during surgical procedures or treatment of injury, or is administered via transfusion of blood procedures. Therapeutic delivery approaches are well known to the skilled artisan, some of which are further described, for example, in US. Patent No. 5,399,363. [00239] To facilitate administration, the TFPI-binding peptide (e.g., TFPI-inhibitory peptide) in one embodiment is formulated into a physiologically-acceptable composition comprising a carrier (i.e., vehicle, adjuvant, buffer, or diluent). The particular carrier employed is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the peptide, and by the route of administration, Physiologically-acceptable carriers are well known in the art. Illustrative pharmaceutical forms suitable for injectable use include without limitation sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U.S. Patent No 5,466,468) Injectable formlations are further described in, e g., Phannaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia. Pa., Banker and Chalmers. eds, pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, Wo1 4th ed, pages 622-630 (1986)). A pharmaceutical composition comprising a peptide provided herein is optionally placed within containers along with packaging material that provides instuctions regarding the use of such pharmaceutical compositions. Generally, such instructions include a tangible expression describing the reagent concentration, as well as, in certain embodiments, relative amounts of excipient ingredients or diluents that may be necessary to reconstitute the pharmaceutical composition. [00240] When appropriate, the TFPI-binding peptide (e.g.. TFPI-inhibi tory peptide) of the invention is administered in combination with other substances and/or other therapeutic modalities to achieve an additional or augmented biological effect. Co-treatments include, but are not limited to, plasma-derived or recombinant coagulation factors, hemophilia prophylaxis treatments, immunosuppressants, plasma factor-inhibiting antibody antagonists (i.e., anti-inhibitors), antifibrinolytics, antibiotics, hormone therapy, anti-inflammatory agents (e.g., Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) or steroidal anti-inflarnmatory substances), procoagulants, and pain relievers. In one aspect, the method is an adjunct therapy to traditional replacement factor treatment regimens involving administration of, e.g., FXITI, FXII, FXI (e.g., HEMOLEVEN@ (Laboratoire francais du Fractionnement et des Biotechnologies, Les Ulis, France) and FXI concentrate (BioProducts Laboratory, Elstree, Herfortidshire. UK)), FX, FIX (e.g., BENEFIX Coagulation Factor IX (Wyeth, Madison, NJ); ALPHANINE® SD (Grifols, Los Angeles, CA); MONONINE® (CSL Behring, King of Prussia, PA); BEBULTN-VHTM (Baxter, Deerfield, IL); PROFILNINEC SD (Grifols, Los Angeles, CA); or PROPLEX TTM (Baxter, Deerfield, IL)), FVIH (e.g., ADVATErM (Baxter, Deerfield, IL); HELIXATE@- FS (CSL Behring, King of Prussia, PA); REFACTO@ (Wyeth, Madison, NJ), XYNTHA TM (Wyeth, Madison, NJ). KOGENATE@ and KOGENATE® FS (Bayer, Pittsburgh, PA); ALPHANATE@ (Grifols, Los Angeles, CA); HEMOPHIL MTM (Baxter, Deerfield, IL); KOATE@-DVI (Talecris Biotherapeutics-USA, Research Triangle Park, NC); or MONARC-MTM (Baxter, Deerfield, IL)), FVIa (e.g., NOVOSEVEN® FVIIa (Novo Nordisk, Princeton, NJ) and FVH concentrate (Baxter Bioscience, Vienna, Austria, or BioProducts Laboratory, Elstree, Hertfordshire, UK)), FV, FVa, FIT, and/or FIH, to a subject. In some instances, the subject also receives FEIBA VH ImmunoTM (Baxter BioScience, Vienna, Austria), which is a freeze-dried sterile human plasma fraction with Factor VIII inhibitor bypassing activity. FEIBA VH ImmunoTM contains approximately equal units of Factor VHII inhibitor bypassing activity and Prothrombin Complex Factors (Factors H1, V11, IX, and X and protein C). Other exemplary co-treatments include, but are not limited to, prekaikrein, high molecular weight kininogen (HMWK), Von WilIebrand's factor. Tissue Factor, and thrombin. Akeratively or in addition, the TFPI-inhibitory peptide is co formulated with one or more different TFPI-inhibitory peptides. In one aspect, administration of the TFPI-binding peptide allows a reduction in the dose of co-therapeutic required to achieve a desired biological response. [00241] The invention thus includes administering to a subject a TFPI-binding peptide (e.g., TFPlI-inhibitory peptide) of the invention (or multiple TFPI-inhibitory peptides), in combination with one or more additionally suitable substances(s), each being administered according to a regimen suitable for that medicament, Administration strategies include concurrent administration (i.e., substantially simultaneous administration) and non--concurrent administration (ie., administration at different times in any order, whether overlapping or not) of the TFPI-inhibitory peptide and one or more additionally suitable agents(s) It will be appreciated that different components are optionally administered in the same or in separate compositions, and by the same or different routes of administration. [00242] In some embodiments, the peptide of the invention is conjugated to a moiety, e.g, a therapeutic or diagnostic moiety, such as the detection moieties and co-treatments described above. Alternatively or in addition, the peptide is administered in combination with an interaction partner (e.g., an antibody, antibody fragment, anticalin, aptamer. or spiegelmer) that (a) binds the peptide and (b) is therapeutically active and/or is linked to a moiety that provides additional functionality to the interaction partner (e.g, a thempeutic, diagnostic, or detection agent). Suitable moieties include, but are not limited to, photosensitizers, dyes, radionuclides, radionuclide-containing complexes, enzymes, toxins, antibodies, antibody fragments, and cytotoxic agents, and, in some instances, the moiety possesses therapeutic activity (i.e., achieves an advantageous or desired biological effect). The peptide conjugates or peptdde-interaction partner pair is suitable for use in any of the methods described herein, such as methods of treating a subject suffering from a disease or disorder or at risk of suffering from a disease or disorder. [00243] The invention further provides a method for identifying a TFPI-binding compound, such as a TFPI-binding peptide. In one aspect, the method comprises (a) contacting a peptide comprising TFPI Kunitz domain I (KD 1) with a TFPI-binding peptide described herein and a test compound under conditions that allow formation of KDI-TFPI binding peptide complexes. The method further comprises (b) measuring KDI-TFPI-binding peptide complexes formed in step (a), and (c) comparing the number of KD1 -TFPI-binding peptide complexes formed in die presence of the test compound with the number of KDI- TFN-binding peptide complexes formed in the absence of the test compound. A reduction in ni1 the number of KDI.-TFPI-binding peptide complexes formed in the presence of the test compound compared to thenumber of KAD 1 .T-FPI--inding peptde complexes formed in the absence of the test compound indicates that theltest compound isa P-binding compound In one aspect, the method further comprises forming KDI -TFPI-binding complexes in the absence of the test compound for comparison in step (c), although this is not required inasmuch as the infonnation may be obtained separately (e.g., from previously prepared reference standards). [00244] KD1, the TPI-b inding peptide, and the test compound are combined simultaneously or sequentially, optionally with washing steps before and/or after addition of the TFPI-binding peptide and/or the test compound. In one embodiment, the peptide comprising KDI is contacted with a TFPI-binding peptide described herein under conditions that allow formation of KD1-TFPI--binding peptide complexes, unbound TFPI-binding peptide is removed, and the remaining KD-peptide complexes are contacted with a test compound. Displacement of the TFPI-binding peptide from the TFPI-peptide complexes is detected, and indicates that the test compound is a TFPI-binding compound. Displacement is detected by, for example, measuring the number of KDI-TFPI-binding peptide complexes before and after exposure to the test compound, [00245] KDI-TFP-binding peptide complexes are detected and/or measured (quantified) using any suitable detection means, including detection means known in the art for detecting peptides in a sample. For example, in one embodiment of the invention, the TFPI-binding peptide comprises a label that generates a signal. Exemplary labels are described herein and include, e.g., radionuclides, fluorescent dyes, isotopes, enzyme substrates, and enzymes. The method comprises measuring signal generated by KDI-TFPI-binding peptide complexes and comparing signal. generated by KDl-TFPI-binding peptide complexes formed in the presence of the test compound with signal generated by KD -TFPI-binding peptide complexes formed in the absence of the test compound. A reduction in signal from a sample comprising KDI TFPI-binding peptide complexes exposed to test compound (compared to signal generated by a similar sample of KDI-TFP-binding peptide complexes not exposed to the test compound) indicates that complex formation has been inhibited or disrupted, and that the test compound is a TFPI-binding compound, [002461 The invention also provides a method of identifying a TFPI-binding compound that interferes with TFPI-FXa interactions. The method is predicated, at least in part, on the surprising discovery that TFPI KDI binds to an exosite of FXa and contributes to TFPI's inhibition of FXa activity, ln one aspect, the method comprises contacting a peptide fil consisting essentially of KDI (i.e. a peptide comprising K D1 in the absence of KD2) with FXa in the presence of a test compound under conditions that allow binding of K11 to FXa. The method further comprises comparing KD1-FXa binding in the presence of the test compound with KOIFXa binding in the absence of the test compound. A decrease in KDI FXa binding in the presencofe test compound compared to KD1-FXa binding in the absence of the test compound indicates that the test compound is a TFP-binding compound. KDI-FXa binding can be detected and/or quantitated using any methodsuch as the methods described herein. For example, KDI or FXa is labeled, and the signal generated by KD1-FXa complexes exposed to the test compound is compared to the signal generated by KD1-FXa complexes not exposed to the test compound. [002471 The methods of the invention to identify TFPI-binding compounds are particularly amenable to the various high throughput screening techniques known in the art, Any "test compound" (e.g, small molecule, peptide, protein (such as an antibody or fragment thereof), peptidomimetic, or polynucleotide (DNA or RNA)) is suitable for screening using the methods described herein. If desired, a collection, population, or library of test compounds is screened for TFPI binding (and, optionally, anti-TFPI activity) using the methods described herein. There are a number of different libraries used for the identification of TFPI inhibitors, including, but not limited to, chemical libraries, natural product libraries, and combinatorial libraries comprising peptides and/or organic molecules. A chemical library, in some aspects, consists of structural analogs of known compounds or compounds that are identified as "hits" or "leads" via other screening methods. Natural product libraries are collections of substances isolated from or produced by microorganisms, animals, plants, or marine organisms, Combinatorial libraries are composed of large numbers of peptides or organic compounds, typically as a mixture. The methods described herein also are useful for screening a display or nucleic acid library, such as a yeast display library, a bacterial display library, a phage display library, a ribosome display library, an mRNA display library, a RNA library, or a DNA library, One method of screening a display library is exemplified in Example 1. High throughput screening methods embraced by the invention include automated procedures allowing screening of tens to hundreds of thousands of test compounds. [00248] In another aspect, the inventive method for identifying a TFPI-binding compound comprises contacting a peptide comprising (or consisting of) KD1 with a test compound, and detecting binding of the test compound to a TFPI binding site defined by KD I amino acid residues corresponding to human TFPI residues Phe2S, Lys29, Ala30, Asp32, 1e46, Phe47, C, -1 A 1 0 ,If l-,P , and Ile55, such as a binding site defined by human TFPI residues Ala27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, Ile38, Ile46, Phe47, and Ie5 5. In one embodiment, the binding site is defined by amino acid residues corresponding to human TFPJ residues Ala27, Phe28, Lys29. Ala30, Asp31, Asp32, Lys36, Ala37, Ile38, Phe44, 11e46, Phe47, and Ile55. The binding site corresponds to the TFPI binding site of JBT1857, a TFPI-binding peptide that inhibits TFPI activity in a number of functional assays. [00249] The TFPI binding site amino acid residues described herein are in reference to the human TFPI amino acid sequence, and the numbering refers to the position of the recited amino acid in relation to the N-terminus of human TFPL Merely for the purpose of illustrating the position of the TFPT binding site, the amino acid sequence of a fragment of human TFPI comprising KDl is provided as SEQ ID NC) 4234 (DSEEDEEHI-IfUTDTEILiPPLKLMHSFCAFKAI)[)GPCKAIMKRFFFNIFTRQCEEFIGG CEGNQNRFESLEECKKMCTRDNA (amino acids 26-75 encoding KDI are indicated in bold)). Corresponding amino acids of other TFPI polypeptides (such as TFPI polypeptides from different organisms, or TFPI polypeptide fragments) are identified, for example, by aligning a polypeptide's amino acid sequence with SEQ I) NO: 4234. While, in one embodiment, the peptide comprising TFPI KDI does not comprise other regions of the TFPI protein responsible for TFPI activity, other embodiments entail the use of a peptide comprising amino acids 1-160 of human TFPI (comprising KD1 and KD2) or comprising full length human TFPI (containing KDI-KD3). [00250] Binding of a test compound to the TFPI binding site defined herein is detected using any of a number methods, including the detection methods described herein, An exemplary method for detecting binding employs nuclear magnetic resonance (NMR) to recognize chemical shifts at amino acid residues within the TFPI binding site. Chemical shifts at TFPI amino acid positions 28-30, 32, 46, 47, and 55, and optionally positions 27, 31, 36-38, and 44, denotes interaction of the test compound with these amino acid contact points on TFPI. To determine the presence or absence of chemical shifts at particular amino acids resulting from test compound binding, NMR data obtained from the KDI -test compound complex is compared to NMR data obtained from free KD1 peptide. Use of NMR to detect binding between a test compound and TFPI KDI is further described in the Examples. [00251] Alternatively, binding of a test compound to the TFPI-binding site defined herein is determined indirectly by detecting alterations in the ability of TFPI KDI to interact with its natural binding partners, e.g., FVIIa or FXa. In this regard, the method comprises contacting the peptide comprising TFP KID I with FVIla in the presence of the test compound urder conditions that allow binding of KDI to FVIa, and KDI-FVTa binding is compared with KDl FVIla binding in the absence of the test compound. Alternatively or in addition, the method comprises contacting the peptide comprising TFPI KD1 with FXa in the presence of the test compound under conditions that allow binding of KI) I to FXa, and comparing KDI FXa binding in the presence of the test compound with KDI-FXa binding in the absence of the test compound. Optionally, the peptide comprising KDI also comprises KD2, and the method comprises contacting the peptide with FXa in the presence of a test compound under conditions that allow binding of KD2 to FXa, and KD2-FXa binding is compared with KD2 FXa binding in the absence of the test compound. A decrease in KDl-FVIa binding, KD1 Fa binding, or KD2-FXa binding in the presence of the test compound (compared to KD1 FVIIa binding, KD1-FXa binding, or KD2-FXa binding in the absence of the test compound) indicates that the test compound is a TFPI-binding compound. The method optionally comprises contacting KD1 andor KD2 to FVHa and/or FYa in the absence of the test compound as a reference for comparing binding in the presence of the test compound. [00252] KD binding to FVJ1a or FXa is determined and/or quantified using any suitable method for detecting protein-protein interactions, such as the methods described herein using detectable labels. Binding of the test compound to the TFPI binding site is, alternatively, detected using an enzymatic assay. FVI1a or FXa enzymatic activity is a suitable surrogate for evaluating binding of the proteins to TFPI KDI or KD2; test compounds that bind the TFPI- binding site defined herein inhibit TFPI activity, resulting in increased FVHa and FXa activity. Enzymatic assays for evaluating FV1a or FXa activity are described in detail herein. [00253] The invention further includes compounds identified as TFPLbinding compounds in the methods of the invention, as well as compositions comprising one or more identified compounds. Methods for isolating or purifying a compound. such as TFPbbinding compound (eg. a TFPI-bindig peptide) identified as described herein are known in the art and described above, In soni aspects, TFPIbinding compounds identified as described herein are TFPI inhibitors that downregulate or ablate one or more TFPI activities. In one embodiment, the invention includes a method for purifying a compound that inhibits FXa activity. The method comprises contacting a peptide comprising TFPI KD with a compound under conditions that allow formation of compound-KDl complexes, removing unbound compound, and dissociating the compound-KD1 complexes to release the compound, which binds TFPI Use of a TFPI inhibitor identified and/or purified as described herein for the manufacture of a medicament, such as a medicament for treating a blood coagulation fl< disorder, is provided, as well as a method for treating a subject suffering from a disease or at risk of suffering from a disease comprising administering the TfPI inhibitor to the subject, [002541 In addition, a method of inhibiting human TFIN is provided, wherein the method comprises contacting human TFP with an inhibitor that binds human TFPI at a binding site defined by amino acid residues Phe28, Lys29, Ala3O, Asp32, le46, Phe47, and 1e5. Another aspect of the invention includes a method for treating a subject suffering from a disease or at risk of suffering fron a disease. The method comprises administering to the subject an inhibitor that binds human TFPI at a binding site defined by amino acid residues Phe2S, Lys29, Ala3O, Asp32, 11e46, Phe47, and e55. In one aspect, the human TFPI binding site is defined by amino acid residues Ala27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, lle38, 1e46. Phe47, and 1155, such as a binding site defined by amino acid residues Ala27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, Ala37, Ue38, Phe4, Ie46, Phe47, and le55. Any inhibitor that contacts the TPI binding site defined herein and inhibits (downregulates or ablates) one or more TFPI activity is suitable for use in the context of the method. The TFP inhibitor is, optionally, a TFI-binding peptide, such as a TFPI-binding peptide having the characteristics described herein. [00255] T1he invention further includes computer storage media and methods for modeling candidate TEP-compounds in the TFPI binding site defined herein, Three dimensional (31)) modeling of proteins can be used in conjunction with 3D models of various test TFPI-binding compounds (e.g., peptides or small molecules) to determine fit between the compounds and targeted amino acids in TFP. Because the effectiveness of a test compound in inhibiting TFPI can be limited if the compound does not remain attached to TFP for a sufficient period of time to effect a biological response, the tendency of the two to remain coupled can be predicted to develop an affinity rating. [00256] By analyzing the 3D surface of the TEN protein and the fit of the corresponding compound to the surface in view of the affinity rating, modifications to the compound (e.g., peptide) can be developed to improve both the number of contact points between the surface and the compound and the strength of the bonds at the contact points. The effectiveness of chemical-based candidates and peptide-based TFPI inhibitors can similarly be modeled using this technique, which facilitates the rational design of TEN-binding compounds. A computer model of the three dimensional (3D) surface of KDi allows testing of the ability of various peptides or chemicals to attach to an identified subset of amino acids that define a TFPI binding site and inhibit KD . A surface of the KD1 protein is modeled in 3D space on a computer, particularly a surface bounded by the targeted amino acids in KDL The 3D 04 models of various peptides, for example, can be matched to the surface to determine how many of the target TFPI amino acids are contacted by the peptide and also to develop an affinity rating predicting how long the peptide will remain attached to the target surface, [00257j By changing the peptide model and repeating the computer modeling, affinity ratings can be quickly generated for a peptide family. The most promising peptide variants (e.g., a second peptide comprising one or more substitutions within the amino acid sequence of a parent peptide) can be singled out for further physical testing, if desired. [00258] The invention provides a computer storage media having computer executable instructions that, when executed on the processor of a computer, implement a method of modeling interaction between selected three dimensional (3D) points in a TFPI KD protein arid a test compound The method comprises obtaining a protein struture 3D model for the TFPI KDi protein; determining a 3D relationship between a selected subset of amino acids in the protein structure, wherein the selected subset of amino acids comprises Phe28, Lys29, Ala3O, Asp32, 1e46, Phe47, and Ie55; modeling a surface bounded by theselected subset of amino acids; obtaining a test compound 3D model of a test compound; matching the test compound 3D model to the surface bounded by the selected subset of amino acids; and identifying contact points between the selected subset of amino acids of the surface and the test compound 3D model. Optionally the method further comprises determining a number of the contact points between the surface and the test compound 3D model; and recording an affinity rating for the test compound 3D model corresponding to die number of contact points, in one aspect, the selected subset of amino acids comprises (or consists of) Ala27, Phe28, Lys29, Ala3, Asp31, Asp32. Lys36. Ala37, le38, Phe44, R1e46, Phe47, and He55. The method further optonally comprises obtaining an updated test compound 3) model based on a second test compound; matching the updated test compound 3D model to the surface bounded by the selected subset of amino acids; and identifying the identified contact points between the selected subset of amino acids of the surface and the updated test compound 3D model on a display of the computer. In one embodiment, the method further comprises determining a number of the contact points between the surface and the updated test compound 3D model; determining a bond type for each contact point between the surface and the updated test compound 31) model; and recording a new affinity rating based on the number of contact points and an aggregate of the bond types for each contact point between the surface and the updated test compound 3D model, The updated affinity rating is then compared with the new affinity rating to determine whether the test compound or the second test compound has a higher affinity rating. if desired, The contact points can be displayed on the computer, thereby facilitating optimization or design of TFPL-binding compounds. [00259] In another embodiment, the computer storage media has computer executable instructions that, when executed on the processor of a computer, implement a method of comparing a peptide to selected three dimensional points (3D) in a TFPI Kunitz domain I protein (KDI), the method comprising creating a protein structure for the KDI protein; determining a three dimensional nodel of a selected subset of amino acids in the KD1 protein, wherein the subset of amino acids comprises Phe28; Lys29, Ala3O, Asp32; le46. Phe47 and he55; determining a three dimensional model of a pepdde; fitting the 3D model of the peptide to the 3D model of the selected subset of amino acids; and generating an affinity of the peptide for the selected subset of amino acids, wherein the affinity is based on a number of amino acids in the subset in contact with the peptide and a bond strength at each contact point. [00260] In addition, a method of comparing a test compound to selected three dimensional points in a TFPI KDI protein is provided. The method comprises creating a protein structure for the KD1I protein in a memory of a computer; determining a three dimensional modelof a selected subset of amino acids in the KDI protein at a processor of the computer, wherein the selected subset of amino acids comprises Phe28, Lys29 AIa30, Asp32, Ile46, Phe47, and 1le55; determining a three dimensional model of a test compound at the processor of the computer fitting the 3D model of the test compound to the 3D nodel of the selected subset of amino acids at the processor of the computer; and generating an affinity of the test compound for the selected subset of amino acids at the processor of the computer, wherein the affinity is based on a number of amino acids in the subset in contact with the test compound and a bond strength at each contact point. The method further comprises, in sotne embodiments, displaying a 3D representation of the fit between the test compound and the 3D model of the selected subset of amino acids and, optionaly, repeating the steps described herein for a plurality of test compounds and saving the respective affinities for each of the plurality of test compounds. [00261] With reference to Figure 58, an exemplary system for implementing the claimed method and apparatus includes a general purpose computing device in the form of a computer 110. Components shown in dashed outline are not technically part of the computer 110, but are used to illustrate the exemplary embodiment of Figure 58. Components of computer 1.10 may include, but are not limited to, a processor 120, a system memory 130,a memory/graphics interface 121 nd an /O interface 122, The system memory 130 and a graphics processor 190 may be coupled to the memory/graphics interface 12L A monitor 191 or other graphic output device may be coupled to the graphics processor 190. [00262] A series of system busses may couple various system components including a high speed system bus 123 between the processor 1.20, the memory/graphics interface 121 and the I/O interface 122, a front-side bus 124 between the memory/graphics interface 121 and the system memory 130, and an advanced graphics processing (AGP) bus 125 between the memory/graphics interface 121 and the graphics processor 190. The system bus 123 nmay be any of several types of bus strutures including, by way of example, and not limitation, such architectures include Industry Standard Architecture (ISA) bus, Micro Channel Architecture (MCA) bus and Enhanced ISA (EISA) bus, As system architectures evolve, other bus architectures and chip sets may be used but often general follow this patten For example companies such as Intel and AMD support the Intel Hub Architecture (IHA) and the HypertransportTM architecture, respectively. [00263] The computer 110 typically includes a variety of computer readable media. Computer readable media can be any available media that can be accessed by computer 110 and includes both volatile and nonvolatile media, removable and non-removable media. By way of example, and not limitation, computer readable media may comprise computer storage media, Computer storage media includes both volatile and nonvoladle; removable and non-removable media implemented in any method or technology for storage of information such as computer executable instructions, data stmetures; program modules or other data. Computer storage media includes RAMI, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices or other physical storage elements that physically embody electronic data and excludes any propagated media such as radio waves or modulated carrier signals. [002641 The system memory 130 includes computer storage media in the form of volatile and/or nonvolatile memory such as read only memory (ROM) 131 and random access memory (RAM) 132. The system ROM 131 may contain permanent system data 143, such as computer-specific configuration data. RAM 132 typically contains data and/or program. modules that are immediately accessible to and/or presently being operated on by processor 120. By way of example, and not limitation, Figure 58 illustrates operating system 134, application programs 135, other program modules 136, and program data 137. nn [00265j The I/O interface 122 may couple the system bus 123 with a number of other busses 126, 127 and 128 that couple a variety of internal and external devices to the computer 110. A serial peripheral interface (SPI) bus 126 may connect to a basic input/output system (BIOS) memory 133 containing the basic routines that help to transfer information between elements within computer 110, such as during start-up. [002661 A super input/output chip 160 may be used to connect to a number of 'legacy' peripherals, such as floppy disk 152, keyboard/mouse 62, and printer 196, as examples The super VO chip 160 may be connected to the I/O interface 122 with a bus 127, such as a low pin count (LPC) busn some embodiments. Various embodiments of the super I/O chip 160 are widely available in the commercial marketplace. In one embodiment, bus 128 may be a Peripheral Component Interconnect (PCI) bus. [00267] The computer 110 may also include other removable/non-removable, volatile/nonvolatile computer storage media. By way of example only, Figure 58 illustrates a hard disk drive 140 that reads from or writes to non-removable, nonvolatile magnetic media. The hard disk drive 140 may be a conventional hard disk drive, [00268] Removable media, such as a universal serial bus (USB) memory 153, firewire (IEEE 1394), or CD/DVD drive 156 may be connected to the PCi bus 128 directly or through an interface 150. Other removable/non-removable, volatile/nonvolatile computer storage media that can be used in the exemplary operating environment include, but are not limited to, magnetic tape cassettes; flash memory cards, digital versatile disks, digital video tape, solid state RAM, solid state ROM, and the like. [002691 The drives and their associated computer storage media discussed above and illustrated in Figure 58., provide storage of computer readable instructions, data structures, program modules and other data for the computer 110. In Figure 58, for example, hard disk drive 140 is illustrated as storing operating system 144, application programs 145, other program modules 146, and program data 147. Note that these components can either be the same as or different from operating system 134, application programs 135, other program modules 136, and program data 137. Operating system 144, application programs 145, other program modules 146, and program data 147 are given different numbers here to illustrate that, at a minimum, they are different copies. A user may enter cornmands and information into the computer 20 through input devices such as a mouse/keyboard 162 or other input device combination. Other input devices (not shown) may include a microphone, joystick, game pad, satellite dish, scanner, or the like, These and other input devices are often connected to the processor 120 through one of the I/O interface busses, such as the SPI 126, the LPC 127, or the PCI 128, but other busses may be used. In some embodiments, other devices may be coupled to paralel ports, infrared interfaces, game ports, and the like (not depicted), via the super 1/O chip 160, [O02701 The computer 110 may operate ini a networked environment using logical communication ports to one or more remote computers, such as a remote computer 180 via a network interface controller (NIC) 170. The remote computer 180 may be a personal computer, a server, a router, a network PC, a peer device or other common network node, and typically includes many or all of the elements described above relative to the computer 110. The logical connection between the NIC 170 and the remote computer 180 depicted in Figure 58 may include a local area network (LAN), a wide area network (WAN), or both, but may also include other networks. Such networking environments are commonplace in offices enterprise-wide computer networks, intranets, and the nternet. [00271] Figure 59 illustrates a 3D model of a TFPI protein 200 showing representative amino acids 202, 204, 206 that comprise the TFPI protein. A specific region of the TFPI protein of interest is KDI, not specifically illustrated, The surface shown is formed by the placement of the amino acids making up the protein. The surface of formed by specific amino acids in the KD1 region are of interest when studying or creating a TFPI inhibitor. As discussed in more detail herein, the biological effects of KDI are inhibited by binding certain amino acids of within the KD1 region. Specifically, these target amino acids include Ala27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, Ala37, 11e38, Phe44, fe46, Phe47, and he55. [00272 Figure 60 illustrates a peptide 300 that binds to at least a portion of the target amino acids listed above. 100273] Figure 6 is an illustration of a method of performing KD and peptide interaction modeling. [002741 A 3D model of a protein may be obtained (block 302) and stored on a memory 140 of a computer 110, The model may be generated locally using a known tool or may be obtained from a public source. In one embodiment the protein is TFPI1 KD 1 200. [00275] A 3D relationship between a selected subset of amino acids in the protein structure may be determined (block 304). In one embodiment, the selected subset of amino acids comprises Phe28, Lys29, Ala30, Asp32, 1e46, Phe47 and fe5; and optionally further comprises Ala27, Asp3l, Lys36, and fle38; and optionally frthe comprises Aia37 and Phe44, although not every amino acid listed here is required for binding to have an inhibitory (eg therapeuticeffect. That is further subsets of this group may also have properties of interest [00276] For the particular subset of amino acids of interest, a surface bounded by the selected subset of amino acids may be modeled. An outer peImeter may be defined by those amino acids not having further amino acids of interest on each side. A texture of the surface may be defined by the 3D location of each amino acid in the subset (block 306). [W277] A 3D model of a candidate TFP-binding compound (eg., peptide) of interest may be generated and stored at a memory 140 of the computer 110 (block 308) [00278] The peptide 3D model may be matched or fitted to the surface bounded by the selected subset of amino acids (block 310). A best fit between the two may be developed at the points of interest, that is, on the selected amino acids of KD. Several computer tool are available for such 3D modeling and fitting and may be used to creat 3D models and match one to another. One example is the HADDOCK tool described in: "de Vries. S L van Dik, A. D, J., Krzeminski, M. van Dijk, M., Thureau, A, Hsu, V, Wassenaar T. and Bonvin, A. M. J. J. (2007), HADDOCK versus HADDOCK: New features and performance of HADDOCK2.0 on the CAPRI targets. Proteins: Structure, Function, and Bioinformatics, 69: 726-733, doi: 10.1002/prot.21723" [00279] The contact points between the model of the surface of the selected subset of amino acids of the surface and the test compound (e.g. peptide) 3D model may be identified, stored, and optionally displayed on a monitor 191 of the computer 110 (block 312). A compound (e.g., peptide) may be modified to increase the number of contact points or the strength of the bonds at the contact points. To facilitate modeling this effect, a metric, described further below, may be developed to measure the affinity of the compound to bind to the protein of interest, in our example, KDL [00280] Further, the contact points between the surface and the compound 31) model may be counted (block 314) and an affinity rating for the compound 3D model may be recorded corresponding to the number of contact points (block 316). For example, if all 14 of the above listed amino acids are targeted and 12 of the 14 are actually contacted, or bound, by the compound 3D model, an affinity rating of 12114 or 0.86 may be calculated and recorded. [00281] However, the affinity rating as a measure of how tightly a candidate compound is coupled, and therefore, how long it may stay coupled to KDl may be more accurately described in terms of not only the number of bonds of interest but also the type of bond. The bond type for each contact point may also be determined (block 318). With respect to TFPI I )l binding peptides, hydrophobic bonds having an intermolecular distant of S 4 angstroms may be differentiated from bonds with an inermolecular distance of 2.6-3.2 angstroms. In one embodiment, bonds less than 3.2 angstroms may be assigned a weight of 1.5 and bonds> than 3.2 angstroms may be assigned a weight of 1.25. The- affinity rating may be updated or recalculated in view of the bond type using this, or another weighting (block 320). For example, if, in the previous example, 5 of the bonds are short bonds and 7 of the bonds are long bonds, the new affinity rating may be (5*1.5 + 7*125)/14= 1.16. [00282] If only 7 amino acids from KDI are targeted and 4 connect with short bonds, the affinity rating may be(4*15)/7=0.86. However, in this case the fewer targeted amino acids will be considered when comparisons are made to other affinity ratings. For example, all ratings could be normalized to a standard based on total desired target sites. [00283] If no more iterations are to be performed the no branch from block 322 may be taken and the results of may be stored for future analysis and decision making (block 324). if additional peptides, or variants of the previously tested peptide, are to be analyzed, the yes branch from block 322 may be taken and a new or updated model of the peptide of interest may be generated or otherwise obtained and stored (block 326), The steps at blocks 310 to 320 may be repeated and the results of the current run may be compared to results from previous runs to determine which peptides/variants have higher affinity ratings and merit more work, including possible physical testing. [002841 The ability to target particular sites with 3D modeling and to generate a comparative rating allows hundreds, if not thousands of samples to be processed and compared with relative ease, avoiding the time and cost of x-ray crystallography. This technique may be particularly applicable to modeling associated with the Phe28, Lys29, Aia3O, Asp32, Ile46, Phe47, e5, Aia27, Asp3l, Lys36, 1e38, Phe2, Ala37 and Phe44 amino acids from TFPI IKD I 100285] All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. In addition, the entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described hereinare contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document. For example. where protein therapy is described, embodiments involving polynucleotide therapy (using polynucleotides/vectors that encode the protein) are I 111 specifically contemplated, and the reverse also is true. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims, The invention includes, for instance, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above. With respect to aspects of the invention described as a genus, all individual species are individually considered separate aspects of the invention. With respect to aspects of the invention described or claimed with "a" or "an," it should be understood that these terns mean "one or more" unless context unambiguously requires a more restricted meaning. With respect to elements described as one or more within a set, it should be understood that all combinations within the set are contemplated. Finally, unless a claim element is defined by reciting the word "means" and a function without the recital of any structure, it is not intended that the scope of any claim element be interpreted based on the application of 35 U.S.C. § 112, sixth paragraph, EXAMPLES [002861 The invention, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of ilustration and are not intended to limit the invention. [00287] The following example describes production, identification, and screening of peptides for binding to TFPI. [00288] Peptides candidates were obtained from commercial suppliers (e.g., PolyPeptide Laboratories SAS (Strasbourg, France) and IPT Peptide Technologies GmbH (Berlin, Germany)), Methods for synthesizing candidate peptides are provided above. Candidate peptides were synthesized as trifluoroacetate (TFA) salts with a purity >90% or >60%. All peptides were solved in DMSO to a stock concentration of 10 mM. TFPI-binding peptide sequences were identified using an mRNA display library. The mnRNA display technology is superior to other library screening techniques for allowing for a diversity of 10 different sequences within a starting pool and avoiding, e the in vivo steps required for phage display. In he the technology involves directly linking m.NA to itsjencoded candidate 1 P\A peptide through a puromycin molecule (Figure 5). 'Te rRNA display method is further described in Intemnational Patent Publication No. WO 2005/051985 and Liu et at, Methods in Enzymology, 318, 268-293 (2000). TEIN was immobilized to a solid support via biotin and exposed to candidate peptide-RNA complexes. TFPI-bound candidate peptide-RNA complexes were isolated, and the RNA reverse transcribed to obtain coding DNA. High affinity binders were obtained following six to ten selection rounds using a competitive elusion strategy. Many of the candidate peptides were 31 amino acids in length (27 randomized amino acids and 2 amino acids flanking both termini) [002891 Selected peptides were synthesized and subjected to peptide optimization using a microarray-based scan analysis to identify peptide fragments retaining TFPI-binding affinity. For example, a microarray-based scan of JBT0047 was performed using a series of 20 amino acid fragments of the peptide, the sequences of which overlapped by 19 amino acids, Briefly, N-terminally, aninooxyacetate-modified peptides were printed on Corning epoxide glass slides. After washing and drying, the slides were treated in a TECAN HS400TMf incubation station. Slides were washed for two minutes in Tris-buffered saline with 0.1% TWEEN 20@ (TBST), and blocked for 30 minutes in Tris-based, T-20 SuperBockTM buffer (5 mM Ca~i 2 ) (Pierce). After blocking, the slides were washed for 2.5 minutes in TBST. The slides were subsequently incubated with DYLIGHTTM 649-labeled TFPI (1 g/ml in Trs-based, T-20 SuperBlockTM buffer(5 mM CaClz)) for 45 minutes, and washed twice with continuous flow TBST for ten minutes The slides were subjected to a final wash with saiine-sodium citrate buffer for two minutes, and ai-dried for four minutes, The slides were scanned in an Axon GienePix@ 4000B scanner, and scans were analyzed using the GenePix® Pro software. N and C-terminal truncation analysis supplemented the scan analysis. The microarray scan results demonstrated that peptide JBT0293 bound TFPI with the highest affinity A series of substitution mutantsbased on the amino acid sequence of JB1T0293 was generated and tested for TFPi binding properties. [00290] 'The affinity of a subset of pep tides for TFPI was demonstrated via an enzyme linked immunosorbent assay (ELISA) -like assay (binding (EC 5 o) ELISA) performed with biotinylated peptides. Ninety-six well MaxiSorp plates (Nunc) were coated with 3 Lg/mL TFPI in coating buffer (15 mM Na 2

CO

3 , 35 mM NaHCO 3 . pH 9.6) over night. Plates were washed three times with 350 gI wash buffer (HNaT: 175 mM NaCl, 25 mM HEPES, 5 mM CaCl 2 , 0.1% Tween 80, pH 7.35), and subsequently blocked with 200 p1l 2% yeast extract in HNaT for 2 hours. Plates were then washed three tirnes with 350 pl HNaT. Biotinylated candidate peptides were diluted from a DMSO stock 1/200 in HNaT. The initial peptide Ii' concentration was 50 pM if no precipitate appeared during the 1/200 dilution of the 10 mM peptide stock solution. Pre-dilutions of the peptide stock in DMS0 were conducted if precipitates formed. The diluted peptides were applied to the Maxisorp plates, serial dilutions (1/3) were generated, and the dilutions were incubated for 1.5 hours at room temperature. Incubation was followed by three wash steps (350 1±1 HNaT). Bound peptide was detected by incubation with horseradish peroxidase-conjugated streptavidin (1 hour), followed by three wash steps with HNaT and a subsequent chromogenic conversion of added 17MB (3,3'5,5'-Tetramethylbenzidin). The assay is illustrated in Figure 6A. [00291] Generally, peptide binding to immobilized TFPI was significantly above background. EC( values for biotinylated peptides are given in Figures 32-39. The binding curve of one TFPI-binding peptide, JBTO132, is depicted in Figure 7. The EC5 0 of JBT0132 was calculated to be about 2.2 nM. [00292] In addition, a competition (TC 5 ) ELISA was performed using biotinylated TFPI binding peptides as "tracers" to compete for TFPI-binding with non-biotinylated candidate peptides. The assay principle is depicted in Figure 6B. Ninety-six well MaxiSorp plates (Nune) were coated with 3 pig/mL TFP]. in coating buffer (15 mM Na 2 CO3, 35 mM NaHCO 3 , pH 9.6) over night. The concentration of TFP can be altered depending on the particular conditions of the assay; in other IC 5 o ELISA assays referenced herein, the coating buffer contained 0.05 pg/ml TFPI. Plates were washed three times with 350 pl wash buffer (HNaT: 175 mM NaCl, 25 rmM HEPES, 5 mM CaCDIN T1% Tween 80, pH 7.35), and blocked with 200 p1 2% yeast extract in H1NaT for 2 hours. Plates were then washed three times with 350 p1 HNaT. Biotinylated tracer peptides were applied at a concentration corresponding to their respective EC 9 o values determined in the binding ELISA (median if n > 2). A competitor stock solution of peptide (10 mM) was diluted 1/33.3 in HNaT without HSA, and a serial 1/3 dilution was prepared with HNaT with 3% DMSO. The dilution strategy employed in a particular assay will depend on the affinity of the peptides. The dilution was further diluted with the biotinylated tracer peptide in a ratio of 1:6 (20 p1 competitor dilution and 100 p1 tracer peptide). The mixture of competitor and tracer peptide was applied to the TFPI-coated microtiter plate and incubated for 1.5 hours, The plates were washed three times with 350 p1 HNaT. Peptide-TFPI binding was detected by applying HRP-conjugated streptavidin to the microtiter plate, incubating the mixture for one hour, washing the plate three times with 350 p1 HNaT, applying TMB (3,3'5,5'-Tetrarethylbenzidin), and detecting the subsequent chromogenic conversion of TMB by HRP. IC 5 o graphs for representative non-biotinylated peptides are provided in Figures 8A-8D. IC 50 measurements of peptides JBT0303, JBT 120, and JBT0224 are set forth in Table 3. TABLE3 -- -- - .. .. .. .. ... ... .. .. ... .. ... .. ... .. . BT330 11 2 0J04 T0]31 0.0409 B118 R ,4 'Tflav4 0NL --------- ------ ---------------- [002931 In addition to the competition ELISA (ICso) assay, a screening assay was employed to measure higher numbers of peptides in parallel The screening ELISA is similar to the competition IC 5 ELSA with the exception that only three different concentrations of the competitor were employed (300 nM, 100 nM and 33.3 nM for the JBT0047 class, and 50000 nM, 16667 nM and 5556 nM for the JBT 0122 class). In some instances screening results were expressed as percent inhibition of the tracer signal in relation to a competitive peptide (competitive peptide JBT0477 for the JBTOO47 family and competitive peptide JBT1697 for the JBT0122 family). The competition ICso assay results and the screening assay results of peptides prepared and screened in accordance with the methods set forth herein are provided in Figures 32-39. The mean IC 50 values presented in Figures 32-39 are based on a greater number of assays than the values presented in Table 3 and, therefore, the values may differ slightly, The results of the screening ELISA are presented as percent inhibiion of tracer peptide JBTO131 binding. Several peptides that were analyzed using the

IC

5 o ELISA are classified in Figures 32-39 according to their binding affinity as set forth in Table 4. TABLE 4 50nM20I A ------- --- - -------------- 50 1 x <-000 M 250 (10 < 1Ki0(00 nM 1003 x 5000 nl 10000 < x < 5000O nM G [002941 Exemplary TFPfbinding peptides identified using the methods described herein are presented in Table 5. Some peptides were biotinylated, and many comprise N- and C terminal lysines to promote solubility. Several peptides exhibited TFPI-inhibitory activity in model and/or plasmatic assay systems, as described below. TABLE 5 -d R Ix I.. D BT0047 QSKKNVFVFGYFERLRA........... JBT1"10471 JBT'0N7'| Ac-'VGRV A FQSK NFVG FLRAK X}S-NH2 253LSN JBT0051JR T0047 - Bitiny Tid-~SVGRLQVAFQSKKNVFFGYPERRAKLTSNH2-96 .................... ...............-------- JBT0055 JBT-047-A--SGVORLQVAFQSKKNVFVFGY-ERLRAKLTS-T-ds-Lys-B- tiny-96 JB B 47 1B y-Td-ASK 964 JNT062"-BT 047 -V AcKKRQVAQSKKNV 2 VFGYFENRLRAKTSKK-NU2 1 JBT16 BTCX047 , tnlTd-KQKNFFYEL KK H 9683 JBT0169V IBOO'c KFQNVFFGFELRKK NH2X 254 BT1 JT0 -Ac--KKQS---\KKNVF----VERLRAKLKK.... N.2 IT294 T047 Ac YQS k"NV FV FGSYERRALI- ANH2 2 J BT0291 15 1 JBTI0047 AeI SKVVGFRRKrH 1 J BT297 JBT04 A8FSNNFFYFRRK NH98 P~\X K\- V- \- "'NY '\ck' JBT29 JTN'047 Ac A' FQKKVFFY "RLA NH24 JBT..N........ -1-'---------x--- - .B4-I T BT33 - B-T- - -A--QSKKNVFVD YPER'-------AKL---NH21 ------------ ---------- L ---------- - - - - - - - - - - - - - - - - - - .2 ... .......... - - ----- ------------------ JT3106 |" JBT "00471 AcS KVFSYERLRAKLNH218 038'TC4 ic~QKNVFVFGYKE '-RLRAKL NH2 41 IBT30 JT047Ac-QSKNFVGY ERRAM NH2 24 JBT010 BT007 A-FQSKK\V\NVFVGYFDRLRAK \N26 --------------...-.----------- - - - B Ac--FQSKKNVFVFGYFER RAKN NH2 748 JBTO049 JBT0049 Ac-SNTFVDERLLYPLTNMGMYAAQKRT NH2 N2 5 .. .. .. . .. .. .. . -N ~ -- --- --- --- -- --- --N "-- -- - -- -- - -- -- - -- -- -- - -- -- - "'NU2 -T21 rCK'KNV''VAIC'.Y NW 10053 JTo -49' BXty~Td- 'NFDRLLYFLNTIGNMGMAAQLKRTSNH2 300 JB'T'019 JBN'NT049tA 2 ' Ac KSOTFVERNYFUGNMMY AKTSKK-NH2 33 JBT019 JBT0049' NASNT XFLLYFLTIO 'ANMGMAALKKKNH NY - - - - - - - --- . . . . . . . . . . . . . . . . . . . . . . . . .. . . --------------------------------------------------- ------ ----------------------- - - ----------------------- J2'1- ,512 " ,c j N ' " A fi A M -4 5-'' -- ........ ... A"-' .. .. .. .. .. .. .. .. .. .. ........... - --------- --- - - -- -- -- ..... .... .... ........-------- r ' ~ A . N ~ ' .' JBIT17 A JBT09 Ac KITV DR-'CY ULTNGKMYAQKRT-NH2-------3- VITP RHNKIYERRY 4 ---------- -f------------------ 31310050 1137050 A& SOiROCTKVIVI'ITRINMAGYFRR'khCTS- NI12 ____ 7B010.54 JBTC1 0 Bkx JO SkKKVFInLIYRYYTS-NK.12 -N 2 (6 SBT, 0: 1. 13T B.FO A S-GRKG iC-TKV I VFT'RiK,IG Y ER RYN CT S- N 12 302 TPin T(' 2 -65 RTO Ac -KKS 6flCV"" fi '7~1NVi... HRNK-R N CTS___ K I-NH2 ~1BT(' 19 WUPV0 AcCVV-? 13,,(Y'YNKtI1 30 11313218 M005T0 A-KVXIF --- 'KIJO 3YRRYN-N112 H <33 JBT'002 JB'1 01)0 Bo 1-Thl~tKKSO VW'KHPYTRYWTMWP IKIFV1PTK-Nf 30043 JBT0219 ITI'101 Ac(SVQTPYWMPDK30VLGk~~l2___ _______ _ ____ ____ A -XKG-----------P----------------KK.- 12- -- a--_ 12 T& TM tKGMSCVX~5 313---' ----- 01--------------------iA---YBrT[0O JRTM1kC 1M CV V E V T-N{ 3043 --- ------ ----------------- sSO SR KW 4 OM_________ 19 JR1 482 1 MT101 Ac-FKSGASTRYK WTGMRDKTMSC WVKFBYDKKVNI 12) SO, HTBIOM 1370120 AKKVCMjDOTSV VFKFR112 N' JK'47 3307W MT01 i20 Ac -KKCGMRDMKGTMSCVWVKFRYI K1 N112. [2173 JaB'!1O" S 1Bfi2'' A-CS YS'TAVQ-:~ ,LHYSFRSKFMI ART .Y - YTSK-N12 MI.__'Nr 1;B IW2 -51 3 20 AC-KKSOYAFPAVQMKT MSKLMARLYYK3M-1---------------------2 JBT0252 1[1.'PW0 ,>K(WSP\Q RVKSEALAKGH 1~ ~ 337(0% IIC0V) k-------------------- ---- ~EM K -N 2 JIV............. ---------------- P------------------K-K--N12-__ ___ B16 JOt § 2 1 &oiK Kc-RYASPLA -AVQLI1VY(RKAK N TIe.ARYKSN i [OO'$] his xamle.povies.eempary.ethds.o.geeratng..d..ar.teriin.TEP mliii try peptides Il p1tie inTbe5weefudtobn0uan3P- .Mtto analysis4 de ostae tha at least one min acd i a FPI-.V I.TSRb--''-2L indin pepid may- be- ---- R412--------22 susiue while reaiin affiniyAfr F'PJ.Tepetds fTbl ese n LSasy bonTFsawt nEQ fls hn1 i (15 x- 1&0 KIS EM)LK an212C 0 oflssta 5 M ..fl O ....... ..... ---- --------- ------- [09296 Selected TFPI-binding peptides were further characterized in terms of "anti target" binding. This example demonstrates that TPI-inhibitory peptides exhibit reduced affinity for non-TFPI- proteins. [00297] TFPI2 was selected as an anti-target because of its similarity to TFP-1. The binding kinetics of TFPI-binding peptides to human TF1- (residues 29-282 fused at the C terminus to a 10 Histag; MW 41 kDa (R&D Systems, Minneapolis. MN; catalog number 2974-P)) muine TEPH1 (residues 29-289 fused at the C-terminus to a 10 His-tag; MW 4ikDa (R&D Systems; catalogue number 2975-P)), and TFP-2 (R&D Systems, Minneapolis, MN) were studied using a BIAcore 3000114 surface plasmon resonance assay (GE Healhcare, Chalfont St Giles, UK). TFPI proteins were immobilized on a C1 cip (GE Healthcare Order Code: BR-100540) by amine coupling chemistry aiming for 500 RU. Several T-P-binding peptides were employed as analytes for interacting with the immobilzed TFPI proteins. A flow rate of 30 p /mi was utilized. After 180 seconds, 180 p1 of peptide solution was injected at six different concentrations ranging from 3.84 nM to 656.25 nM, followed by a dissociation time of 480 seconds. The chip was regenerated with 45 pl 10 mM NaOH. Each binding experiment was preceded and followed by four measurements with HBS-P buffer (10 mM HEPES, pH 7.4, 150 mM NaCi, 0.005% P20) plus 1% DMSO and 0.8% P80, BIAevaluation@ Version 4.1 software (GE Healthcare) was employed to analyze the data. Sensorgrams were fitted to a 1:1 Langmuir binding curve to determine kw and kg and calculate KaD [002981 Certain tested peptides, e.g., JBT0050, JBT0121, JBT0205 and JBTO21I, bound to the blank cell and binding constants from those sensorgrams could not be determined, JBT0133 showed weak binding to TFPFI. Sensorgrams from other peptides gave reliable binding constants Results from BlAcore analysis of several TEP-inhibitory peptides is provided in Table 6 and Figures 19-21. Each of the peptides listed in Table 6 presented a K, of less than 10 M. In addition to the peptides listed below, JBT0375 and 1BTO477, substitution mutants of 3BT0293 at amino acid position 5 (JBTO375) or amino acid positions 5 and 10 (JBT0477) also exhibited a K 0 of less than 10 pM. Sensorgrams of two of the peptides are provided as Figures 9A and 913 TABLE 6 pfd ' X (Is' KD As' jT04 403 x 10 1,0 1 7 x' 10X 4io JBT224 61 3 x 107 x 10 3 05 x 10 ElTD3 6 1 x 10 6, x 0 9 5 x 10 JBT303 8- x 10 2 7 x3 1 .BT.30 7.5.x 1.. 31x_6 00299] Interaction with the TFPI--2anti target also was examined. The maximum signal generated from candidate peptide interaction with human TFPI-2 was much lower than the signals obtained with TFPI as an interaction partner, Kinetic analysis of the low TFPI-2 binding signals was prone to error; therefore, visual comparison of sensorgrams was used to estimate binding affinity. A sensorgram illustrating JBTO 120 binding to TFPI-1 and TFPI-2 is provided as Figures I0A and 10B. JBT012O binds TFPI-2 with 10-fold lower affinity compared to its binding affinity for TFPI-1 JB3TO132 also was found to exhibit at least 10 fold greater affinity for TFPM--I than TFPI--2 [00300] The data provided by this example confirm that TFPL-inhibitory peptides specifically bind TEPI-1. [003011 The following example describes the characterization of TFPI-inhibitory activity of select peptides identified in Example 1 using FXa inhibition and extrinsic tenase inhibition assays. Roth assays are predictive of activity in plasmatic systems. The extrinsic tenase assay gives insight into the influence of the peptides on (a) the interaction of FXa anid TFPI and (b) the interaction of the FXa-TFPI complex with the TF-FVIIa complex. The FXa inhibition assay measures a peptide's influence on the interaction of FXa and TFPI only. [90302] The extrinsic tenase complex is responsible for FX an-d FIX activation upon initiation of the coagulation process. The extrinsic complex is composed of FVIIa, Tissue Factor (TF), and FX substrate, To determine the iuence of peptides on the TFI-mediated inhibition of the extrinsic tenase complex, a coupled enzyme assay was established. Peptides were diluted 116.25 from 10 mM stocks (in DMS0) and further diluted by serial 114 dilutions in buffer or DMs to prevent unwanted precipitation. TFPI was diluted in HNaCa-HSA or BSA 025mM|HEPES; 75mM NaC%)mM CaId-0S1% HSA or BSA; pH 7,35), FVIla, lipidated TF, phospholipid vesicles (DOPC/ POPS 80/20), and chromogenic substrate specific for FXa (S-2222 (available from DiaPharma, West Chester, OH)). all diluted in HNaCa-HSA, were added to 96-well plates, After an incubation period, TFPI and peptide dilutions were added, resulting in a final concentration of 25% DMS0 (if present in the peptide stock), FX activation was initiated by adding FX to the wells. FXa-mediated chromogenic substrate conversion was detcnined by observing an increase in absorbane using a micro-plate reader, The amount of F a generated at certain time points was calculated from the OD readings. FTXa generated at|20 minutes after start of the reaction was considered for calculation of EC 5 from plots of peptide concentration versus the inhibition of TFPI (%). [00303] The functional inhibition of TFPI also was examined using a FXa inhibition assay. A Fa-specific chromogenic substrate (8.-2222) and TFPF, both diluted in HNaCa1HSA. were added to 96 well plates. eptides were diluted 1/6.25 from 10 mM stocks (in DMSO or Aqua-Dest) and further diluted by serial 1/4 dilutions in buffer er DMS to prevent unwanted precipitation. The peptide dilutions (25p ) were added to the 96 well plates, resulting in a final concentration of 25% DMSC (if present in the peptide stock). The conversion of chromogenic substrate was triggered by the addition of FXa, and the kinetics of the conversion were measured in a micro-plate reader. Because TFPI inhibits FXa slowly, 01D readings after 115 minutes were considered for calculation of the EC 50 from plots of peptide concentration versus the inhibition of TE1 (%) [00304] Results from the extrinsic tenase assay and FXa inhibition assay are provided in Table 7 and Figures 22-27. TABLE 7 ---- ----------------- - ---------------- . ... ...--------------------- N" , MUNI ........... --------- ------------------------------------------ .......... %T2 ( 94 5 N5 9 ---------- ---------------- -A ----------------- - - - - ------------ ---- -..........-.-------------------- ---- [00305] Refeiring to Table 7, JBTO120, JBT01 3 and JBT0224 restored extrinsic complex-mediated FX activation in the presence of TFPI-1 with an ECso of < 2 gM, resulting in between about 20% to about 60% inhibition of TFPI. activity, JBT0047 (ECs 0 =1.4 pM), JBT0131 (EC 5 0 =12 pM), and JBT0293 (EC 5 o=2,9 pM) also restored extrinsic complex activity in the presence of TFPJ-l In addition, JBT0120, JBTOI132, J3T0224, and JBTO303 restored FXa activity in the presence of TFPI- with an EC 5 o of <5 pM, resulting in between about 5% to about 50% inhibition of TFPI activity, in the FXa inhibition assay JBT047

(EC

5 0 =O.7 pM), JBTO131 (EC 5 o =8.2 p M), JBTh293 (EC 50 =1,3 pM), JBT0297 (EC 50 =0.6 piM), and JBTO305 (EC 50 =2,3 pM) alsorestored activity of FXa in the presence of TFP- I in the FXa inhibition assay. This example confirms that peptides of the invention are TFPI antagonists. Example 4 [00306 In this example, the TFPI inhibitory activity of peptides is established using a plasma-based assay, [00307] The influence of peptides on thrombin generation was measured in duplicate via calibrated automated thrombography in a Fluoroskan Ascent@ reader (Thermo Labsystems, Helsinki., Finland; filters 390 nm excitation and 460 nm emission) following the slow cleavage of the thrombin-specific fluorogenic substrate Z-GlyGiy-'Arg-AMC (Hemker, Pathophysioi Haemst Thronb . 33, 4-15 (2003)) Plasma from patients with FVIII or PD deficiency (George King Rio-Medical Inc, Overland Park, RN) was obtained for testing. The residual coagulation factor activity for each of the plasmas was lower than 1%. As a model for antibody-mediated FVIII deficiency, frozen pooled normal plasma (George King Bio-Medical Inc., Overland Park, KN) was incubated with high titer, heat inactivated, anti human FVIII plasma raised in goat (4490|BU/mt Baxter BioScience, Vienna, Austria) giving rise to 50 BU/mL. The plasmas were mixed with corn trypsin inhibitor (CTI) (Hematologic Technologies, Inc. Essex Junction, VT) to inhibit Factor X1Ha contamination, resulting in a final concentration of 40 ig/mL. [00308] Pre-warmed (37C) plasma (80 4) was added to each well of a 96 well micro plate (Immulon 2HB1, clear U-bottom; Therno Electron, Waltham, MA). To trigger thrombin generation by Tissue Factor, 10 L of PPP low reagent containing low amounts (12 pM) of recombinant human Tissue Factor and phospholipid vesicles composed of phosphatidylserine, phosphatidyicholine and phosphatidylethanolamine (48 pM) (Thrombinoscope BV, Maastricht, The Netherlands) were added, Peptides were diluted 1/7.5 from 10 mM stocks with DMSO, 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 mix. Just prior putting the plate into the pre-warmed (37*C) reader, 5 pL of HEPES buffered 1 12 saline with 5 mg/mL human serum albumin (Sigma-Aldrich Corporation, St. Louis. Missouri, USA) or 12% DMSO in Aqua-Dest was added, followed by addition of the peptide dilutions or reference proteins (PVtill mmunate reference standard (Baxter BioScience, Vienna, Austria); Factor VIII Inhibitor By-Passing Activity (FEIBA) reference standard (Baxter BioScience, Vienna, Austria); NovoSeven (Novo Nordisk, Denmark); and purified human plasma FIX (Enzyme Research Laboratories, South Bend, IL)). Thrombin generation was initiated by dispensing into each well 20 pL of FluCa reagent (Thronbinoscope BV, Maastricht, The Netherlands) containing a fluorogenic substrate and HEPES-buffered CaCl 2 (100 mM). Fluorescence intensity was recorded at 37 0 C. [00309] The parameters of the resulting thrombin generation curves were calculated using ThrombinoscopeM software (Thrombinoscope BV, Maastricht, The Netherlands) and thrombin calibrator to correct for inner filter and substrate consumption effects (Herker, Pathophysiol Haenost. Thromb., 33, 4-15 (2003)). For calculating the thrombin generating activity of certain peptide concentrations equivalent to the reference proteins (e.g, FVIII Immunate@ reference standard, FEIBA reference standard), the thrombin amounts at the peak of each thrombin generation curve (peak thrombin, nM) were plotted against the standard concentrations, and fitted by a non-linear algorithm. Based on this calibration, FVIII Irmiunate, FIX, FEIBA or NovoSeven equivalent activities were calculated. Results for various peptides are provided in Figures 12-18 and 28-30, Representative results are provided in Table 8. (* denotes that FVRI deficient plasma was obtained from a different donor.) TABLE S % FVIII-equivalent activity FEIBA-equivalent activity in in FVIII deficient plasma @ FV11! inhibited plasma @ 10 pM -10 M peptide ptide[mU/ml] JBT0120 37A* 298 JBT0132 5.3 41 JBT0224 16.2 191 IJBT0303 20.825 [00310] With reference to Table O 8JB 120, JBT30)132, JBTO022A. and JBTf03'03 mr'pro-ved' TFP-dependent thrombin generation in FVIII-depieted plasma to levels exceeding 1% of the level of thronbin generation in plasma containing FVH (% FVIF-equivaient activity). The tested peptides exhibited approximately 5%-40% FVll-equivalent activity in FVl deficient plasma. JBT0120 and JBTOI 32 improved peak thrombin and peak time, dose dependently, as illustrated in Figures 1 IA and 11B. 1 A [003.11 Substitution mutants based on the amino acid sequeneof JB10293 also vere tested in a plasma-based assay, as wel as the FXainhibition and extrinsic tense inhibition assay described in Example 3. Representative results are provided in Table 9. TABLE 9 Extrmice FT1 FXa Ensi equivalent Inhibition activity (nU/md in (pMTh) ttt plasma @ 1 I1104 j (M)7 97 P3 29 48 . M 2 3 4 7.. .... ..... . . . .. ... ... . .

4- - ... T0303 34 12 NA .25 JETO500 | 3.2 0.12 - ____372 111T0740 |2,4 0.07 - ____{ 333 | [00312] Additionally, JBTO477, which comprises the amino acid sequence of JBT0293 but for substitutions at amino acid positions 5 and 10 of the JBT0293 sequence, improves thrombin generation equivalent to 413 mU/mi of FVIII (at I [M of peptide) in FVIII deficient plasma. Substitution mutation of JBT0293 resulted in highly optimized peptides with respect to affinity for TFPI and improved activity in FXa inhibition, extrinsic tenase inhibition, and plasma-based assays. ExamIe5 [00313] The following example demonstrates that the peptides of the invention can be modified by the addition of moieties that enhance physicochernical or pharmacokinetic properties of the peptides. As ilustrated below, the addition of 40 kDa PEG to peptides described herein dramatically improved the pharmacokinetic behavior of the peptdes. The example also describes optimization of a TEPI-binding peptide, JBT1857, to reduce susceptibility to proteolysis. [00314] Methods of conjugating chemical or biological moieties to peptides are known in the art. To add PEG (polyethylene glycol) to the peptides describe herein, a functional group (AOA aminooxy acetate) was added to the N-terminus of the peptides for coupling to aldehydes and ketones. Alternatively, a cysteine was added to Othe C-terminal part of the peptide for coupling with maleimid (Hermanson, Bioconju gate Techniques, Academic Press 1 C (1996)). The peptides (BT1586) AOA-FQSKGNVFVDGYFER L-Aib-AKLNH2 (SEQ 1T) NO: 166) and (JBT1587) Ac-FQSKGNVFVDGYFERL-Aib-AKLC-NH2 (SEQ ID NO: 167) were used for N-terminal and C-terminal modification with PEG, respectively. AOA FQSKGNVFVDGYFERL-Aib-AKL.NH2 (SEQ ID NO: 166) and Ac FQSKGNVEVDGYEERr-Aib-AKLC-NH2 (SEQ ID NO: 167) were incubated with excess 0 kDa rnPEG-Propionaldehyde (SUJNBRIGHT ME-400AL2, NOF, Japan) and 40 kDa mPEG-maleimide (SUNBRIGHT ME-400MA, NOF, Japan), respectively. The resulting PEGylated peptides, JBTI1852 and JBT1855, show similar affinities compared to the starting structure Ac-FQSKGNVFVDGYFERL-Aib--AKL--NH2 (J BT0740) (SEQ ID NO: 66). [00315] The resulting PEGylated peptides demonstrated significantly increased plasma stability and prolonged plasma half-life in mice. Figure 31 illustrates the results from a phannacokinetic analysis of the free peptide JBT0740 (Ac-FQSKGNVFVDGYFERL-Aib AKL-NH2) (SEQ ID NO: 66) compared to the C-terminally PEGylated peptide JBT1855 (-FQSKGNVFVDGYFERL-Aib-AKLC(PEG(4kD))-NH2) (SEQ I) NO: 252) following intravenous administration to mice. Tn contrast to the unPEGylated peptide, the PEGylated peptide is present at high concentrations in mouse plasma at 100 minutes post-administration. The unPEGylated peptide is rapidly cleared from the plasma. Figure 40 illustrates the results from a phannacokinetic analysis of JBT1855 following subcutaneous injection. JBT1855 also strongly improved thrombin generation in the assay described in Example 4 (Figure 41). 1003161 The JBTI 852 and JBT1 855 peptides also were characterized in the assays described in Examples 1-4 and compared to JBT074) and other peptides in the JBT0047 family. Representative results are provided in Table 10 set forth below TABLE 10 Plasma Stability TFPI, I Solubility equivalent (haf'ifti Affiny a activity (half life in (nM) (irnhib i\g/tionnd; minutes) PBS FVIII I'CLe Its! without deficient C+ plasma @ 1 a mouse human Mg) pM peptide JBT0I71-7 | L1I 0,105 41097212 -- ----------------- ~ JBTA74 006 33 ) 50 >120 JBT1582 1L 0 7 8 L16* >1202 JBTK855 .. 000i K** r n 2--mm --- E----H-7----17 5 M Na U, I 111411,1 1.-1 % ,* > 1*'(N, 2 -------------- N---------------- . ..- -------------------------- ) ------ ------ ------ ------ :-- -- ---- ------------ . ..... . . .......... . ..............------ ------------------t ----------- ----- -- [00317] The peptides listed in Table 10 also were assayed for interaction wjth the TFPI2 anti-target, and generated signals too low for reliable affinity measurement. The data suggest that PEGylation does not ablate the inhibitory activity of the inventive peptides or negatively affect selectivity for TFPI- 1. Cell-based extrinsic tenase assay [00318] The ability of the TFPI-binding peptides described above to restore extrinsic tenase complex-mediated conversion of FX to FXa also was determined using a cell-based extrinsic tenase assay. The cell-based extrinsic tenase assay also was employed to explore the influence of PEGylation on an exemplary TPI-binding peptide of the invention, JBT0740. Human umbilical vein endothelial cells (HUVEC) were counted and seeded in complete growth medium in a 96-well plate (black flat with clear bottom) at a density of L.5x10 4 cells per well. Cells were grown overnight (for approximately 16 to 18 hours), washed twice with pre-warmed basal medium, stimulated with I ng/ml recombinant TNFa (Sigma Aldrich (Cat.No. T6674)) in 200 pl of basal medium for four hours at 37"C, and washed twice with 200 pA of pre warmed cell culture buffer. Buffer (50 W) containing FVIIa (Enzyme Research Laboratories), TFPI-binding peptides (dissolved in either DMSO or Hepes buffered saline with or without 0.1% Tween-80), or aTFPI antibodies were applied to the cells and incubated for 20 minutes at 37C, allowing FVIIa/TF complex formation and binding of TFPI antagonists to TFPl. After the incubation period, 50 ld of cell culture buffer containing FX and a FXa-specific substrate (Fluophen FXa (HYPHEN BioMed)) was applied, resulting in a final volume of 100 pl cell culture buffer mix on the cells. The final concentrations were: 39 pM FVIla; 170 nM FX; 250 pM Fluophen FXa, and 2.5% DMSO (when peptides were dissolved in DMSO). [00319] The 96 well plate was transferred to a pre-warmed (37*C) fluorescence reader for detecting FXa-specific fluorogenic substrate conversion by FXa, which is generated by the TF/FVIIa complex on the surface of stimulated HUVECs. Readings taken after nine minutes of incubation were used for calculation of the TITI inhibitory effect of the TFPI-binding peptides or antibodies. The approximate percent inhibition of TFPI observed at various concentrations of the following peptides (belonging to the JBT0047 family) is set forth in Table 11: JBT0717 (Ac-FQSK-Nmg-NVFVDGYFERLRAKL-NH) (SEQ ID NO: 61), JBT0740 (Ac-FQSKGNVFVDGYFERL-Aib-AKL-NI-H2) (SEQ ID NO: 66), JBT1584 (Ac FQSK-Nmg-NVFVDGYFERL-Aib-AKL-NH 2 ) (SEQ ID NO: 164), and JBT1857 (Ac FQSKpNVHVDGYPERL-Aib-AKL-NH2) (SEQ ID NO: 178). 1 1-? TABLE 11 ~ _____ % TFPI inhibition 40pM j M 16 M 032 M 64nM JBT0717 6% 50% 32 % .28% '._20% JBT0740 70% 39% 281% 14% 3 JBTS 743% 62% ~ 1 ~ 40% 29%___ ------------ -_ -_ ..... I-_ _ _ -------- - 80% j 57% 41% 35% 15% [00320] PEGylated peptides also were tested using the cellased extrinsic tense assay JBT0740 (SEQ ID NO 66) was conjugated to a 1 kD PEG moiety at the N-terminus to produce JBT1853 or at the C-terminus to produce JB1854. JBT1853 and JBT 854 inhibited TPi by 20% or less depending on the amount of peptide used in the assay. JBT1855, which comprises a 40 kD PEG moiety at the C-terminus (parent peptide, JBT0740) performed better in the cell-based assay than 1BTI 852, which comprises a 40kD PEG moiety at the N-terminus. JBT185 mediated 20-30% TFPI inhibition, while JBT1852 inhibited TFPI activity by 10% or less. [00321] Peptides of the JBTO120 family, JBT0120, JBTO415, JBT0444, JBT1426, and JBT1837, also were tested in the cell-based extrinsic tenase assay and found to inhibit TFPI to a lesser degree compared to peptides of the JBT0047 family. The reduced or partial inhibitory activity may be desired in some embodiments of the invention. Similar to the peptides of the JBT0047 family, peptide optimization increased TFPI inhibitor activity of JBT0120 family peptides. [00322] In the course of examining the stability and inhibitory activity of JBT857 it was determined that the amino acid sequence of the peptide contained a protease cleavage site between VaI9 ard Asp10. Substitution of Te at position 9 (generating JBT2431) and substitution of Pro at position 10 (generating 1BT2432) blocked cleavage of the peptide and enhanced the plasma stability of the peptide by about three-fold from 27% (JBTI857) to 82% (JBT2431) and 76% (1BT2432), An additional puative cleavage site was identified between Glyl I and Tyr12. A GI la substitution (generating JBT2414) further improved the stability of the peptide to 100%. All stabilties were determined by quantitative ELISA after 24 hour incubation in human plasma. 110 [00323] The results described above demonstrate that optimization of the TFPI-binding peptides described herein utilizing non-conventional amino acids improved TFPI inhibition and plasma stability. Additionally, PEGylated peptides of the invention inhibit TFP1 activity in a cellbased extrnsic tenase assay, with C-terninal PEGylated peptides performing better than N-tenninal PEGylated peptides. The TFP-binding peptides of the invention inhibit the activity of both free TFPI and cell-bound TFPI. [00324] The following example illustrates the abiity of peptides described herein to reduce bleeding in an animal modeh [00325] Ten week old C57Bf6NCrd mice were housed for two weeks prior to the study, Thirty minutes before the nail clip, the animals were administered (a) JBT1 855 (10 mg/kg) intravenously (i.v.) via the tail vein or subcutaneously (s.c.) in the neck region, (b) anti-TFPI antibody (18 mg/kg; i-v), or (c) vehicle (175 mM NaC , 25 mM HEPES, pH 7.35; 10 n/kg; i.v.). The animals were anaesthetized with 80 mg/g pentobarbital ten minutes prior to the nail chp. To achieve bleeding the nail of the small toe of the right hind paw was removed The paw was submerged in a 0.9% NaCI solution for blood collection for a period of 60 minutes. Blood loss was quantified after lysis by spectrophotometry. The temperature was kept constant at 37C C over the course of the experiment. The results of the study are illustrated in Figure 42 and summarized in Table 11L TABLE 11 B185 JBT1855 cuTFPI Vehicle isv. s.c. i~v. Lv. Mean (in pj) 29,9 3 17 19 74.9 (SD) (71 4) (31,5) (14) (74.6) # office 12 12 12 12 p-value 0.07 0.04 0.001 I ______ [00326] Intravenous or subcutaneous administration of JBT1855, a PEGylated peptide of the invention, reduced blood loss in mice compared to treatment with vehicle alone, 1 i Exampnle 71 [00327] The following example describes characterization of TFPI-peptide interactions via nuclear magnetic resonance and x-ray crystallography. in particular, the TFPI binding site of the antagonistic peptides JBTO3O3, JBTl122 and JBT0415; the residues of JBT0303, 3BT0122 and JBTO4I5 interacting with TFPlTi6; and the secondary structure of completed and free JBT0303, JBTO122 and JBTO415 were investigated at a molecular level using 2D isN-heteronuclear single quantum coherence (HSQC) spectra. The interaction of JBT1857 and KDI of TFPI was examined using x-ray crystallography, and the residues of TFPI KD1 that mediate JBT1857 binding were mapped. Identification of the binding site of JBTO303 on TFPI60 [00328 A Nabeled preparation of TFH 160 was used for iataion experiment of TPI6O with 31T0303 HSQC spectra of a= -500 tM ' 5 N-TFPl6O sample without and with increasing amounts of peptide were recorded at 30*C on a Varian 600 MHz spectrometer The peptideprotein interaction showed slow exchange behavior (k < A<), meaning that each TFPI residue results in a defined signal for the free protein and the protein.peptide complex. Unlike fast exchange behavior (km > Aco), where a mixture results in only one peak with averaged position according to the population of the species, slow exchange behavior does not allow tracking of the signals upon peptide binding. Thus, in order to locate the binding site, the shifted peaks of the TFPl60-BT0303 complex needed to be assigned. This required the preparation of a sample of "C/N: TFPIi60 and JBTO3O3 [003291 initially, a sample was prepared with 992 iM 'CNTFPUl and 1190 p M iBT0303 However, the NMR sample resulted in poor quality spectra which did not allow assignment of the complex The sample gelled, lkely due to the formation of high molecular weight aggregates. Thus, the acquired NMR data predominantly showed signals arising from the most flexible parts of the isotopelabeled TFPI6O. Therefore, sample conditions were reinvestigated for further experiments From a series o 5 N-HSQC experiments conducted on the TFPJ!60JBTO3O3 complex, it was concluded that gel formation could be avoided by sample dilution and data acquisition at elevated temperature. The final concentration of "C/'N-TFPl160 was 331 pM and that of JBT0303 was 397 pM. Spectra quality was unproved. Due to the lower concentration and reduced signalto-noise ratio, assignment had to be performed based on HNCA, HNCO and HNCOCA experiments. [00330] Except for four previously assigned residues, all residues that could be assigned in the apo-TFPI160 could be assigned in the TFPI160-JBT0303 complex. Assignment of some I11Af residues was ambiguous due to the lack of peaks in the 3D spectra. Furthermore, the peaks of three residues were only visible in the HSQC spectrum from the original titration experiment, However, all peaks in the vicinity where unambiguously assigned and, therefore, the assignment of these residues is likely to be correct 100331] Chemical shift changes of the HSQC signals of N-TFPI.60 bound to JBTO3O3 compared to free TFPII6O is illustrated in Figure 43. Residues undergoing the strongest chemical shift were exclusively on Kunitz domain 1, Chemical shifts of residues F25, F28, D32, A37, T48 and Y56 shifted the most (> 2 ppm). Residues 138, 146, F47 and F54 also shifted more than 1.5 ppm. It is unclear whether residues N-terminal of F25 are involved the interaction with JBT0303 because residues 20-24 are not assigned. L19 shows a change of chemical shift amounting to - 0,6 ppm. Thus, in contrast to previous beliefs that amino acids within residues 1-18 of TFPI are involved in peptide binding, the present data suggest that there is little, if any, peptide binding to the N-terminal tail of TFPl A ribbon model of the secondary structure of TFPI protein illustrating regions of chemical shift changes of HSQC signals of TFPI60 bound to JBT0303 compared to free TFPU60 is set forth in Figure 44. [003321] To more particularly identify the binding site of JBT0303 on TFF]l60, the amide exchange rates of ' 5 N-TPI60 and 'N-TP60+1BT0303 were determined. The amde exchange experiment mainly detects changes in the environment of the peptide backbone by measuring H exchange of aide groups. The HO frequency is irradiated with a power high enough that it is not dissipated by relaxation, resulting in a complete saturation and suppression of the HfO signal. A side effect of this method of Hf signal suppression is that the suppression is transferred to exchangeable amide NHs which exchange with solvent (-I exchange) The saturation transfer is dependent on the H/H exchange rate which is semi quantitative. The effect is reduced for more protected NI groups (i.e., unprotected Ns are attenuated more than protected NTIs). If a protected NH lies in proximity to H-alphas of a Hgand- a higher exchange rate is observed compared to the apo form. Similarly H exchanges can be mediated by the OH groups of Ser, Thr or Tyr. [00333] HSQC spectra without and with water suppression of apo "N-TFPIl60 and the 1 N-TP 60-JBT0303 complex were recorded. The relative exchange rate of each residue of TFPII60 was determined by calculating the ratio of the peak intensities in the HSQC spectra vith and without water suppression. A comparison of the data sets of N-TP160 and 1 NTFPR60iBT0303 revealed that TFPI residues 25, 26, 36, 62, 63, 127, 132 and 152 exhibited greater than 10% decreased anide exchange rate in the complex, whereas residues 2, 30, 42, 45 49,0 50,56 66 and exhibited more than 10% increased exchange rate [00334] Constraints derived from the amide exchange experiment were included for the calculation of refined HADDOCK models: (a) torsion angles are taken from the calculations of TALOS for K4, K5, V7, F8, Y12-AlS of JBT0303 (chemical shift experiments); (b) residues of KDI with chemical shift changes of more than L 5 ppm are involved in binding JBT0303: F25, F28, D32, A37, 138, 146, F47, T48, F54 and Y56 (chemical shift experiments); (c) the hydrophobic side of the amphipathic helix of JBT0303 is bound to KDI: Y12 or L16 or L20 of JBT0303 bind to D32 or A37 or 138 or F54 or Y56 of KDI (chemical shift experiments); (d) R15 or K19 of JBT0303 bind to D31 or D32 or E60 of KDI (chemical shift experiments); (e) F8 or V9 of JBT0303 bind to F25 or F28 of KDI (chemical shift experiments); (f) Y12 or F13 of JBT0303 bind to 146 or F47 or T48 of KDI (chemical shift experiments); (g) Q2 of JBT0303 binds to Y56 of KDI (chemical shift experiments); (h) F1 of JBT0303 binds to M39 or F66 of KDI aidee exchange experiments); (i) S3 or K4 or K5 of JBT0303 bind to F66 (amide exchange experiments); (j) V7 or F8 or V9 of JBT0303 bind to F25 or C26 or N62 or Q63 of KD1 (amide exchange experiments); (k) V9 or DIO or GI1 or R15 of JBT0303 bind to F28 or K29 or A30 of KDi aidee exchange experiments); () YI2 or P13 of 3BT0303 bind to N45 of KDl amidee exchange experinments|);(n) Y12 or F13 or E14 or R15 bind to R9 or QSO of KDI (amide exchange experiments) and (n) L20 of 1BT )303 binds to K36 of KDI (aide exchange experiments) The data convegedto essentially one model of the KDI+JBT0303 complex. Idendification of the binding site of JBT0J122 on T FPII6L) (90335] As with the i 3

C/J

5 N-TP160 + JBT0303 complex, the "CI/N-TFPI160 + JBTI 122 NMR sample resulted in spectra of poor quality due to the formation of a gel The concentration of 723 pM CT/ 15 NVTFPII160 + JBTO 122 lead to formation of higher order aggregates. The sample was diluted to 361.5 pM and spectra recorded at 37C, resulting in improved spectra quality. HNCO, HNCA and HNCOCA spectra were acquired. Except for five residues, all of the previously assigned peaks of apo-TFP1160 could be assigned in the TFPI I 6OJBTO 1.22 complex. Residues undergoing the strongest chemical shift changes and likely to interact with the peptide often did not result in peaks in the 3D spectra. Peaks in the linker region between Kunitz domain 1 (KDi) and Kunitz domain 2 (KD2), however, also exhibited low intensities Hence, the assignment of these peaks isambiguous. Some peaks were only visible in the HSQC of the original titration experiment. Their assignment was in most cases certain, as the peaks overlapped in the TFPI6 and the TFPI60 + JBTO122 HSCQ spectra.

[00336] Chemical shift changes of the HSQC signals of 'N-TFPI160 bound to JBT1I22 compared to free TFPI160 is illustrated in Figure 45, Significant chemical shift changes were exclusively found for residues of KD2. In general, the extent of the chemical shift changes caused by binding of JBT0122 to TFPI160 was less pronounced than that of JBT0303. Residues with the strongest perturbation of chemical shift were F96, G128, G129, G132, N133 and N136, C97, E101, T111, F114, N135 and F137 were perturbed, exhibiting chemical shift changes of more than 0,5 ppm. A ribbon model of the secondary structure of TFPI protein illustrating regions of chemical shift changes of 1-SQC signals of TFPH6 bound to JBT1 22 compared to free TFPII60 is set forth in Figure 46. Identification residues of JB10122 that interact with 71FPU160 [00337] For the sequential backbone signal assignment of JBT0122, '-C/'N-labelled peptide was produced recombinantly. Briefly, the peptide was expressed as a fusion protein with thioredoxin in E. coli. 1 3C/ 1 N-labelled peptide was prepared using M9 medium containing 3.0 gil IC-glucose and 1.0 g/l1 'NH4Ci, The fusion protein was affinity purified using a Ni-chelating column and a poly-histidine tag. The peptide was cleaved by thrombin. The thioredoxinlhis-tag and thrombin was removed using a Ni-chelating column and a benzamidine column, respectively, The peptide was then purified by reverse phase chromatography. Purity, integrity, and identity were verified by SDS-PAGE, RPIPLC and mass spectrometry. Recombinant JBT0i22 was named 3BT0788 and had two additional residues at its N-terminus, glycine and serine, which represent the remains of the thrombin cleavage site. [00338] The assignment of JBT0788 was done on the basis of HSQC, HNCACB, HNCA, HNCO and HNN spectra recorded at I 0 0 C on a Varian 600 MHz spectrometer and assigned using the SPARKY software, The temperature was reduced compared to NMR experiments with TFPI160 to improve spectra quality. From the recorded spectra, the carbonyl carbon (C), the alpha caton (CA), the beta carbon (CB) the amide proton (Fit and the amide nitrogen (N) of most residues were assigned. The assignment for residues H13 and R17 was ambiguous. An HNCOCA led to an unambiguous assignment for these residues. [00339] An assignment table for JBTO788 is provided in Figure 47 Two sets of signals for residues 4-12 were observed in the spectra of JBT0788. Considering that the primary structure of JBT7SS is not compromised, the two sets of signals likely result from a cis/trans isomerization of the peptide bond between F6 and P7 A ratio of 76:24 was determined for majorminorconformation based on|the intensities of the corresponding signals in the -ISQC spectrum. As judged from the Ca shift of the proline the major conformationisikely tans, as its Ca value of 63.16 ppm is higher than of the minor conformation (62.49 ppm). [003401 One purpose of the assignment was to extract the secondary structure of the peptide from Cc chemical shifts, Ca chemical shifts are influenced by the angles p and yjF and, thus, by the secondary structure of the peptide, in fbstrands, Ca are general shifted to lower ppm; in i-helices, Ca are general shifted to higher ppm. By subtracting the measured Ca value from a tabulated random coil value, negative values are calculated for residues in n-strands and positive values for residues in a-helices. Thus, a batch of consecutive negative values indicates a Q-strand while a batch of consecutive positive values indicates an a-helix. [00341] JBT0788 exhibited a broad patch of increased Ca values (A6(Ca) Caiomn) ~ indicating an a-helix comprising residues 8 to 26. A6(Ca) values for stable a.

helices within tertiary structures of native proteins are typically between 3-4 ppm. A6(Ca) values of the a-helix of JBT0788 rise up to about 1.7 ppm, indicating more flexibility than an average helix within a protein. Another feature of JBTO788 is the proline at position 7, directly N-terminal to the ahelix, which fits well as aihelices in proteins are frequently terminated by a proline at the N-terminus. Residue 6 has a strong negative value, which is caused by the neighboring prone known to force its N-terminal neighbor into a p-strand-ike conformation. The strong positive value of C-terminal residue 31 is also typical for residues without a C-terminal neighbor. The peptide bond between F6 and P7 in JBT0788 adopts two confirmations, a trans (76%) and a cis conformation (24%). The conformation at this position impacts the conformation of the consecutive residues. In the trans isoforma, the a helix starts immediately after P7; the a-helix of the cis isoform does not start untilresidue L12. A ribbon model ilustrating the secondary structure of free JBT0788 is set forth in Figure 48. [00342] The chemical shifts within JBT0788 can also be employed to calculate the torsion angles using TALOS software. TALOS is a database system for empirical prediction of (p and W backbone torsion angles using a combination of five kinds (HA, CA, CB, CO, N) of chemical shift assignments for a given protein or peptide sequence. The TALOS approach is an extension of the observation that many kinds of secondary chemical shifts (i.e., differences between chemical shifts and their corresponding random coil values) are correlated with aspects of protein secondary structure. The goal of TALOS is to use secondary shift and sequence information in order to make quantitative predictions for the protein backbone angles p and y, and to provide a measure of the uncertainties in these predictions. TALOS uses the secondary shifts of a given residue to predict p and yj angles for that residue. TALOS also includes the information from the next and previous residues when making predictions for a given residue. The idea behind TALOS is that if one can find a triplet of residues in a protein of known structure with similar secondary shifts and sequence to a triplet in a target protein, then the <p and v angles in the known structure will be useful predictors for the angles in the target. In practice, TALOS searches a database for the 10 best matches to a given triplet in the target protein. [00343] in order to assign the H4SQC spectrum of JBT0788 complexed with TFPI1 60, a sample consisting of 400 1 iM "C/' 5 N-JBT0788 and 400 pM TFPTI160 was prepared. As with previous NMR samples of peptide and TFPL 60, the sample gelled. The sample was diluted and the pellet dissolved in deuterated DMSO, resulting in a final concentration of -300 gM "3C/"N-JBT0788 + TFP160 and 5% DMSO. Measurements were performed at 40C. This improved the quality of the acquired spectra. Experiments were acquired in the TROSY mode to account for the relaxation properties of a partially aggregated sample. Cryo-probe technology on the Vadan 600 MI-lz spectrometer was employed due to the low concentration of the protei-peptide complex in the sample. 'Iheresulting data quality was sufficient to obtain the backbone shifts of JBT078S when utilizing the cryo-probe technology and acquirng the triple-esonance experiments in duplicate. The assignment of JBT0788 in complex with TFP 60 was performed on the basis of HNCA, HNCOCA and INGO spectra. From the recorded spectra, the carbon cacarbon (CO), the alpha carbon (CA), the amide proton (-), and the amide nitrogen (N) of most residues were assigned. An assignment table for JBTO78 cormplexed to TFPl60 is provided in Figure 49. 1003441 A feature of apo-JBT0788 was the presence of two sets of signals for amino acid residues 4-12, likely resulting from a cis/trans isomerization of the peptide bond between F6 and P7. In the JBT0788-TFPI160 complex, only one set of peaks is observed, implying that only one of the conformations binds to TFPI160. Apo-JBT0788 also exhibited a broad patch of increased Ca values (A6(Ca) = Cameansud - Cmardom coi = positive) indicating an a-helix reaching from residue 8 to residue 26, As mentioned above, A6(Ca) values for stable a helices within tertiary structures of native proteins are typically between 3-4 ppm. A6(Ca) values of the a-helix of apo-JBT0788 increase to about 1.7 ppm, indicating more flexibility than an average helix within a protein. When complexed with TFPI, residues 8 to 26 exhibited values of between 3-5 ppm, indicating the formation of a stable a-helix or helices. A ribbon nodel illustrating the secondary structure of JBT0788 when complexed with TFP 60 is set forth in Figure 50. Large chemical shift changes within JBTO788 caused by binding with TFPA60 are evenly distributed over the length of the peptide. Residues undergoing the strongest perturbation of chemical shift were residues S5, A9. QII Y28, and K29 with more than 4 ppm. Residues Y3, A4, V01, L12, S15, M21, A22, L23, and A24 were perturbed by more than 3 ppm. Identrifcation of residues of JBT0303 that interact with TFPII6 [003451 JBT0303 was produced recombinantly using the same procedure as described above for JBT0122 and isotope-labeled with ' 3 C and 1N, The recombinant JBT0303 was named JBT0616 and had an additional glycine and serine at its N-terminus. The assignment of JBT0616 was performed on the basis of .HSQC, HNCACB and HNN spectra, which were recorded at 10'C on a Varian 500 MHz spectrometer and assigned using SPARKY software. The quality of the spectra of JBT0616 was better than that of JBT0788, although the experimental conditions with respect to buffer, temperature, NMR tube, and NMR parameters were identical. The alpha carbon (CA), the beta carbon (CB), the amide proton (H), and the amide nitrogen (N) of most residues were assigned. The assignment was mainly based on the less sensitive but more informative HNCACB instead of the HNCA. In combination with the I-INN spectrum, this resulted in an unambiguous assignment of all JBT0303 derived residues. [00346] An assignment table for JBT0616 is provided in Figure 51. The secondary structure was extracted from Ca chemical shifts and determined by TALOS using the assignments of H, CA, CB, CO and N. Like JBT0788, JBT0616 exhibited a patch of positive A(Ca) values indicative of a-helical conformation. The helix was located at the C-terminal part of the peptide and comprised residues 10-18. As for JBT0788, A6(Ca) values up to about 1.8 ppm were calculated, qualifying this helix as relatively stable for such a short peptide. A ribbon model illustrating the secondary structure of JBT0616 is set forth in Figure 52. The strong positive value of the C-terminal residue 20 is, like residue 31 in JBT0788, typical for residues without a C-terminal neighbor. The N-terminal residues 1-9 of J1BT0616 exhibited slightly positive AKCQ.) values, suggesting a preference for an a-helical structure. [003471 The assignment of JBT0616 in complex with TFPI160 was performed using a "C/'N-labelled peptide sample with an excess of unlabelled TFPI160. HSQC, HNCA, HNCOCA. and HNCO spectra were recorded on a Varian 800 MHz spectrometer and assigned using the SPARKY software. The spectra were recorded at 30*C. Using these spectra, the alpha carbon (CA), the beta carbon (CO), the amide proton (H), and the anlide nitrogen (N) of most residues were assigned, as set forth in the table in Figure 53. The secondary structure of BT016 n complex wih TFP160 was extracted from Ca chemical shifts and calculated by TALOS. Like the free peptide, J13T0616 in complex with TFPIl60) exhibited a C-terminal patch of positive Ad(Ca) values indicative of a-helical conformation. The stability of the a-helix is increased upon complex formation, This finding suggests that the C-terminal region of JBT0616 is the core binding motif. The A6(Ca) values for the N terminal residues also changed, but to a lesser extent. The secondary structure of JBT0616 when complexed with TFPI is illustrated in the ribbon modei in Figure 54. [00348] The nost significant changes of chemical shifts upon complex formation were observed for residues Q2 K5, FS, V9 and AIS of JBTO6I6 with more than 7 ppm. Residues F13, R17, K19 and L20 also were perturbed and demonstrated chemical shift changes of more than 4 ppm. The strong chemical shift changes of residues at the N-teminus indicated that it is not only the amphipathic C-terminal a-helix which drives binding of the peptide to TFPI160. [00349] Results from the NMR experiments in combination with analysis of JBTO477 substitutions were used to create a model of KD I in complex with JBT0303 using HADDOCK (High Ambiguity Driven protein-protein DOCKing) software. HADDOCK is an information-driven flexible docking approach for the modeling of biomolecular complexes. HADDOCK distinguishes itself from ainiti docking methods in the fact that it encodes information from identified or predicted protein interfaces in ambiguous interaction restraints (AIRs) to drive the docking process, identification of the binding site on TFPIU6O and the peptides as revealed by chemical shift data, the torsion angles of the peptides as determined by the software TALOS, and the substitution analysis of J1BT0477 provide the restraints for the calculation of the models. [003501 For the calculation of the KDI1-JBTO303 HADDOCK models, the frollowing restraints were employed: (a) torsion angles were taken from the calculation of TALOS for K4, K5, V7, F8, Y12-A1 of JBT0303; (b) residues of KD1 with chemical shift changes of more than 15 ppm are involved in binding to JBT0303: F25, P28, D32, A37, B8 146, F47, T48, P54 and Y56; (c) the hydrophobic side of the amphipathic helix of JBT0303 is bound to KDI: Y12 or L16 or L20 of JBT0303 bind to D32 or A37 or 138 or F54 or Y56 of KDI; (d) R15 or K19 of JBT0303 binds to D31 or D32 or E60 of KDF; (e) PS orV9 of JBTQ303 binds to F25 or F28 of KD1; (f) Y12 or F13 of J1BT0303 binds to 146 or F47 or T48 of KD1; and (g) Q2 of JBTO303 binds to Y56 of KDI The Q2 JBTO3O3 - Y56 KD1 interaction also was taken as a restraint for model calculation.

[003511 Strong chemical shift changes were observed for K5 of JBTO303 upon complex formadon. For the remaining residues of JB1T0303 considered to drive the peptide-protein interaction, the models are in good agreement with the data The model of. -D1JBTO3O3 with the lowest energy places F8 of JBT0303 in proximity to F25 and F28 of TFPL explaining the observed chemical shift changes and the data from the subs titution analysis, V9 of JBT0303 interacts with the hydrophobic patch of the KDI including F54 Y12, F13, L16 and L20 of JBT0303 also face the hydrophobic patch of the KD1. The proximity of Y12 to 128, 146, T48 of F13 to F47, T48, of L16 to P54 and of L20 to A37, 138 causes the observed perturbations of NMR chemical shift of those residues in the complex; the conservation of Y12 and L16 maybe due to the extensive interactions of these residues with the protein K19 of BT0303 is in a position allowing interaction with D32 of KD1 The role of R15 of JBT0303 seems to be an interaction with the hydrophobic patch of KD as well as with 132 Moreover, the model explains why a negatively charged aspartate is preferred at position 10 of JBT0303; it can interact with the positively charged K29 of KDL. A glycine at position 11 of JBT0303 is present due to the steric and conformational restraints at this position. A HADDOCK model of KDi (TPPI residues 22-79 comprising KD1) in complex with JBTh303 is provided in Figure 5. Models of 1BT0740 and JBT1857 bound to KDP [00352] Peptides JBT0740 and JBT1857 (FQSK-dP-NBHBDGYFERL--Aib-AKL (SEQ ID NO: 178)), both derivatives of JBT0303, demonstrate significantly enhanced ECso values in the FXa-TFPI inhibition assay (0.11 gM and 0.0023 gM, respectively) and lower K& s as determined by Biacore. Models of JBT0740 and JBT1857 in complex with TFPI KDI (residues 22-79 of TFPI160) were calculated by HADDOCK using similar constraints as for JBT0303: (a) the constraints for the torsion angles of residues 4 and 5 of JBT0740 and JBT1857 were amended in order to take account of the substitutions at position 5 of the JBT0303 derivatives; (b) torsion angles were taken from the calculations of TALOS for V7, FS, Y12--A18 of JBT0303 and, in contrast to 3BT0303, no fixed values for Phi and Psi were given for K4 and for NmetG5/dP5; (c) NmetG5 and dP5 are in the cis conformation; (d) residues of KDI with chemical shift changes of more than 1.5 ppm are involved in binding to JBT0303: F25, F28. D32, A37, 138, 146, F47, T48, F54 and Y56; (e) the hydrophobic side of the amphipathic helix of JBT0303 is bound to KDI; (f) Y12 or L16 or L20 of JBT0303 bind to D32 or A37 or 138 or F54 or Y56 of KD1; (g) residues R15 or K19 of JBT0303 bind to D31 or D32 or E60 of KD1; (h) residues F8 or V9 of JBT0303 bind to F25 or F28 of KD1; (i) : - Q residues Y12 or F3 of JBTO303 bind to 146 or F47 or T48 of K-'D1 and )residue Q2 of 1BT0303 binds to Y56 of KD 1 [00353] The energetically most favorable HADDOCK models of JBT0740 and JBT1857 illustrated a different mode of binding compared to JBT0303. The most obvious differences were in die region of residues 5 to 11. Less dramatic deviations were observed at the N teninus and the C-terminus of the peptides. However, the different binding of the termini also might contribute to the optimized binding of the JBT0303 derivatives to TFPI. X-ray Crystal Structure of JBT1 857 bound to KD [00354] The crystal structure of KD1 in complex with a KD1 binding peptide, JBT1857, was determined. TFPI was recombinantly expressed in .coli and oxidatively refolded from inclusion bodies. TFPT amino acids 1-150 comprising a thrombin cleavage site within the TFPI linker sequence joining KD1 and KD2 (TFPI1-150-Thrombin (MADSEEDEEHTIITDTELPPLKLMHSFCAFKADDGPCK AIMKRFFFNIFTRQCEEFI YGGCEGNQNRFESLEECKKM CTRDNANRLVPRGSQQEKPDFCFLEEDPGICRGYI TRYFYNNQTKQCERFKYGGCLGNMNN'ETLEECKNICEDG (SEQ ID NO: 4235)) was cloned into an . coli expression vector (pET19b). The TFPI 1-150-Thronbin sequence comprises two amino acids at the N-terminus that are artifacts of recombinant expression, and are not part of the wld-type TFPI amino acid sequence. The sequences encoding Kunitz domain I and 2 are bolded. E coli (BL24DE3) pLysS) was cultivated in MagicMediam and TFPI 1-150-Thrombin was expressed as insoluble inclusion bodies. Inclusion bodies were harvested by lysis of F. coIJ by incubation with BugBuster Master Mix and purified upon washing with 50 mM TrIs/HCI pH 8 01% Tween 20, inclusion bodies ere dissolved in 8M urea, 50 mM Thirs/HCI pH- 8.0 and TFPI I l50drhrombin was reduced upon addition of 20 mM DTT. Oxidative refolding was performed by rapid 1/10 dilution into a buffer containing 50 mM Tris/HCI pH 10 and 1.1 mM oxidized Glutathion, followed by excessive dialysis against 20 mM Tris/RCI pH 7, Refolded TFPII -150-Thrombin was purified by a sequential purification protocol using a Q Sepharose FF anion exchange and a peptide affinity (JBT131) media. Purified TFPI1-150-TFP] was proteolytically digested by incubation with thrombin (IU thrombin / tg TFPI -150-Thrombin, cleavage site, LVPR/GS) resulting in the generation of Nterm 11-Thrombin (MADSEEDEEHThTDTELPPLKLMHSFCAFKADDGPCKAIMKRFFFNIFTRQCEEFI GGCEGNQNRFESLEECKKMCTRDNANRLVPR (SEQ I) NO: 4236)) and KD2 Thrombin (GSQQEKPDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLE I Inl ECKNICEDG (SEQ ID NO: 4237)). Nterm KDI-Thrombin was purified from the digestion mixture using benzamidin sepharose for removal of thrombin, followed by a JBTI3 I peptide affinity column, Purified Nterm KDI-Thrombin was used for complex formation with JBT1857 and further crystallization. [00355] The antagonistic peptide, JBT1857, was prepared by solid phase synthesis. Successful co-crystallization of equinolar complexes was obtained under 100 mM MIS pH 6.5, 20% PEG 4000, 600 mM NaCL. Crystals diffracted to better than 2.5 A resolution, albeit with some non-merohedral twinning. Diffraction data were processed with iMosfim and SCALA from the CCP4 program package, revealing a monoclinic crystal form with unit cells dimensions of a= 113.67 A, b= 69.32 A, c= 42.37 A, a= 90.0*, [= 92.97', Y= 90.0', spacegroup C2 (Leslie, Acta Crystallogr D Biol Crystallogr, 62(Pt 1), 48-57 (2006); Evans, Acra Ciystallogr D Biol Crystallogr, 62(Pt 1). 72-82 (2006)). Self-rotation calculations indicated an approximately two-old non-crysalographic symmetry. Consistent herewith, two molecules were localized in the asymmetric unit related by a 170 rotation. The Patterson search was carried out by using the program PHASER and a structure ensemble of the available Kunitz domain 2 crystal structures as search model (McCoy et al, JApt Crystallogr, 40(Pt 4), 658-674 (2007)). The unit cell contained approximately 64% solvent. Non-crystallographic electron density averaging and model building and model refinement was carried out with Coot, Refmac, MAIN and CNS programs. The current model was completely defined for both copies of the JIBTI 857 peptide and the interaction with the protein with current R=0.257, Rfree=0.298, deviation from ideal geometry rns(bond) = 0.008 A, rms(angle) = 1.8'. [00356] 11T1857 surucure: The structure of JBT857 can be segmented into (i) the N terminal anchor consisting of acetylated PhelAp-Gln2AF; (ii) an 2-shaped loop comprising Ser3.y-Asn6,4y; (iii) an intermediate segment built from Val7,v and HisSAp; (iv) a tight glycine-loop containing Val9.-Gly11A; and (v) the C-terrninal a-helix comprising Tyri 2p Leu20Ap. As used herein, the subscript AP indicates the sequence numbering in the 'antagonistic peptide" JBT1857. The conformation of the a-helix is stabilized by a non natural a-methyl alanine positioned at the center of the helix (position 17Ap); a C-terminal amide that completes the 1-4 hydrogen bonding pattern of the a-helix; and a stacked cluster by the aromatic side chains of His8Ap, Tyrl2Ap and Phe13AP. These effects cooperate to stabilize the C-terminal a-helix spontaneously in solution, consistent with circular dichroism data on the peptide. The observed aromatic side chain stacking (His8Ap, Tyrl2Ap, Phel3Ap) enforces a tight tum that can be only accomplished by glycine at position P T his structural constraint is reflected by dramatic losses in binding affinity upon replacement of Glyl 1,p by any other amino acid. The conformation of the N-terminal loop segment is partly stabilized by a D-proline. known to induce a tight turn conformation, and a 1-4 hydrogen bond by the carbonyl oxygen of Ser 3 AP with the aide nitrogen of Asn6, . All ring side chains (TyrI p, Pro5AP, His8p, Tyr12,T. Phe 3 AP) point towards the same direction, enabling them to interact with the KD 1 domain of TFPI [00357] Interaction qf JBT7857 and KD: The interactions between JBT1857 and KDi were determined, Hydrophobic contacts are interactions having an intermolecular distance of 5 4 A, while hydrogen bonds have a distance between 2.6 - 3.2 A. Phel AP interacts non specifically with TFPI making contacts with Phe2 and Ala27. In contrast, Gln2A contacts a deeply buried pocket of TFPI and makes hydrophobic interactions with Phe28, Lys29, Ie46 and Phe47. Moreover, the anide group of Gln2p forms three H-bonds with Phe28-CO, Phe44-CO and Ie46-NH. The D-loop of JBTI1857, comprising Ser3A-Asn6AP, mediates rather limited hydrophobic interactions with the protein; Ser3a, PrO5Ap and Asn6e interact with Lys29 and Phe47. Valp of JBTI857's intermediate segment also binds to Lys29 and Phe47. HisSAp mainly contributes intramolecular aromatic stacking interactions with Tyr12,x and partly with Phe13,w, and exhibits a hydrophobic interaction with Ala3O of TFPL Similarly, the glycine-loop VaI9M-Glyl I contributes few contacts with the Kunitz domain. Val9" interacts directly with KDI by forming a hydrogen bond with the carbonyl group of Ala30 and a hydrophobic interaction with Asp32. Tyr2p mediates a hydrogen bond via its hydroxyl group with the amide nitrogen of Ile55 and a hydrophobic interaction with Asp3O. Leu16Ap is part of a hydrophobic contact with Ile55. Beside the largely hydrophobic interactions of the C-terminal helix of the peptide with the protein, there are electrostatic interactions between Argl5&x and Asp32. Furthermore, Lys 19 A contributes to binding with TFPI by forming a hydrogen bond to the carbonyl group of Ala37 and contacts with Lys36 and 11e38. The TFPI contact surface has an overall hydrophobic character with some charted hot spots, and a driving force of complex formation with JBTI 857 is the steric surface complementarity. [00358] This example describes characterization of the secondary structure of exemplary peptides of the invention and correlates the structure. with inhibitory function of the peptides. The example also identifies the TFPI amino acid residues that interact with JBT1857, a TFPI binding peptide that inhibits TFPI activity. 1'21 100359] The following example describes additional TI-binding peptides modified by the addition of moieties that enhance physicochemical or pharmacokinetic properties of the peptides. The example further describes a method for assessing clot formation in whole blood using rotation thromboelastography. [00360] JBTI1857 (3BT0047 peptide family) was conjugated to different PEG moieties, and the binding affinity and 'FlPI inhibitory activity of the PEGylated peptides were examined. JBT1857 was modified by addition of a C-terminal cysteine to produce JBT2315 (Ac-FQSKpNVHVDGYFERL-Aib-AKLC-NH2 (SEQ ID NO: 4077)), which was conjugated at the C-terminus with linear maleimide PEG moieties of increasing size: 5 kD, 12 kD, 20 kD, 30 kD, and 40 kD, using the methods desfnbed in Example 5. The resulting PEGylated peptides were designated as follows: TABLE 12 Pcptnh.I PEG(k)Seunc SEQ 11) ___ ____ ____NO JBTI857 Ac FQSKpNVIiVDGYFERL- Aib-AKL-NH2 4020 JBT2325 5 c-FQSKpN VHiVDGYFERL-Aib-AKLC.(PEG)-NH12 4086 JIBT232' 1 Ac-FQSKpNVHVDGYFERL-Aib-AKLC(PEG)-NH2 4087 JET227 2 ~0 Ac FQSKpNVHVDGYPERL-Aib-AKCPG H48 JBT2328 I 29 1 Ac FQSKpNVH VDGYFERL-Aib-A.KLC(PEG) NH2 4089 JBT2329 4L5 Ac-FQS NpNVH VDGYFERL-Aib-AKLC( PEG)-NH2 4090 Sabity, binding affAncy and TFP-inhibitoy activity of PEGylated pepids 4 [00361] The PEGylated peptides demonstrated significantly increased plasma stability in mouse and human plasma. The peptides were added to samples of rnouse or human plasma, and the percentage of the initial amount of peptide remaining in plasma 24 hours after the addition was measured by IC 5 0 ELISA on Maxisorp plates coated with 0.05 mg/iMl TFPI (2.26 M tracer peptide JBT227 1). Less than approximately 10% of the initial amount of JBT1857 and JBT2317 remained in plasma, while 40% or more of the initial amount of the PEGylated TPPIbinding peptides remained after 24 hours. Approximately 60% or more of PqAJ~'. 1 PA1 1BT2327 and JBT2329 was detected. PEGylated peptides also are significantly more stable in human plasma compared to unmodified peptides. Approximately 60% or more of PEGylated peptide remained after 2.4 hours. The unmodified peptides were more stable in human plasma than mouse plasma; about 20% or more of the initial amount remained after 24 hours of incubation. [00362] The PEGylated peptides also were characterized in the assays described in Examples 1-4 and compared to JBT 1857. Representative results are provided in Table 13 set forth below. The thrombin generation assay was performed as described in Example 4, and the results are provided as ECqo, corresponding to the concentration of peptide which improved peak thrombin (nM) half maximal, TABLE 13 I Thrombin FExtrinsic generation Bicr Competition 1 i Tenase in human ELSA Inhibition FV111 PEC ICQ inhibited (nMf) EC0 (kD ) (nM)M plasm (nM) (nM EC6 nRM) JBT1857 0,061 3,0 37 6.9 JBT2317 0054 29 38 78 88 JBT2325 5 3 0.71 6 6 10 7 35 JBT2326 1 1 11 93 93 93 34 JBT2327 21,0 L-3 10,9 72 7 2 2-4 JTB1328t29 I 12.3 1 0 691 'jBT2329 4I~ 11 .5 -2-- 60 J, 0 Competition ELISA performed with tracer J3T2271 (1 nM) and 05 ONgn/ml TFPI in the coating buffer. [00363] Addition of the C-terminal cysteine blocked with NEM did not significantly influence the binding affinity of JBT2317 or the activity of the peptide in the FXa inhibition, extrinsic tenase assay, or thrombin generation assay compared to JBT1857. PEG size did not significantly impact the TFPI-binding peptides' ability to restore activity of FXa in the presence of TFPI-1. In the extrinsic tenase assay of Example 3, inhibitory activity increased with higher molecular weight PEG moieties up to 20 kD PEG. Activity did not further improve for 30 kD or 40 kD PEG moieties, In the thrombin generation assay of Example 4 using human plasma, EQ 3 decreased with PEG size, and maximal inhibition of TFPI(as measured by peak Fla (nM)) increased with PEG size. In mouse plasma, attachment of 40 kD PEG to a TFPI binding peptide increased maximal inhibition of TIPL. [00364] The ability of PEGylated TFPI-binding peptides to restore exrnsic tenase complexactivity forlconverting|FX to FXa also lwas determined using a ceibbased extrinsic tenase assay using the method of Example 5. Addition of the C-tenninal cysteine blocked with NEM did not significantly influence the activity of JBT2317 in the cell-based extrinsic tense assay compared to JBTI857. Conjugation of PEG moieties (5 kD, 20 D), 30 kD, or 40 kD) to PBT2317 increased TFPI inhibitory activity by 5-20%. Rotational thromboelastography [00365] Continuous viscoelastic assessment of human whole blood clot formation am firmness was performed by rotation thromboelastography with whole blood preparations in the presence or absence of peptides. Blood samples from a healthy individual were drawn into citrated Sarstedt Mono S (0.106 M or 3.2% (w/v) Na-citrate) (5 ml), mixing one part of citrate with nine parts blood, using a 21. gauge butterfly needle. A portion of the blood samples was incubated with high titer, heat-inactivatedanti-human EV! antiserumraised in goat (3876 RU/mi; Baxter BioScience, Vienna, Austria) resulting in 51 BU/mL. Test samples were prepared by dissolving quantities of peptides in either DMSO or HEPES buffered saline (with or without 0 1% Tween 80). [00366] Recordings were made using a ROTEM thromboelastography coagulation analyzer (Pentapharm, Munich, Germany) at 37*C. Briefly, blood is added into a disposable cuvette in a heated cuvette holder. A disposable pin (sensor) is fixed on the tip of a rotating axis. The axis is guided by a high precision ball bearing system and rotates back and forth. The axis is connected with a spring for the measurement of elasticity. The exact position of the axis is detected by the reflection of light on a small mirror on the axis, The loss of elasticity when the sample clots leads to a change in the rotation of the axis. The data obtained are computer analyzed and visualized in a thromboelastogram. The thromboelastogram shows elasticity (mm) versus time (s). An elasticity of approximaely zero is observed before clot formation begins. Mirror image traces above and below the zero line indicate the effect of clot formation on the rotation of the axis. [00367] Before starting each experiment, the citrated whole blood was mixed with com trypsin inhibitor (CTI) (Hematologic Technologies, Inc., Essex Junction, VT, USA) providing a final concentration 62 pg/mL for specific inhibition of FXIIa, in order to inhibit FXlla-mediated contact activation. The analytical set-up was as follows: tol20 pL of test sample or control 300 p L of pr-warmed (37*C) CTI treated citrated whole blood was added, followed by 20 p L of a 1:15 dilution of TF PRP reagent containing recombinant human tissue factor (rTF, 3 pM) (TS4O, Thrombinoscope BV, Maastricht, The Netherlands). Coagulation was initiated by the addition of 20 pL 200 mM CaCi (star-TEM@, Pentapharm, Munich, Germany) and recordings were allowed to proceed for at least 120 min. The final concentration of rTF in the assay was 11 or 44 ff4 [00368] The thromboelastographic parameters of clotting time (CT), cl ot formation time (CR1') and maximum clot firmness (MCF) were recorded in accordance with the manufacturer's instructions. CT is defined as the time from the start of measurement to the start of clot formation. CF is defined as the time from the start of clot formation until an amplitude of 20 mm is reached. MCF is the maximum difference in amplitude between the two traces during the assay. The first derivative of the data of the thrormboelastogram are plotted to obtain a graph of velocity (mm/s) against time (s). From this graph, the maximum velocity (maxV) is determined. The time at which the maximum velocity is obtained (maxV t) is also determined, [00369] Exemplary results are illustrated in Figures 56 and 57. JBT1857 and JBT2317 restored coagulation parameters in Hem A blood. PEGylated (40 kID) TFPI-binding peptide JBT2329 also restored prolonged coagulation parameters in Hem A blood, as illustrated in Figure 57. PEGylation of JBT23 17 reduces clot time anid clot formation time, Nail Clip Study [003701 The effect of JBT2329 on blood loss in naive mice also was studied, C57BL6 mice were administered vehicle, 1 mg/kg JBT2329, or 0.1 mg/kg JBT2329 (N=19 or 20 for each group) intravenously 30 minutes prior to nail clip at 10 mL/kg. Animals were anaesthetized 10 minutes before nail clip with 80 mg/kg pentobarbital (i.p) At time=0 minutes, the nail of the small toe of the right hind paw was cut just before the nail bed. The paw was transferred to a vial prefilled with 0 r,% NaCI solution. Samples of blood were collected for analysis during the first 30 minutes following the nail clip and the next 30 minutes thereafter and mean collected volume for the groups was calculated and compared. Mean blood loss in vehicle treated mice was about 30.5 1 over the first 30 minutes, 52.1 p1 over the second 30 minute period, resulting in about 826 pl of blood loss over 60 minutes. In contrast, administration of 01 mg/kg JBT2329 reduced blood loss by about 50% over the first 30 minutes (16.0 p1) and about 64% over the second 30 minute period (18.7 1), resulting in about a 60% reduction of total blood loss over 60 minutes (34.7 P1) compared to I 1,Z vehicle-treated mice. Increasing the dose of JBT2329 to LO. mg/kg further reduced blood loss by at least about 10%; 12.2 pl was collected over the first 30 minutes, 10.6 pd was collected over the second 30 minute period, resulting in 22.8 pI collected over the entire 60 minute collection period. 1BT2329 also efficiently reduced bleeding when administered subcutaneously compared to vehicle-treated naive mice; subcutaneous injection of 10 mg/kg JBT2329 reduced blood loss during the 60 minutes following nail clip by approximately 58% compared to vehicle-treated subjects. [00371] The results described above were generated using a JBT1857 derivative comprising a linear PEG moiety attached to the C-tenninus of the peptide and a JBTI386 derivative comprising a PEG roiety at the N-terminus. Peptides comprising an altemate conjugation site or alternative chemical moiety aso were generated. A 40 kD linear PEG moiety was conjugated to residue 14 of JBT1 857 to generate JBT2404. The linear 40 kD PEG moiety of JBT2329 was replaced with a 40 kD branched PEG moiety to generate 1BT2401. JBT1.857 also was modified to comprise K(Ttds-Maleimidopropionyl) (JBT2374) at the C-terminus. JBT2374 was used to generate JBT2410, an HSA conjugate of JBT2374. JBT2375, a K(AOAy-comprising derivative of JBTI857 was used to couple PSA aldebyde to tde peptide JBT 857 resulting in JBT2430 JT240 JBT2404, JBT241 0 and JT2450 were characterized using the assays desi bed above. Representative results are summarized in Table 14: TABLE 14 T hrombin ESA ma generation Biacore affmity FPa mhuma KIa Inhbition Stabihty, 24 FIH

EC

0 nihM) JBT2329 < 1 266 67,' BT44 <1 18,2 134 6,77 JET2K Z41 n d. 4, 7678 JBT243u nd 5,6 9.,0\...... 135S.6 [00372] T his example demonstrates that an exempla y TFPI-binding peptide of the invention, JBT] 857, is a potent inhibitor of TFPI and can be functionalized and conjugated with PEG without loss of activity. PEGylation increased TFPI-inhibitory activity in several functional assays. Surprisingly, peptides conjugated to higher weight PEG moieties demonstrated enhanced TFPI inhibitory activity. JBT2329, comprising a 40 kD linear PEG moiety, significantly reduced blood loss in a clinically-relevant animal model. PEG conjugation within the amino acid sequence of JBT] 857, use of a branched PEG moiety, and attachment of HSA and PSA did not destroy the activity of the peptide. Exam e_ 9 [00373] The following example describes the characterization of two TFPI-binding peptides of the invention, JBT1837 and JBT1857. JBT1837 (Ac SYYKWU[CAMRDMKGTMTC]VWVKF-NH) (SEQ ID NO: 1044) is a cyclic peptide of the JBT0120 family that binds KDI and KD2 of TFPL JBT1857 (Ac FQSKpNVHV)GYFERL-Aib-AKLNH2) (SEQ ID NO: 178) is a linear peptide of the JBT0047 family that binds K~I of TFPL The affinity and TFPI-inhibitory activity of 1BT1837 and JBT1857 were examined using the assays described in Examples 1-4, the results of which are summarized in Table 15. TABLE 15 FXa trinsic Thrombin Thrombin E LISA Inhibition;- eaeeato eeao i reLSAfit R&hitin Inhibition; i human in human Biaore t PR R&D FVI- FIX T(nM) ET TFPI/ITFPI inhibited defeient (nM)) 0 ECEC plasma plasma (p)EC50 EC50 (nM) (HM) T874,8 32/59 C5/9 1 16 [003741 Affinity of the peptides to human TFPI measured via BiaCore was less than iM, Affinity measured by ELISA (IC 5 ,) was 4.8 nM for JBT1837 and 2.5 nM for JBT1857. JBT1837 dissociated from human TFPI more slowly than JBT1857 (i.e., JBT1837 remained bound to human TFPI for a longer period of time compared to JBT1 857). A FXa inhibition assay was performed using both full length human TFPI ("fITFPI") and truncated human TFPI (254 amino acids "R&D TFP) (0.1 nM FXa, 0.5 nM TFPI, 0.25% DMSO). Activity of the truncated TFPI was fully inhibited by both JBT1837 and JBT1857 at 0.5nM TFPI, while full length TFPI was inhibited 85% and 95% by JBT1857 and JBT1837, respectively. I1 787 At higher concentrations of fTP(e.g., 10 nM ITFPI), JBTIS137 fully inhibited TFP! activity, while JBTI857 partially inhibited TFPI activity. EC 5 's also were higher when f1TFPI was used in the FXa inhibition study. 1093751 Lithe extrinsic tenase assay, about 85% of truncated TIPI was inhibited by both peptides. Full length TFPT activity was inhibited about 56% and 48% by JBT1837 and JBT 857, respectively Surprisingly, in the cell-based extrinsic tense assay, JBT11837 inhibited the activity cell-associated TFPI by about 50% whereas JBT1857 almost fully inhibited cell- bound TFPI activity. In the plasma-based functional assay, iBTl837 inhibited TFPI more efficiently than JBT1857 in human FVIII-inhibited plasma and FIX-deficient plasma. JBTI 837 corrected blood coagulation parameters in FVIII-inhibited blood in the ROTEM assay described in Example 8. JBT1857 also positively impacted blood coagulation parameters, but performed less efficiently than JBT1837 in the assay. [00376] This example compared the affinity and TFPI-inhibitory activity of cyclic and linear TFPI-binding peptides that target different regions of the TFPI protein. JBT1837 (a cyclic peptide belonging to family JBT0120) and JBT1857 (a linear peptide belonging to family JBT10047) efficiently bind hunan TFPI with affinities less than I nM and are potent inhibitors. FXa-TFPI interaction is fully blocked at low TFPI concentrations by both peptides, while TFPI inhibition bV JBT1857 is reduced in the presence of higher concentrations of TFPL Both peptides partially inhibit the activity of full-length 1FF in the extrinsic tense assay, and IBT1857 inhibits TFPI activity to a greater degree in the cell based extrinsic tenase assay compared to .BT1837. Compared to 1BT1857, JBT1837 more efficiently inhibits TFPI in FYIIH-deficient plasma. Both peptides improve coagulation parameters of FVIIIbinhibited human whole blood by reducing clot time, while JBT 1857 improves clot formation velocity to a lesser degree compared to JBT1 837. [003771 This example lustrates the in vivo activityof TFPI-binding peptides of the invention in a clinically-relevant animal model, As described below,an exemplary TFPI binding peptide significantly reduced blood loss in an animal when administered with suboptimal doses of FVIII and FIX. [00378] JBT2329, a PEGylated (40 kD) TFPI-binding peptide (1BT0047 family) that cross-reacts with hunan and murine 1FFL, was tested in tail-tip bleeding model in EVI knock-out mice and FIX knock-out mice. FVIII knock-out mice closely mirror the condition of hemophilia A patients, and the tail-tip bleeding model is widely used in research to assess efficacy of drugs by measuring, e.g., bleeding time, blood loss or survival. ADVATE, a commercially available rFVIII, served as a reference, and ADVATE buffer-treated animals served as negative controls. Each group contained 16 FVIl knock-out mice (8 female + 8 male). 1BT2329 (1 mgkg or l mg/kg) or antiTFPI antibody (maTPI; 18 mg/kg) was administered 30 minutes before the tail-ip was cut. ADVATE (0 I/kg or 50 I mg/g) or ADVATE buffer was administered five minutes before the tail was cut off. Test and control substances were administered as an intravenous bolus via a lateral tail vein injection. Animals were anaesthetized by an intraperitoneal injection of 100 ng/g ketamine and 10 mg/kg xylazine. Approximately 10 minutes later, 2 mm of the tail-tip was cut off The tail tips were placed in warm saline (approximately 37 'C) and blood was collected over an observation period of 60 minutes. The amount of blood was determined gravimetrically. At the end of the observation period of 60 minutes the animals were humanely killed by cervical disocation before recovery from anesthesia, [003791 Median total blood loss in buffer-treated animals was 930 mg, Median total blood loss in subjects treated with murine anti-TFPI antibody (maTFPI) was 724 mg. The reduction in median total blood loss was more pronounced when the subjects were administered maTFPI with ADVATE. A combination of maTFPI + 10 Ri/kg ADVATE led to a median total blood loss of 136 mg, animals treated with maTFPI + 50 IU/kg ADVATE experienced a median total blood loss of 13 mg. Median blood losses of animals treated with either 10 or 50 IU&g ADVATE alone experienced median blood loss of 798 and 364 mg, respectively. The superiority of the combination treatment of maTFPI + ADVATE over ADVATE alone was statistically shown for maTFPI + 50 IU/kg ADVATE versus 50 IU/kg A DVATE (p = 0.0010). Although not statistically significantly superior, blood loss in animals treated with maTFPI + 1.0 IU/kg ADVATE was distinctively lower than in animals treated with 10 IU/kg ADVATE alone. [003801 Efficacy, defined as statistically significant superiority over buffer at a 2.5% level, was shown for JBT2329 dosed at 1 mg/kg in combination with 10 and 50 RI/kg ADVATE and dosed at 0.1 mg/kg in combination with 50 RI/kg ADVATE (p <0,0004). Animals treated with JBT2329 in combination with ADVATE showed a clinically-relevant reduction in blood loss, although the results were not statistically significant (p >0.0506). Administration of 1 mg/kg JBT2329 without ADVATE did not reduce median total blood loss over that observed in buffer-treated animals (930 mg).

(003811 J3T2329 also was tested in a FIX knock-out tail-tip bleed mouse model, which is a clinically-relevant model for hemophilia B human patients. The methodology was substantially similar to that described above with respect to the FVIII knock-out model. Instead of ADVATE, a recombinant FIX (rFIX) served as a reference. Median total blood loss in buffer-treated animals was 935 mg. Median total blood loss in animals treated with a murine anti-TFPI antibody (maTFPI) was 774 mg. Median total blood loss was reduced further when the animals received combined treatment of maTPPI and rEX A combination of maTFPI +10 U/kg rFiX led to a median total blood loss of 25 ug, while animals treated with maTFPI+± 50 lU/kg rFiX exhibited a median total blood loss of 10 mg Median blood loss of animals treated with either 10 or 25 lU/kg rFIX alone experienced a median blood loss of 888 and 774 mg, respectively [00382] Efficacy, defined as statistically significant superiority over buffer at a 2.5% level. was shown for JBT2329 when dosed at 1mg/kg in combination with 1.0 IU/kg rFIX and at 0.1. mg/kg in combination with 10 IU/kg rPIX. The superiority of JBT2329 in combination with rFIX over administration of rFIX alone was observed (p <0,0172), while treatment with I mg/kg JBT2329 alone did not lead to a significant reduction in median total blood loss compared with buffer-treated animals (p = 0321). 100383] In sunnary, JBT2329 promoted a clinically-relevant reduction of blood loss when co-adminitered with suboptinal doses of FII and rFIX at all doses tested. Furthermore intravenous administration ofBT2329 was welltolerated in all subjects across al treatment groups without any signs of acute toxicity. Exawple I11 [00384] The TFPI-binding peptides described herein are suitable for detecting TFPI in a sample, such as biological sample. This example describes a method for detecting TFPI using the inventive peptides in an ELISA-like assay format. [00385] The peptide sequence of BT85T was N terminally modified by the addition o f a biotinyl-Ttds moiety to generate JBT227 1 (Biotinyl-Ttds-FQSKpNVHIVDGYFERL-Aib AKL-NH2 (SEQ ID NO: 403|3)). A 96-well microtiter plate (Maxisorp, Nunc) was coated with 50 pl per well coating buffer (15 mM NaCOs 35 mM NaHCO, pH 93) containing a range of TFPI concentrations (0-3 pg/nl huania recombinant TFP, R&D Systems)for 1 hour at room temperature. The plate was washed three times with 350 pl/well wash buffer (175 mM NaCl 5 mM CaCl 2 25 mM HEPES, 01% Tween 80, pH 75)7 The plate was then blocked with 100 p1 blocking buffer (2% yeast extract, 175 mM NaCL 5 rM CaCI, 25 mM HEPES. 0.1% Tween 80, pH 7.35) f"or 1 hour at room temperature. The plate was then washed three times with 350 p1 wash buffer. Fifty p1 of differently concentrated JBT227 1 solutions in wash buffer (100-0 iM) were added to each well The plate was incubated for I hour and washed three times with 350 p1 wash buffer. To each well, 50 1 streptavidin horseradish peroxidase conjugated(R&D Systems, 1:200 in wash buffer) is added. After an incubation period of 1 hour at room temperature, the plate was washed three times with wash buffer. Fifty pl TMB solution (SerarnunBlau fast, Seramun) was added to each well. After a 1.5 minute incubation at room temperature, the reaction was stopped by adding 50 p1 1 M 112804 per well. Absorbance was measured in a photometer (Molecular Devices Spectramax M5) at 450 and 620 nm, [00386] JBT2271 allowed detection of as little as 4.1 x 1014 mole of TFPI per well. The results of the assay described above illustrate that the inventive peptides are powerful tools for identifying and/or quantifying TFPI in a sample. [003871 This example describes conditions for an exemplary k 4 assay for characterizing TFPI-binding peptides. [00388] Wells of a microtiter plate (96 wells, Maxisorp, Nunc) are coated with 1.6 nM TFPI in coating buffer (15 mM Na 2

CO

3 , 35 mM NaHCO 3 , pI 9.3) for two hours at room temperature. The plate is then washed three times with 350 pl wash buffer (175 mM NaCI, 5 mM CaCI 2 , 25 mM HEPES, 0.1% Tween 80, pH 7,35), and wells are blocked with 100 p1 blocking buffer (2% yeast extract, 175 mM NaCl, 5 mM CaClI 2 , 25 mM HEPES 0.1% Tween 80, pH 7.35). If an incubation period of 24 hours is employed, the wells are blocked for at least one hour. Control wells used for a15 minute incubation period are blocked for an additional 23.5 hours [00389] For a 24 hour incubation period, the wells are washed three times with 350 pI wash buffer and are incubated with 50 pl test peptide in wash buffer. The concentration of test peptide depends on the individual IC% 0 concentration determined in, e.g., the TFPI ICs ELISA assay described herein, The TFPI-coated wells are exposed to test peptide for approximately 15 minutes. The wells are subsequently washed three times with 350 pl wash buffer and 50 pl tracer peptide (competitor) is added. An exemplary tracer peptide is JBT22 113 nM in wash buffer) Control wels (maximumsignal) are incubated with tracer only. Blank wells tacking TFPL are incubated with tracer only. Addition of the tracer peptide commences the 24 hour incubation period. [003901 A 15 minute incubation period is employed as a control if the IC 90 concentration of the test peptide leads to a 90% reduction of the maximum signal. Wells blocked for an additional 23.5 hours are washed three times with 350 pul wash buffer to remove the blocking buffer, Subsequently, 50 plI analyte in wash buffer is added and the wells are incubated for 15 min, The concentration of test peptide utilized depends on the peptide's IC 90 concentration determined using, e.g., a TFPI ICso ELISA assay. The 15 minute incubation is followed by three washes with 350 pl wash buffer and addition of 50 pl tracer peptide. Control wells (maxium signal) are incubated with tracer only. Blank wells lacking TFPI also are incubated with tracer only. [00391] The plate is washed three times with 350 pi wash buffer, and 50 p streptavidin horseradish peroxidase conjugate (R&D Systems, 1:200 in wash buffer) is added to each well. After an incubation period of one hour at room temperature, the plate is washed three times with wash buffer. TMB solution (50 pl per well; Seramunflau fast, Seramun) is added. After a 1.5 minute incubation at room temperature, the reaction is stopped by the addition of 50 p1 1 M H 2 S0 4 per well. Absorbance is measured using a photometer (Spectramax M5, Molecular Devices) at 450 and 620 nm. The assay results are presented as a percentage of the corrected optical density (OD450-OD620) of wells exposed to test peptide and tracer peptide in relation to TFPI-coated wells exposed only to tracer. Example 13 [003921 TFPI inhibits FVIIa/TF activity by binding to FVTa via Kunitz domain i (KDi). This example describes an exemplary method for evaluating the influence of TFPI-binding peptides on TFI' s inhibition of FVIla/TF. [00393] Kinetic measurements were performed in 25 mM HEPES, 175 mM NaCl, 5 mM CaC12, 0.1% BSA, pH 7.3 at 25 C in 96-well microtiter plates. Twenty pl soluble tissue factor (residues 33-251; Creative Biomart) and 20 pl FVIIa (ERL) at final concentrations of 100 nM and 5 nM, respectively, were mixed and incubated for 15 minutes, Twenty si of TFPI-binding peptide in varying final concentrations (0-2 pM) were added to the mixture and incubated for a further 15 minutes. In order to measure the residual activity of the FVIIa/sTF complex, the reaction mixture was incubated for 60 minutes with 20 pl TFPI (200 nM). The reaction was initiated by the addition of a chromogenic substrate, Chromozym-tPA (Roche) I A4 11 (1 mM). The change in absorbance at 405 nm was monitored by using a Labsystems iEMS ELISA Reader for 30 minutes. FVIIa/sTF activity measured in the absence of TFPI was considered "100% activity" in the context of the assay. By plotting peptide concentration against residual activity, EC 50 values were determined. [00394] JBT1857 and JBT1837 were screened against TFP160, TFPI1-150-Thrombin, NTermKD1, KDI, and KD2 (negative control). JBT1857 demonstrated an EC 50 of approximately 0.21-0.23 pM for TFPI160, TFPII-150-Thrombin, NTermKD1, and KD1. JBTI 837, which binds KD1 and KD2, demonstrated an EC 50 of approximately 0.17-0.19 PM for TFPII.60 and TFPII-150-Thrombin, while activity in assays involving NTermKD1 and KD1 was approximately background. [003951 The results described above demonstrate that TFPI-binding peptides efficiently inhibit TFPI-FVIa/TF interaction. JBT1857 efficiently inhibited TFPI fragments containing KD1 as a minimal functional entity. Thus, this enzymatic assay confirms X-ray crystallographic data placing the binding site of JBT1857 within KDl. JBT1837 inhibited TFPI fragments containing the first two Kunitz domains, suggesting that the JBT1837 binding site(s) are located within KD-linker-KD2 region of TFP. A combination of Kunitz domains and fragments of a thrombin cleaved TFPI (1-150) did not restore inhibitory activity of JBT1837 in the chromogenic assay. The enzymatic assay described herein is a suitable surrogate for detecting binding of a TFPI-binding peptide (or a test compound) to TFPI, and is useful for examining the TFPI-inhibitory effect of TFPI-binding compounds. Exa1mle 14 [00396] This example describes the influence of PEG and HSA conjugation on exemplary TFPI-binding peptides in vivo. [00397] For pharmacokinetic analysis, C57B16 mice were treated with various TFP binding peptides conjugated to different molecular weight PEGs and HSA. The dose of the peptide-PEG and peptide-HSA conjugates was normalized to 1 mg/kg (peptide content). Normalization assures comparability between the conjugates of different molecular weight. The peptide conjugates were dissolved in 175 mM NaCl, 25 mM IEPES pH 7.35 and administered intravenously via the tail vein or subcutaneously in the neck region. Blood draws were taken from three animals (retro bulbar) and collected in heparinized vials at several time points following administration. The samples were centrifuged, and the peptide conjugate content in plasma was quantified by ELISA.

[00398] Figure 63 lustrates the concentration of PEGylated TP-peptides deleted in plasma at several time points following administration, and Table provides detailed information about the terminal half life and bioavailability of JBT2325-JBT2329, JBT2401, JBT2404 and JBT24-10. TABLE 16 T2325 BT2326 BT2327 BT2328 T2329 BT240 IT240 B410 1L x 7 [h] 06 0.35 42 10 198 20.7 12.3 (intravenous) Bitavailabili y [% 58.2 76.0 89, 52.0 73. 58.4 59-3 46,6 [00399] JBT2329, JBT2401 and JBT2404 are peptides conjugated to 40 kDa linear PEG (JBT2329 and JB12404) or 40 kDa branched PEG (3BT2401). The 40 kDa conjugates exhibited a longer terminal half-life (HLXz) compared to peptides conjugated to smaller PEGs following intravenous administration, The area under cume (AUC) of the concentration-time curve resulting from subcutaneous administration of the peptides was compared to the AUG generated following intravenous administration to calculate the bioavailability of the peptides. Results are shown in Table 16. The data demonstrate that TFPI-binding peptide conjugation to higher molecular weight molecules allows a subcutaneous bioavailabilitv of more than 30%. [004001 Figures 64AC illustrate the pharmacokinetic profile of JBT2401, JBT2404, and JBT2410 resulting fom subcutaneous and intravenous administration of the peptides to mice JB3T2404 comprises a PEG conjugated to cysteine in position X401 relative to formula (Xl). JBT2401 comprises a branched PEG, and JBT2410 is conjugated to HSA. Figure 64A demonstrates that fusion of a higher molecular weight molecule to a TFPI-binding peptide at an internal position increases half life., Half life also is increased if using branched PEG (JBT2401) and HSA, which increased the in vivo half life of JBT2410 compared to conjugates having smaller-sized PEGs (e.g., JBT2325) or free peptide (see Figure 31). [00401] This example illustrates that the in viv properties of various peptides described herein can be improved by conjugation wvith higher molecular weightmolecles (like PEG) and/or with n~eR ligands (ike HISA). 1 A

Claims (13)

1. 2. The peptide according to claim 1, wherein X4001 is an amino acid selected from the group consisting of F, Y, I Ni, Bta, and Dopa; wherein X4003 is an amino acid selected from the group consisting of D, E, and S; wherein X4004 is K; wherein X4005 is an amino acid selected from the group consisting of p, Nmg, NpropylG, aze, pip, tic, oic, and hyp; wherein X4006 is an amino acid selected from the group consisting of C, E, K, R, S, V, C(Acm), Nie, C(NEM), I and Cit; wherein X4007 is V or TIle; wherein X4008 is an amino acid selected from the group consisting of H, 1Ni, 2Ni, and Pmy; wherein X4009 is an amino acid selected from the group consisting of V, Abu, and Tie; wherein X4010 is an amino acid selected from the group consisting of D, P, C, and T; wherein X4011 is an amino acid selected from the group consisting of G, a, c, hey, and Sar; wherein X4012 is Y; wherein X4013 is an amino acid selected from the group consisting of F, 1Ni, and Bta; wherein X4014 is an amino acid selected from the group consisting of Ab, C, B, and Hey; Ilarl-UPIOCIC~d e-e P7 r l;cIl)01 -stn fA ,E wherein X4016 is an amino acid selected from the group consisting of L, An, He, and Hey; wherein X4017 is an amino acid selected from the group consisting of A, Aib, C, c, Aic, Eca, and Deg; wherein X4018 is an amino acid selected from the group consisting of A, Aib, C, c, L, and Hey; wherein X4019 is K; and wherein X4020 s an amino acid selected from the group consisting of L, Am, and HeIy.
2. 3. The peptide according to claim I further comprising N-terminal amino acids) and/or moieties linked to X4001 and selected from the group consisting of FAM-Ttds, PE, Palm, 2 phenyl acetyl, 3-phenyl propionyl, 2-(naphtha-2-yl) acetyl, hexanoyl, 2-methyl propionyl, 3 methyl butanoyl, 2-naphthylsulfonyl, and 1-naphthylsulfonyl.
3. 4. The peptide according to claim I further comprising X4021 linked to X4020, wherein X4021 comprises C-terminal amino acid(s) and/or moieties selected from the group consisting of C, c, C(NEM), K(Ttds-maleimidopropionyl(EtSH)), FA19205, FA19204, FA19203, FA03202, K(Tdts-maleimid), K(AOA), and Cea. The peptide according to claim 3 further comprising X4021 linked to X4020, wherein X4021 comprises C--terminal amino acid(s) and/or moieties selected from the group consisting of C, c, C(NEM), K(Ttds-maleimidopropionyl(EtSH)), FA19205, FA19204, FA19203, FA03202, K(Tdts-maleimid), K(AOA), and Cea.
4. 6. The peptide according to any one of claims I to 5, wherein the peptide comprises a cyclic structure.
5. 7. The peptide according to claim 6. wherein the cycli structure is formed between X4018 and X4021. I AID
6. 8. The peptide according to claim 7 wherein (a) X4018 is C ore and (b) X4021 is Cea.
7. 9. The peptide according to claim 6, wherein the cyclic structure is formed between X4011 and X4014,
8. 10. The peptide according to claim 9, wherein (a) X401 1 is c or hcy and (b) X4014 is C or Hcy. 1. The peptide according to any one of claims I to 5, comprising an intramolecular disulfide bond.
9. 12. The peptide according to any one of claims 1 to 5, wherein the IC 50 of the peptide is less than 1000 nM. 13 The peptide according to any one of claims 1 to 5, wherein the ICm of the peptide is less than 250 nM.
10. 14. The peptide according to any one of claims 1 to 5, wherein the IC 53 of the peptide is less than 50 nM.
11. 15. The peptide according to any one of claims I to 5, wherein the IC 0 o of the peptide is less than 10 1M1
12. 16. A peptide consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 4022, 4024, 4032, 4036-4047, 4049-4078, 4086-4097, 4100-4127, 4129-4170,
13. 4173-4195, 4200-4214, 4217-4225, 4228, 4230, 4231, 4238, and 4239. 1 A V 17. A peptide consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1294-1336, 4002, 4013, 4021, 4023, 4025-4031, 4033-4035, 4048, 4079-4085, 4098, 4099, 4128, 4171, 4172, 4196-4199, 4215, 4216, 4226, 4277, 4229, 4232,. and 4233. 18. A TFPI-binding peptide comprising a homo-dimer or homo-multimer of two or more peptides according to any one of claims 1-5, 16, or 17. 19. A TFPL-binding peptide comprising a hetero-dimer or hetero-multimer of two or more peptides according to any of the claims 1-5, 16, or 17. 20, The peptide according to any one of claims 1-5, 16 or 17, wherein the peptide inhibits TFPI activity and binds to TFPI 1-alpha with a dissociation constant of less than 10 pM. 21, The peptide according to any one of claims 1-5, 16, or 17, wherein the peptide is conjugated to a polyethylene glycol (PEG) moiety. 22. The peptide according to any one of claims 1-5, 16, or 17, wherein the peptide is conjugated to human serum albumin (HSA), an antibody or fragment thereof, hydroxyethyl starch, a proline-alanine-serine multimer (PASylation), a Cl 2-C 18 fatty acid, or polysialic acid. 23. The peptide according to any one of claims 1-5, 16, or 17, wherein the peptide is conjugated to a moiety selected from the group consisting of a photosensitizcr, dye, a fluorescence dye, a radionuclide, a radionuclide-containing complex, an enzyme, a toxin, an antibody or fragment thereof, and a cvtotoxic agents 24. A peptide according to any one of claims 1-5, 16, or 17 for use in a method for the treatment of a subject. 25. The peptide according to claim 24, wherein the method is for the treatment of a blood coagulation disorder. 26. Use of the peptide according to any one of claims 1-5, 16, or 17 for the manufacture of a medicament. 27. Use of the peptide according to any one of claims 1-5, 16, or 17 for the manufacture of a medicament for the treatment of a blood coagulation disorder. 28. A pharmaceutical composition comprising the peptide of any one of claims 1-5, 16, or 17 and a pharmaceutically acceptable carrier. 29. The pharmaceutical composition according to claim 28, wherein the composition comprises a further pharmaceutically effective agent. 30. The pharmaceutical composition according to claim 28, wherein the pharmaceutical composition is for use in a method of treating a blood coagulation disorder. 31. A method for targeting a cell displaying TFPI, the method comprising contacting the cell with the peptide of any one of claims 1-5, 16, or 17. 32. The method of claim 31, wherein the cell is in a mammal, and contacting the cell comprises administering the peptide to the mamma. 33 The method according to claim 31 further comprising detecting peptide binding to TFPI displayed on the cell. 34. The method according to claim 33, wherein peptide-TFPI binding is detected by detecting a moiety conjugated to the peptide and selected from the group consisting of a dye, 1 Cl a fluorescence dye, a radionuclide, a radionulidecontaining complex, an enzyme, a toxin, an antibody, and a cytotoxic agent. 35. The method according to claim 33, wherein peptide-TFPI binding is detected by detecting an interaction partner complexed with the peptide or a moiety conjugated to the peptide. 36. The method according to claim 35, wherein the interaction partner is selected from the group consisting ofan antibody or fragment thereof, an anticalin, aptaer, streptavidin avidin, neutravidin, and a spiegeimer. 37. The method according to claim 35, wherein the interaction partner comprises a detection moiety. 38. The method according to claim 37, wherein the detection moiety is selected from the group consisting of a dye, a fluorescence dye, a radionuclide, a radionuclide-containing complex, and an enzyme. 39. A method for treating a subject suffering from a disease or being at risk of suffering from a disease, the method comprising administering to the subject the peptide of any one of claims 1-5, 16, or 17, wherein the peptide is conjugated to a therapeutic agent. 40. The method according to claim 39, wherein the disease is a blood coagulation disorder. 41. A method for treating a subject suffering from a disease or being at risk of suffering from a disease, the method comprising administering to the subject the peptide of any one of claims 1-5, 16, or 17, and administering to the subject an interaction partner that (a) binds the peptide and (b) is a therapeutic agent or is conjugated to a therapeutic agent. 42 The method according to claim 41 wherein the therapeutic agent is selected from the group consisting of a photo sensitizer, a radionuclide, a radionuclide-containing complex, an enzyme, a toxin, an antibody or fragment thereof, and acytotoxic agent. 43. The method according to claim 42, wherein the disease is a blood coagulation disorder. 44. The method according to claim 41, wherein the interaction partner is selected from the group consisting of an antibody or fragment thereof,a anticalin, an aptamer, streptavidin, avidin, neutravidin, and a spiegelmer. 45. The method according to claim 44, wherein the therapeutic agent is selected from the group consisting of a photosensitizer, a radionuclide, a radionuclide-containing complex, an enzyme, a toxin, an antibody or fragment thereof, and a cytotoxic agent. 46. The method according to claim 41, wherein the disease is a blood coagulation disorder. 47. A method for diagnosing a subject suffering from a disease or being at risk of suffering from a disease, comprising (a) administering to the subject the peptide of any one of claims 1-5, 16, or 17 conjugated to a detectable moiety and (b) detecting the detectable moiety. 48. The method according to claim 47, wherein the detectable moiety is selected from the group consisting of a dye, a fluorescence dye, a radionuclide, a radionuclide-containing complex, an enzyme, and an antibody or fragment thereof. 49. The method according to claim 47, wherein the disease is a blood coagulation disorder. 1 G 50. A method for diagnosing a subject suffering from a disease or being at risk of suffering from a disease, comprising (a) administering to the subject the peptide of any one of claims 1-5, 16, or 17, (b) administering to the subject an interaction partner conjugated to a detectable moiety, and (c) detecting the detectable moiety. 51. The method according to claim 50, wherein the interaction partner is selected from the group consisting of an antibody or fragment thereof, an anticalin, an aptamer, streptavidin, avidin, neutravidin. and a spiegelmer. 52. The method according to claim 50, wherein the detectable moiety is selected from the group consisting of a dye, a fluorescence dye, a radionuclide, a radionuclide-containing complex. an enzyme, and an antibody or fragment thereof. 53, The method according to claim 50, wherein the disease is a blood coagulation disorder. 54. A method for purifying TFPI, wherein the method comprises a) contacting a sample containing TFPI with the peptide of any one of claims 1 5, 16, or 17 under conditions appropriate to form a complex between TFPI and the peptide; b) removing the complex from the sample; and, optionally, c) dissociating the complex to release TFPL 55. The method according to claim 54, wherein the peptide is immobilized to a support. 56. The method according to claim 55, wherein the peptide immobilized to a chromatography stationary phase, and step (c) comprises eluting TFPI bound to the immobilized peptide. 57. The method according to claim 56, wherein TFPI is purified via affinity chromatography. 58. A method for identifying a TFP-binding compound, the method comprising (a) contacting a peptide comprising TFPI Kunitz domain I (KD1) with a TFPI binding peptide of any one of claims 1-5, 16, or 17 and a test compound under conditions that allow formation of KDI.-TFPI-binding peptide complexes, (b) measuring KD1-TFPI-binding peptide complexes formed in step (a), and (c) comparing the number of KDI -TFPI-binding peptide complexes formed in the presence of the test compound with the number of KD1-TFPI-binding peptide complexes formed in the absence of the test compound, wherein a reduction in the number of KDI-TFPI-binding peptide complexes formed in the presence of the test compound compared to the number of KD1-TFPI-binding peptide complexes formed in the absence of the test compound indicates that the test compound is a TFPT-binding compound. 59. The method of claim 58, wherin the TFPIbinding peptide comprises a label that generates a signal; tep (b) comprises measuring signal generated by KD1 FhHbinding peptide complexes; and step (c) comprises comparing signal measured in step (b) with signa[ generated by KD 1-TFPI-binding peptide complexes formed in the absence of the test compound, wherein a reduction in signal generated by KD I.-TFPI-binding peptide complexes forced in the presence of the test compound compared to signal generated by KDI-TFPI binding peptide complexes formed in the absence of the test compound indicates that the test compound is a TFPIbinding compound. 60. The method of claim 58 or claim 59, wherein step (a) comprises I CC (al) contacting the peptide comprising KDI with the TFPI-binding peptide under conditions that allow formation of KDI-peptide complexes, and (a2) contacting KD1-TFPI-binding peptide complexes formed in step (al) with the test compound. 61. A method for identifying a TFPI-binding compound, the method comprising (a) contacting a peptide comprising TFPI Kunitz domain 1 (KD1) with a test compound, and (b) detecting binding of the test compound to a TFPI binding site defined by KD1 amino acid residues corresponding to human TFPI residues Phe2S, Lys29, Ala3O, Asp32, fe46, Phe47. and 1e55. 62. The method of claim 61, wherein the binding site is defined by amino acid residues corresponding to human TFPI residues Ala27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, fle38, 1e46, Phe47, and I1e55. 63. The method of claim 62, wherein the binding site is defined by amino acid residues corresponding to human TFPI residues Ala27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, Aia37, 11e38 Phe44, fe46, Phe47, and e 55. 64. The method of any one of claims 61-63, wherein step (b) comprises determining the presence or absence of a nuclear magnetic resonance (NMR) chemical shift within the TFPI binding site. 65. The method of any one of claims 61-63, wherein step (a) comprises contacting the peptide comprising TFPI KD1 with FVIIa in the presence of a test compound under conditions that allow binding of KDI to FVI1a, and step (b) comprises comparing KDI -FVJIa binding in step (a) with KDl-FVHa binding in the absence of the test compound, wherein a decrease in KD I -FVIIa binding in the presence of the test compound compared to KD I -FVIIa binding in the absence of the test compound indicates that the test compound is a TFPI-binding compound. I CC 66. The method of claim 65, wherein binding of the test compound to the TFPI binding site is detected using an enzymatic assay. 67. The method of any one of claims 61-63, wherein step (a) comprises contacting the peptide comprising TFPI KDI with FXa in the presence of a test compound under conditions that allow binding of KD1 to FXa. and step (b) comprises comparing KD1-FXa binding in step (a) with KDI-FXa binding in the absence of the test compound, wherein a decrease in KD1-FXa binding in the presence of the test compound compared to KD1 -FXa binding in the absence of the test compound indicates that the test compound is a TFPI-binding compound. 68. The method of clain 67, wherein binding of the test compound to the TEPI binding site is detected using an enzymatic assay. 69. The method of any one of claims 61-63, wherein the peptide comprising TFPI KDI further comprises Kunitz domain 2 (KD2), step (a) comprises contacting the peptide comprising TFPI KD1 and TFPI KD2 with FXa in the presence of a test compound under conditions that allow binding of KD2 to FXa, and step (b) comprises comparing KD2-FXa binding in step (a) with KD2-FXa binding in the absence of the test compound, wherein a decrease in KD2-FXa binding in the presence of the test compound compared to KD2-FXa binding in the absence of the test compound indicates that the test compound is a TFPL-binding compound. 70. The method of claim 69, wherein binding of the test compound to the TFPI binding site is detected using an enzymatic assay. 71, The method of claim 61, wherein the peptide comprising TlP KD1 comprises amino acids 1-160 of human TFPh 72, The method of claim 61, wherein the peptide comprising TFPI KD1 is full length human TFPL 73. A method of identifying a TFPJ inhibitor, the method comprising (a) contacting a peptide consisting essentially of TFPI Kunitz domain I (KDI) with FXa in the presence of a test compound under conditions that allow binding of KD1 to FXa, and (b) comparing KDI FXa binding in step (a) with KDI-FXa binding in the absence of the test compound, wherein a decrease in KD1-FXa binding in the presence of the test compound compared to KD 1 -FXa binding in the absence of the test compound indicates that the test compound is a TFPI inhibitor. 74, A composition comprising a TFPI inhibitor identified by the method of claim 58 75. Use of a TFPI inhibitor identified by the method of claim 58 for the manufacture of a medicament. 76. Use of a TFPI inhibitor identified by the method of claim 58 for the manufacture of a medicament for treating a blood coagulation disorder. 77. A composition comprising a TFPI inhibitor identified by the method of claim 61. 78. Use of a TFPI inhibitor identified by the method of claim 61 for the manufacture of a medicament, 79. Use of a TFPI inhibitor identified by the method of claim 61 for the manufacture of a medicament for treating a blood coagulation disorder 80. A method for treating a subject suffering from a disease or being at risk of suffering from a disease, the method comprising administering to the subject a TFPI inhibitor identified by the method of claim 58, 81. A method for treating a subject suffering from a disease or being at risk of suffering from a disease, the method comprising administering to the subject a TFPI inhibitor identified by the method of claim 61. 82. A method for inhibiting human TFPI, the method comprising contacting human TFPI with an inhibitor that binds human TFPI at a binding site defined by amino acid residues Phe28, Lys29, Ala30, Asp32, Ile46, Phe47, and He55. 83 A method for treating a subject suffering from a disease or at risk of suffering from a disease, the method comprising administering to the subject an inhibitor that binds human TFPI at a binding site defined by amino acid residues Phe28, Lys29, Ala30, Asp32, fe46, Phe47, and 11e55. 84. The method of claim 82 or claim 83, wherein the human TFPI binding site is defined by amino acid residues AIa27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, 11e38, Ile46, Phe47, and Ile55. 85. The method of claim 84, wherein the human TFPL binding site is defined by amino acid residues Aia27, Phe28, Lys29, Ala3O, Asp3l, Asp32, Lys36, Ala37, 11e38, Phe44, 1le46, Phe47, and le55. 86 A method for purifying a compound that inhibits FXa activity, the method comprising (a) contacting a pepdde comprising TFPJ Kunitz domain 1 (KDi) with a compound under conditions that allow formation of compound-KDI complexes, (b) removing unbound compound, and (c) dissociating the compoundKDI complexes to release the compound. 87. The method of claim 86, wherein step (a) comprises contacting the peptide comprising KD11 with a population of compounds. 88. A computer storage media having computer executable instructions that, when executed on the processor of a computer, implement a method of modeling interaction between selected three dimensional (3D) points in a TFPI Kunitz domain I (KD Iprotein and a test compound, the method comprising: obtaining a protein structure 3D model for the TFPI KDI protein; determining a 3D relationship between a selected subset of amino acids in the protein structure, wherein the selected subset of amino acids comprises Phe28, Lys29, Ala30, Asp32, Ile46, Phe47, and Ile55; modeling a surface bounded by the selected subset of amino acids; obtaining a test compound 3D model of a test compound; matching the test compound 3D model to the surface bounded by the selected subset of amino acids; and identifying contact points between the selected subset of amino acids of the surface and the test compound 3D model. 89. The computer storage media of claim 88, wherein the selected subset of amino acids comprises Ala27, Phc28, Lys29, Ala30, Asp3l, Asp32, Lys36, fle38, 11e46 Phe47, and Ie55. 90. The computer storage media of claim 88, wherein the selected subset of amino acids comprises Ala27, Phe28, Lys29, Ala30, Asp3l, Asp32, Lys36, Ala37, 11e38, Phe44, Re46, Phe47, and Re55. 91. The computer storage media of claim 88, further comprising: determining a number of the contact points between the surface and the test compound 3D model; and recording an affinity rating for the test compound 3D model corresponding to the number of contact points. 92. The computer storage media of claim 88, wherein the test compound is a peptide. 93. The computer storage media of claim 92, further comprising: determining a bond type for each contact point between the surface and the test compound 3D model; and updating the affinity rating based on an aggregate of the bond types for each contact point between the surface and the test compound 3D model. 94, The computer storage media of claim 93, further comprising: obtaining an updated test compound 3D model based on a second test compound; matching the updated test compound 3D model to the surface bounded by the selected subset of amino acids; and identifying the identified contact points between the selected subset of amino acids of the surface and the updated test compound 3D model on a display of the computer. 95. The computer storage media of claim 94, further comprising: determining a number of the contact points between the surface and the updated test compound 3D model; determining a bond type for each contact point between the surface and the updated test compound 3D model; and recording a new affinity rating based on the number of contact points and an aggregate of the bond types for each contact point between the surface and the updated test compound 3D model, 96, The computer storage media of claim 95, further comprising: comparing the updated affinity rating with the new affinity rating to determine whether the test compound or the second test compound has a higher affinity rating. 97. The computer storage media of claim 96, wherein the second test compound is a variant of the test compound. 1 t21 98. The computer storage media of claim 92, further comprising displaying the contact points on a display of the computer. 99. The computer storage media of claim 92, further comprising modifying the peptide to increase the number of contact points with the selected subset of amino acids or increase bond strength between amino acids of the peptide and the selected subset of amino acids. 100. A method of comparing a test compound to selected three dimensional points in a TFPI Kunitz domain 1 (KD1) protein, the method comprising: creating a protein structure for the KD1 protein in a memory of a computer; determining a three dimensional model of a selected subset of amino acids in the KD1 protein at a processor of the computer, wherein the selected subset of amino acids comprises Phe28, Lys29, Ala30, Asp32, 1e46, Phe47, and le55; determining a three dimensional model of a test compound at the processor of the computer; fitting the 3D model of the test compound to the 3D model of the selected subset of amino acids at the processor of the computer; and generating an affinity of the test compound for the selected subset of amino acids at the processor of the computer, wherein the affinity is based on a number of amino acids in the subset in contact with the test compound and a bond strength at each contact point. 101. The method of claim 100, wherein the selected subset of amino acids comprises Ala27, Phe28, Lys29, Ala30, Asp3l, Asp32, Lys36, le38, 1e46, Phe47, and 1e55. 102. The method of claim 100, wherein the selected subset of amino acids comprises Ala27, Phe28, Lys29, Ala3O, Asp31, Asp32, Lys36, Ala37, 11e38, Phe44, ]1e46, Phe47, and Be55. 103. The method of claim 100, further comprising: I1 4n~ displaying a 3D representation of the fit between the test compound and the 3D model of the selected subset of amino acids. 104. The method of claim 103, further comprising: repeating the steps of claim 100 for a plurality of test compounds; and saving the respective affinities for each of the plurality of test compounds. 105. A computer storage media having computer executable instructions that, when executed on the processor of a computer, implement a method of comparing a peptide to selected three dimensional points (3D) in a TFPI Kunitz domain 1 protein (KD1), the method comprising: creating a protein structure for the KD1 protein; determining a three dimensional model of a selected subset of amino acids in the KDi protein, wherein the subset of amino acids comprises Phe28, Lys29, Ala30, Asp32, 1e46, Phe47 and Ile55; determining a three dimensional model of a peptide; fitting the 3D model of the peptide to the 3D model of the selected subset of amino acids; and generating an affinity of the peptide for the selected subset of amino acids, wherein the affinity is based on a number of amino acids in the subset in contact with the peptide and a bond strength at each contact point. 106. The computer storage media of claim 105, wherein the selected subset of amino acids comprises Ala27, Phe28, Lys29, Ala30, Asp3l, Asp32, Lys36, 1e38, le46, Phe47, and Ile55. 107. The computer storage media of claim 105, wherein the selected subset of amino acids comprises Ala27, Phe28, Lys29, Ala30, Asp3l, Asp32, Lys36, Ala37, Ile38, Phe44, Ie46, Phe47, and Be55.