WO2006049580A1 - Anticoagulant peptides - Google Patents

Abstract

The present invention provides an isolated peptide having an amino acid sequence of: (a) [Σ]nNYYYVHQNFDRVAYDYDI[U]m ; or (b) [Σ]nKIX3X4X5X6KX8X9[U]m, wherein X3, X4 and X8, and are independently an amino acid selected from A, F, I, L, M, V, W and Y; each of X5, X6 and X9 is any amino acid; [Σ] is any amino acid, and n is an integer from (0) to (15); [U] is any amino acid, and m is an integer from (0) to (15). Further, the use of these peptides as anticoagulants is also provided.

Description

Anticoagulant Peptides

Field of the invention

The invention relates to peptides which inhibit the assembly of the prothrombinase complex. These peptides are useful as anticoagulants. The present invention also relates to the identification of peptides which inhibit the assembly of the prothrombinase complex.

Background of the invention

In the blood coagulation cascade, thrombin is directly involved in the formation of fibrin, activation of platelets and feedback activation of other components of the cascade. Thrombin is formed when prothrombin is activated by prothrombinase complex, an enzymatic complex assembled from factor Xa (FXa), factor Va (FVa), phospholipids (PL) and calcium ions (Ca²⁺). While FXa alone has very low prothrombin-activating ability, the formation of prothrombinase enhances the rate of the reaction by six orders of magnitude (Joseph et al., 1999).

Current established anticoagulants (heparin and warfarin) have some major limitations, including narrow therapeutic window, highly variable dose- response relation and extensive drug-drug or drug-food interactions. This is mainly due to their mechanisms of action that affect multiple steps in the coagulation cascade. Consequently usage of the drugs has to be under close monitoring. There is a pressing need for a more efficacious and safer anticoagulant.

Newer anticoagulants are emphasizing on selectivity and specificity in the inhibition of coagulation factors. FXa is one of the most targeted coagulation factors. However, FXa and the other serine proteinases in the coagulation cascade possess multiple roles both within the cascade, as well as in other physiological processes. FXa induces the release of growth factors from the endothelium (Gajdusek et al., 1986) and the proliferation of vascular smooth muscle cells (Gasic et al., 1992; Ko et al., 1996) and endothelial cells (Nicholson et al., 1996). It triggers an increase in cytosolic free Ca²⁺ ions in Madin-Darby canine kidney cells (Camerer et al., 1999), activates nitric oxide synthase in endothelial cells (Papapetropoulos et al., 1998) and induces the synthesis of cytokines and the expression of adhesion molecules (Papapetropoulos et al., 1998; Senden et al., 1998). Therefore even the use of specific active site inhibitors of FXa as anticoagulants would result in undesirable interference with the pleiotropic functions of FXa.

Summary of the invention

The present invention addresses the problems above, and in particular provides new peptides which are able to interact with the Factor Va (FVa) and inhibit the assembly of prothrombinase complex. Accordingly, the peptides of the invention are useful as anticoagulants.

According to one aspect, the invention provides an isolated peptide having an amino acid sequence of:

(a) [Σ]_nNYYYVHQNFDRVAYDYDI[U]_m ; or

(b) [Σ]_nKIX³X⁴X⁵X⁶KX⁸X⁹[U]_m wherein

X³, X⁴ and X⁸, and are independently an amino acid selected from A, F, I,

L, M, V, W and Y; each of X⁵, X⁶ and X⁹ is any amino acid;

[Σ] is any amino acid, and n is an integer from 0 to 15;

[U] is any amino acid, and m is an integer from 0 to 15. For example, m and/or n may be an integer from 1 to 14, 2 to 13, 3 to 12, 4 to 11 , 5 to 10, 6 to 9 or 7 to 8.

The peptides (a) and (b) may be obtained by fragmentation and/or amino acid derivation of the protein trocarin or of other proteins. They may also be prepared by chemical synthesis.

In particular, the peptide (b) has the following amino acid sequence: [Σ]_nKIYVX⁵X⁶KFX⁹[U]_m.

More in particular, in the peptide of the invention: X⁵ is H or A; X⁶ is T or A; and X⁹ is V or A.

For example, the peptides have the following amino acid sequence: [Σ]_nKIYVX⁵TKFV[U]_m and X⁵ is H or A; [Σ]_nKIYVHX⁶KFV[U]_m and X⁶ is H or A; or [Σ]_nKIYVHTKFX⁹[U]_m and X⁹ is H or A.

Preferably, the peptide of the invention is: [Σ]_nKIYVHTKFV[U]_m.

In particular, [∑]_n and [U]_m are such that: [∑]_n is selected from ETRRLLSVD₁ GPLLSVD, LLSVD, SVD, D, P, PP, PPP, PPPP, PPPPP, VP, VPP, VPPP, VPPPP, F, FV, FVP, FVPP and [U]_n, is selected from P, PP₁ PPP, PPPP, PPPPP, PN, PPN, PPPN, PPPPN.

Preferably, the amino acid sequence of the peptide of the invention is selected from: SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25 and SEQ ID NO:26.

According to another aspect, the invention provides an isolated nucleic acid molecule encoding any peptide of the invention, a vector comprising the nucleic acid molecule, and a host cell comprising the vector or the nucleic acid molecule. It is further provided a method of producing a peptide comprising cultivating the host cell and expressing the peptide.

According to another aspect, the invention provides a composition comprising at least one peptide of the invention, the nucleic acid molecule encoding the peptide of the invention, or the vector. In particular, the composition is a pharmaceutical composition. The composition may further comprise a pharmaceutically acceptable excipient, diluent and/or carrier.

According to another aspect, the invention provides any peptide of the invention for use in medicine. In particular, for use as an anticoagulant. The invention also relates to the use of at least one peptide of the invention for the preparation of an anticoagulant.

According to another aspect, the invention provides a method for inhibiting the assembly of prothrombinase complex in a subject comprising administering to a subject at least one peptide and/or a composition according to the invention.

Further, the invention provides a method for the prevention and/or treatment of formation of intravascular blood clots in a subject comprising administering to the subject at least one peptide and/or a composition according to the invention. In particular, the method is for the prevention and/or treatment of ischemic events. More in particular, the ischemic event is coronary syndrome, myocardial infarction, stroke, venous thromboembolism and/or peripheral arterial occlusion. The subject may be a mammal, for example human.

According to another aspect, the invention provides a method for identifying anticoagulant peptides, comprising:

(a) providing at least one peptide;

(b) determining the minimum interaction site (MIS) between the at least one peptide and Factor Va (Fva); (c) contacting in vitro the peptide comprising the MIS with the prothrombinase complex; and

(d) determining the ability of the peptide to inhibiting the assembly of the prothrombinase complex.

The method may further comprise the step of contacting the peptide comprising the MIS to plasma, and determining the ability of the peptide to interfere with the blood coagulation cascade in plasma.

The method may further comprise the step of selecting the peptide comprising the MIS, modifying at least one amino acid residue inside and/or outside the MIS, and determining the ability of the modified peptide to interfere with the assembly of the prothrombinase complex and/or with the blood coagulation cascade in plasma.

The method may further comprise the step of isolating or synthesising the modified peptide capable of interfering with the assembly of the prothrombinase complex and/or with the blood coagulation cascade in plasma. In particular, the at least one peptide provided which comprises the MlS is obtained from fragmentation of a protein. The protein may be trocarin. The peptide which is used for the identification of derived or improved peptide is preferably at least one peptide according to the invention.

Brief description of the figures

Figure 1 : Alignment of amino acid sequence of trocarin with bovine (β taurus) and human (H sapiens) FXa

The sequences are taken from following sources: trocarin - SWISS-PROT no. P81428; bovine FXa - SWISS-PROT no. P00743; human FXa - SWISS- PROT no. P00742.

Identical residues are marked with (*) while homologous residues are marked with (:) and (.). Shaded segments represent the two regions whereby trocarin is markedly different from the other two FXa. The first region is in the EGF-II between Arg101 and GIn 107 and the second region is in the heavy chain between Glu246 and Ala278.

In the light chain sequences, the amino acid denoted by ¹J' refers to gamma carboxy glutamic acid, which is a special modification of glutamic acid residue, E.

Figure 2: Synthetic peptides compete in the formation of FXa-FVa complex

(A) Dose response curves of FVa at varying concentrations of TroHCpepi peptide: (♦) 0 μM, (■) 50 μM, (•) 100 μM, and (A) 200 μM. (B) Dose response curves of FVa at varying concentrations of TroHCpep2 peptide: (■) 0 μM, (•) 50 μM, and (A) 100 μM. In both (A) and (B), the Kd for FXa-FVa remains the same irrespective of the amount of inhibitor present, while the V_max decreases with increasing concentration of inhibitor. Thus TroHCpepi and TroHCpep2 compete in the formation of FXa-FVa complex.

Figure 3: Non-competitive inhibition of FXa-FVa complex by synthetic peptides

(A) Dose response curves of bovine prothrombin at varying concentration of TroHCpepi peptide: (■) 0 μM, (•) 50 μM, (A) 100 μM. (B) Dose response curves of bovine prothrombin at varying concentrations of TroHCpep2 peptide: (■) 0 μM, (•) 50 μM, and (A) 100 μM. In both (A) and (B), it can be observed that the V_max remains constant at different concentrations of inhibitor while the K_m varies with the concentration of inhibitor. This is characteristic of non-competitive inhibition. The experiment was done three times in triplicate each time.

Figure 4: Mechanism of inhibition of FXa-FVa complex by trocarin D peptides

(A) Normal prothrombinase complex. In the physiologic system, FXa binds to FVa and converts prothrombin to thrombin. TroHCpepi (B) and TroHCpep2 (C) bind specifically to FVa. Both peptides compete with FXa for the formation of the complex, indicating that they bind to the same site as FXa. Thus, the complex formation and thrombin generation is inhibited by the peptides.

Figure 5: Anticoagulant effects of synthetic peptides on human plasma

The recalcification time of citrated plasma was prolonged by 50% of the control clotting time at 50 μM concentration of TroHCpepi (■) and 100 μM concentration of TroHCpep2 (D). The recalcification time was ~250% of the control clotting time at a peptide concentration of 500 μM. Figure 6: Dose response curve of 13VP_A7_PN and 13VP_A8_PN, compared to 13VPK_VPN

IC₅₀ of the peptides shown in Table 1 and Table 3 were all obtained from such dose response curves. In this curve, (■) are data points for 13VP_A7_PN (VPKIYVATKFVPN). (O) are data points for 13VP_A8_PN

(VPKIYVHAKFVPN). (•) are data points for 13VPK_VPN

(VPKIYVHTKFVPN). 13VP_A7_PN (IC₅₀ = 0.25 μM) and 13VP_A8_PN (IC₅₀

= 1.25 μM) showed much higher activity compared with 13VPK_VPN (IC₅₀ =

300 μM). 13VP_A7_PN and 13VP_A8_PN are good leads for further development of anticoagulant peptides due to their high potency.

Detailed description of the invention

Definitions and Nomenclature

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting in nature.

The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A or Ala, alanine; B, asparagine or aspartic acid; C or Cys, cysteine; D or Asp, aspartic acid; E or GIu, glutamic acid; F or Phe, phenylalanine; G or GIy, glycine; H or His, histidine; I or lie, isoleucine; K or Lys, lysine; L or Leu, leucine; M or Met, methionine; N or Asn, asparagine; P or Pro, proline; Q or GIn, glutamine; R or Arg, arginine; S or Ser, serine; T or Thr, threonine; V or VaI₁ valine; W or Trp, tryptophan; Y or Tyr, tyrosine; Z, glutamine or glutamic acid. It should also be noted that amino acid referred to as 'J' denotes gamma carboxy glutamic acid, which is a special modification of glutamic acid residue, E. A "conservative amino acid substitution" is one in which the 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 in the art. These families include hydrophobic, hydrophilic, basic and acid amino acids as described below. Thus, a predicted nonessential amino acid residue in the peptide of the invention is preferably replaced with another amino acid residue from the same category. For example, a hydrophobic amino acid is replaced by another hydrophobic amino acid, etc. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of the coding sequence of the nucleic acid encoding the peptide of the invention, such as by saturation mutagenesis, and the resultant mutants can be screened for anticoagulant activity to identify mutants that retain that activity.

"Hydrophobic" amino acids are: A, V, L, I₁ P, W, F and M; "hydrophilic" amino acids are: G, S, T, Y, C, N and Q; however Y has a hydrophobic aromatic group and may be considered to be acting as a hydrophobic amino acid depending on the circumstances and uses; "basic" amino acids are: K, R and H; and "acidic" amino acids are: D and E.

"Peptide" as used herein refers to and encompasses any amino acid molecule, a peptide or polypeptide. The "peptide" can be obtained as a gene product, a purified and/or isolated product, an expression product, from fragmentation of protein(s) or a synthetic peptide. An "isolated peptide" encompasses naturally occurring, a gene expression product and a synthetic peptide.

The phrase "nucleic acid" or "nucleic acid molecule" as used herein refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing. Nucleic acids of the invention can also include nucleotide analogs (e.g., BrdU), and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof.

As used herein, "reverse analog" or "reverse sequence" refers to a peptide having the reverse amino acid sequence as another reference peptide. For example, if one peptide has the amino acid sequence ABCDE, its reverse analog or a peptide having its reverse sequence is as follows: EDCBA.

The position of the amino acids in a peptide is denoted by the superscript. For example, one of the basic peptide of the invention is denoted by K¹I²Y³V⁴A⁵T⁶K⁷F⁸V⁹. In V²P-¹K¹I²Y³V⁴A⁵T⁶K⁷F⁸V⁹P¹⁰N¹¹, the negative superscript indicates an amino acid positioned to the left of the first amino acid of the basic peptide.

Compounds, Compositions, Uses and Methods of the Invention

Snake venom is a rich source of exogenous haemostatic factors, including prothrombin activators. Prothrombin activators from snake venoms proteolytically activate prothrombin and are classed into four groups based on their structural characteristics, functional properties and cofactor requirements in prothrombin activation (Kini et al., 2001). Among these, Group D prothrombin activators are very similar to mammalian blood coagulation FXa. Group D prothrombin activators, like FXa, cleave two peptide bonds of prothrombin (Arg-274-Thr-275 and Arg-323 and lle-324) to yield thrombin. Their activities are comparable to FXa in the presence of FVa, phospholipids and Ca²⁺ (Kini et al., 2001 ). One of the Group D prothrombin activators, trocarin, a venom procoagulant of the Australian rough-scaled snake Tropidechis carinatus, is a structural homologue of FXa. Functionally, trocarin is also similar to FXa. Like FXa, trocarin cleaves both peptide bonds of prothrombin, and the activation of prothrombin is at a comparable rate with FXa in the presence of FVa, phospholipids and Ca²⁺.

In WO 03/082914, Masci, Paul Pantaleone et al. describes snake venom protease polypeptides that are prothrombin activators and that are useful, e.g., as reagents to increase coagulation, to promote haemostasis and prevent blood loss such as during surgery or for treatment of wounds.

The amino acid sequence of trocarin was determined recently (Kini et al., 2001 ) and was found to be highly similar to that of mammalian FXa.

The present inventors have further investigated trocarin, and based on kinetic studies, have found that trocarin, when compared to bovine FXa, has higher affinity to bovine FVa. Short peptides which contain the sequence of residues involved in the binding to FVa but lack the functional prothrombin activating catalytic residues are able to inhibit the assembly of prothrombinase complex.

The peptides can be either synthetic or obtained from naturally occurring protein(s) or polypeptide(s). As a result, the activation of prothrombin can be minimized and the propagation of coagulation cascade can be inhibited.

Based on this principle, the peptides can be used as anticoagulants which prevent spontaneous formation of intravascular blood clots for the prevention and treatment of ischemic events, including coronary syndromes, myocardial infarction, stroke, venous thromboembolism and peripheral arterial occlusion.

Accordingly, the present inventors have found that two fragments of trocarin bind to FVa and inhibit the prothrombinase complex. Accordingly, these two peptides may be particularly useful as anticoagulants. These two peptides are identified as TroHCpepi (ETRRLLSVDKIYVHTKFVPPN) (SEQ ID NO:1) and TroHCpep2 (FVPPNYYYVHQNFDRVAYDYDI) (SEQ ID NO:2). These two peptides are obtained from portions of trocarin (as shown in Figure 1) and bind the FVa in different positions (as shown in Figure 4). The ability of TroHCpepi and TroHCpep2 inhibiting the prothrombinase complex assembly is shown in Table 1.

TroHCpepi and TroHCpep2 have been synthesized based on the sequence of trocarin and used for the determination of the minimum interaction segment (MIS) for FVa. It was found that the MIS on trocarin for FVa is located within this sequence. Kinetic studies and binding studies confirmed the peptide interfered with prothrombinase complex by binding to cofactor FVa. In assays done using human plasma, the anticoagulant effect was retained. TroHCpepi was used to further delineate the MIS which was identified to be KIYVHTKFV based on in vitro activity of synthetic peptides. This segment corresponds to residues 255-263 on Trocarin.

Substitutions, deletion and/or modifications of the amino acids of TroHCpepi and TroHCpep2 have been carried out according to the standard technologies. For example, Y, V and F, which are mostly hydrophobic have been substituted with other hydrophobic amino acids (overall Y is hydrophilic but has a hydrophobic aromatic group). Substituting amino acids with other amino acids of similar properties is acceptable, as the overall properties of the peptide are not altered. For example, substituting one hydrophobic or hydrophilic amino acid with another hydrophobic or hydrophilic amino acid respectively is acceptable as the overall property of the peptide is not altered.

Further, it has been found that the attachment of one or more residue of praline, further attached to one or more amino acidic residue, for example N and/or V, at either side of the selected peptide improves the prothrombinase complex inhibitory activity. In particular, the attachment of one proline at either side of the MIS (VPKI YVHTKFVPN) to form 'Proline bracket' was found to improve the activity of MIS by 3-folds (Evans and Kini, US patents 6,100,044 and 6,258,550, herein incorporated by reference). Alanine scans performed on MIS (K¹IYVHTKFV⁹) indicate that side chains of K¹, 1² and K⁷ are essential for the peptide's inhibitory activity.

Side chains of Y³, V⁴ and F⁸ are important, although not essential for interaction.

Side chains of H⁵, T⁶ and V⁹ are not important for activity. Among these three, H⁵ and T⁶ hindered the optimum binding between peptides and FVa. When H⁵ and T⁶ are replaced with alanine separately, the activity increased 1200-folds and 240-folds respectively.

For example, two peptides 13VP_A7_PN (VPKIYVATKFVPN) and 13VP_A8_PN (VPKIYVHAKFVPN) are good leads for further development of anticoagulant peptides due to their high potency (IC₅₀ = 0.25 μM and 1.25 μM respectively).

Accordingly, the invention provides an isolated peptide having an amino acid sequence of:

(a) [Σ]_nNYYYVHQNFDRVAYDYDI[U]_m ; or

(b) [Σ]_nKIX³X⁴X⁵X⁶KX⁸X⁹[U] J_nm

wherein

X³, X⁴ and X⁸, are independently an amino acid selected from A, F, I, L, M, V, W and Y;

each of X⁵, X⁶ and X⁹ is any amino acid;

[Σ] is any amino acid, and n is an integer from 0 to 15;

[U] is any amino acid, and m is an integer from 0 to 15. More in particular, n may be an integer from 0 to 15, from 1 to 14, from 2 to 13, from 3 to 12, from 4 to 11 , from 5 to 10, from 6 to 9, or from 7 to 8. Further, m 0 to 15, from 1 to 14, from 2 to 13, from 3 to 12, from 4 to 11 , from 5 to 10, from 6 to 9, or from 7 to 8.

The peptides (a) and (b) may be obtained by fragmentation and/or amino acid derivation of the protein trocarin or from other proteins. The peptides (a) and (b) may also be prepared by chemical synthesis according to standard methods known in the art.

In particular, the peptide (b) has the following amino acid sequence:

[Z]_nKI YVX⁵X⁶KFX⁹[U]_m.

More in particular, in the peptide of the invention:

X⁵ is H or A;

X⁶ is T or A; and

X⁹ is V or A.

For example, the peptides have the following amino acid sequence:

[Σ]_nKIYVX⁵TKFV[U]_m and X⁵ is H or A;

[Σ]_nKIYVHX⁶KFV[U]_m and X⁶ is H or A; or

[Σ]_nKIYVHTKFX⁹[U]_m and X⁹ is H or A.

Preferably, the peptide of the invention is: [Σ]_nKIYVHTKFV[U]_m.

In particular, [I]_n and [U]_m are such that: [∑]_n is selected from ETRRLLSVD, GPLLSVD, LLSVD, SVD, D, P, PP, PPP, PPPP, PPPPP, VP, VPP, VPPP, VPPPP, F, FV, FVP, FVPP and [U]_n, is selected from P, PP, PPP, PPPP, PPPPP, PN, PPN, PPPN, PPPPN. Preferably, the amino acid of the peptide of the invention is selected from:

SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25 and SEQ ID NO:26.

Peptide Production - peptide of the invention may be produced by any method known in the art. One method of producing the disclosed peptides is to link two or more amino acid residues together by protein chemistry techniques. For example, peptides are chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycar- bonyl) or Boc (tert-butyloxycarbonoyl) chemistry (Applied Biosystems, Inc., Foster City, Calif.). A peptide can be synthesized and not cleaved from its synthesis resin, whereas the other fragment of a peptide or protein can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group, which is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, (Grant G A (1992) Synthetic Peptides: A User Guide. W. H. Freeman and Co., N.Y. (1992); Bodansky M and Trost B., Ed. (1993) Principles of Peptide Synthesis. Springer-Verlag Inc., NY). Alternatively, the peptide is independently synthesized in vivo. Once isolated, these independent peptides may be linked to form a peptide or fragment thereof via similar peptide condensation reactions.

According to another aspect, the invention provides an isolated nucleic acid molecule encoding any peptide of the invention, a vector comprising the nucleic acid molecule, and a host cell comprising the vector or the nucleic acid molecule. It is further provided a method of producing a peptide comprising cultivating the host cell and expressing the peptide. Nucleic Acid and Vectors - The invention is also directed to an isolated nucleic acid encoding any one or more of the peptides disclosed herei n. In one embodiment, the nucleic acid comprises DNA, RNA and/or cDNA. It would be routine for one with ordinary skill in the art to make a nucleic acid that encodes the peptides disclosed herein since codons for each of the amino acids that make up the peptides are known. As non-limiting examples, the nucleic acids of the invention can be produced by recombinant, in vitro methods, or by chemical synthetic means using machines and standard chemistry which would be known to one with skill in the art, or by in vivo cellular synthesis. Methods of synthesizing nucleic acids would be well known to one with skill in the art. For example, US 6,472,184 entitled "Method for producing nucleic acid polymers" and US 6,444,111 entitled "Electrochemical solid phase synthesis of polymers describes such synthesis" describe such synthetic methods. These references are hereby incorporated by reference in their entireties.

Additionally, the invention provides a vector comprising the nucleic acid encoding any one or more of the peptides described herein. In certain embodiments, the invention provides a vector comprising a nucleic acid encoding at least one of the peptides of the present invention. The vector can be a viral vector, a plasmid vector, a cosmid vector, an adenoviral vector, a phage vector, a retroviral vector, an adeno-associated viral (AAV^ vector, or any other vector capable of including a nucleic acid encoding a peptide of the invention. The vector can be an expression vector that is intended and capable of integrating into a cell genome. Other useful virus vectors include retroviruses such as Moloney murine leukemia virus ^MoMuLV); papovaviruses such as JC, SV40, polyoma, adenoviruses; Epstein -Barr Virus (EBV); papilloma viruses, e.g. bovine papilloma virus type I (BPV); vaccinia and poliovirus and other human and animal viruses. Useful vectors and their construction are disclosed in Sambrook and Russel, (2001 ) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, US.

Host cell -The invention also provides for a host cell containing the nucleic acid, polypeptide, peptide, and/or the vector of the invention. Such a host cell is a eukaryotic cell or a prokaryotic cell. In the case of eukaryotic cells, retrovirus or adenovirus based vectors can be used to put the nucleic acid of the invention into the host cell. Methods known to one with skill in the art to insert the nucleic acids or polypeptides in host cells are encompassed within this invention. The following are non-limiting examples of such methods: naked DNA transfection, lipofectin-mediated transfer, transformation, micro¬ injection of nucleic acid into a cell, or calcium-phosphate precipitation transfection methods. Host cells can be obtained from commercial sources such as the American Type Culture Collection (ATCC). Host cells can be grown in liquid media culture or on tissue culture plates. The growth conditions will be dependent upon the specific host cells used and such conditions would be known to one with skill in the art. Transfection and growth of host cells is described in Sambrook and Russel, id. The invention provides for a recombinant cell expressing a nucleic acid encoding the polypeptide of the claimed invention. The invention also provides for a recombinant cell producing the polypeptide of the invention.

According to another aspect, the invention provides a composition comprising at least one peptide of the invention, the nucleic acid molecule encoding the peptide of the invention, or the vector. In particular, the composition is a pharmaceutical composition. The composition may further comprise a pharmaceutically acceptable excipient, diluent and/or carrier.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier, excipient and/or diluent, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for administration to humans, the person skilled in the art will understand that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modifications. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates.

According to another aspect, the invention provides any peptide of the invention for use in medicine. In particular, for use as an anticoagulant. The invention also relates to the use of at least one peptide of the invention for the preparation of an anticoagulant. The anticoagulant peptides and/or polypeptides of the present invention may block only the hemostatic function but not other non-hemostatic roles of the coagulation enzymes, overcoming the limitations of other anticoagulants currently available or under development.

According to another aspect, the invention provides a method for inhibiting the assembly of prothrombinase complex in a subject comprising administering to a subject at least one peptide or a composition according to the invention.

Further, the invention provides a method for the prevention and/or treatment of formation of intravascular blood clots in a subject comprising administering to the subject at least one peptide or a composition thereof according to the invention. In particular, the method is for the prevention and/or treatment of ischemic events. More in particular, the ischemic event is coronary syndrome, myocardial infarction, stroke, venous thromboembolism and/or peripheral arterial occlusion. The subject may be a mammal, for example human.

According to another aspect, design and development of the peptides and/or polypeptides of the invention may used for the identification of further and/or improved anticoagulants peptides and/or polypeptides, which specifically interfere with the assembly of prothrombinase complex by preventing the binding between FXa and FVa.

Accordingly, the invention provides a method for identifying anticoagulant peptides, comprising:

- providing at least one peptide;

- determining the minimum interaction site (MIS) between the at least one peptide and FVa;

- contacting in vitro the peptide comprising the MIS with the prothrombinase complex; and

- determining the ability of the peptide to inhibiting the assembly of the prothrombinase complex.

The method may further comprise the step of contacting the peptide comprising the MIS to plasma, and determining the ability of the peptide to interfere with the blood coagulation cascade in plasma.

The method may further comprise the step of selecting the peptide comprising the MIS, modifying at least one amino acid residue inside and/or outside the MIS, and determining the ability of the modified peptide to interfere with the assembly of the prothrombinase complex and/or with the blood coagulation cascade in plasma.

The method may further comprise the step of isolating or synthesising the modified peptide capable of interfering with the assembly of the prothrombinase complex and/or with the blood coagulation cascade in plasma.

In particular, the at least one peptide provided, which comprises the MIS is obtained from fragmentation of a protein. The protein may be trocarin. The peptide which is used for the identification of derived or improved peptides is preferably at least one peptide according to the invention.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention.

Examples

1.1.1. Peptide synthesis Peptides used were chemically synthesized using solid phase peptide synthesis (SPPS) based on 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry. SPPS is based on sequential addition of α-amino and side chain protected amino acid residues to an insoluble polymeric support. Synthesis of peptides was carried out on PerSeptive BioSystems Pioneer Peptide Synthesis System. Fmoc amino acid derivatives, chemical reagents and resins used were of peptide synthesis grade purchased from Applied Biosystems. Temporary α-ami no-protecting Fmoc group was removed using 20% v/v piperidine in N,N-dimethyformamide (DMF), coupling reagent used was 0.5 M of N-[(dimethylamino)-1 H- 1 ,2,3-triazolo[4,5-jb]pyridin-1 -ylmethylene]-Λ/- methylmethanaminium hexafluorophosphate Λ/-oxide (HATU) in DMF. Side- chain protecting groups were cleaved simultaneously with detachment of the peptide from the resin. The cleavage was carried out in a cleavage cocktail of trifluoroacetic acid (TFA): ethanedithiol (EDT): thioanisole: water (45:2:2:1) for 3 hours. All sequences of peptides synthesized are listed in Table 1 and Table 3.

1.1.2. Purification of peptides

Synthesized peptides were subjected to purification using reverse-phase high performance liquid chromatography (RP-HPLC) system on a Phenomenex Jupiter C18 (3OθA, 5μ) 250 x 10 mm column. The RP-HPLC systems were equilibrated with two solvents. Solvent A was 0.1 % v/v TFA in water and Solvent B was 80 % v/v acetonitrile (HPLC grade) with 0.1 % v/v TFA in water.

Masses of peptides were verified using electrospray ionization mass spectrometry (ESl-MS). The spectra were then analyzed using software BioMultiview to obtain the mass of the peptides.

1.1.3. Assay of inhibition of prothrombinase complex assembly

To test for the inhibitory effects of synthetic peptides on the assembly of prothrombinase complex, an inhibition assay modified from previously reported method (Joseph et. ai., 1999) was carried out. Pure bovine FXa, bovine FVa and bovine prothrombin were purchased from Haematologic Technologies Inc., bovine serum albumin (BSA) was purchased from Sigma Chemical Co., H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline dihydrochloride (S-2238) was purchased from Chromogenic and all other chemicals and reagents used were of the highest quality available.

1.1.3.1. Principle The principle of the assay is based on the observations that assembly of the prothrombinase complex is an important step in the activation of prothrombin to thrombin. The thrombin formed will then be added to S-2238, a chromogenic substrate for thrombin. The rate of coloured product formation (i.e. the increase in absorbance per second) at 405 nm is proportional to thrombin activity. This increase in absorbance can be monitored using an ELISA plate reader.

1.1.3.2. Assay

Reactions were carried out in 96-well microtiter plates. 8 wells of reaction mixtures, each made up to a final volume of 50 μl, were prepared. Lyophilized peptides were reconstituted in buffer containing 50 mM Tris-HCI, 50 mM CaCI₂, 0.5 mg/ml BSA, pH 8.0. Suitable amount of peptides were added to the 8 reaction wells such that eight different concentrations of peptides were obtained in the final reaction mixtures: 0 μM, 0.1 μM, 1.0 μM, 10 μM, 100 μM, 500 μM, 800 μM and 1000 μM. FVa was then added to the reaction wells such that each well contained 13 nM of FVa in the final mixture of 50 μl. The reaction mixtures were incubated at room temperature for 20 minutes. FXa was then added to each reaction well such that a concentration of 11 nM of FXa in the final mixture of 50 μl was obtained in each well. The reaction mixtures were incubated at room temperature for 20 minutes. Prothrombin was then added to each well such that a concentration of 15 μM of prothrombin in the final mixture of 50 μl was achieved. At sampling time of 5, 10 and 20- min after addition of prothrombin, 2.5 μl aliquots of each reaction mixture was taken out and added to 197.5 μl of buffer containing 50 mM of Tris-HCI, 100 μM of S-2238, 100 μM of EDTA (to inhibit further conversion of prothrombin to thrombin). The OD was monitored for 5 min at 405 nm using ELISA plate reader SpectraMax 340 and the thrombin activity was obtained from the gradient of the absorbance plotted against time. Averages of the readings obtained in three independent experiments were taken.

1.1.4. Binding sites on Trocarin

Despite being the structural homologue of FXa, amino acid sequence of trocarin has only 70 % similarity with that of FXa. There are mainly two regions where the sequences share minimum similarity. For trocarin, the first region is in the EGF-II between Arg101 and Gln107 and the second region is in the heavy chain between Glu246 and Ala278 (Figure 1). These two sites were predicted to be the possible binding sites on Trocarin for FVa and account for the higher binding affinity compared to that of FXa. Three peptides were synthesized based on the sequences of these two regions and assayed for activity. TroLCpep (LyslOO to Glu107: KRVQSETQ) was synthesized based on the first region (light chain EGF-II domain). TroHCpepi (Glu246 to Asn266: ETRRLLSVDKIYVHTKFVPPN), and TroHCpep2 (Phe262 to Ile283: FVPPNYYYVHQNFDRVAYDYDI), were synthesized based on the second region (heavy chain). Both TroHCpepi and TroHCpep2 effectively inhibit FXa- mediated formation of thrombin while TroLCpep had no significant inhibitory effects. TroHCpepi recorded IC₅₀ of 70 μM and 93% inhibition at 500 μM while TroHCpep2 recorded IC₅₀ of 150 μM and 94% inhibition at 500 μM (Table 1 ). TroLCpep did not show any inhibition even at 500 μM.

1.1.5. Kinetic studies of peptides

To verify if TroHCpepi and TroHCpep2 interfered with the FXa-FVa interaction by competing for FXa binding site on FVa, competitive inhibition studies were carried out using the same system as the assay of inhibition of prothrombinase complex assembly, varying concentrations of FVa, peptides and prothrombin accordingly. The dissociation constant (K_d) and maximum velocity (V_max) for the interaction between FXa and FVa in the presence of a fixed concentration of a peptide inhibitor was estimated from double reciprocal plots of reaction velocity versus increasing concentrations of FVa. The inhibition constant, Kj, for the peptide was also determined from the double reciprocal graph using the formula: Kj = xKd [l]/(1 - xKd), where x = x-intercept, [I] = inhibitor concentration, K_d = affinity constant for FXa- FVa interaction.

Both peptides competitively inhibited the interaction between FXa and FVa; since the K_d values for interaction between FXa and FVa were different at different concentrations of peptides while the V_max values were equal, a characteristic of competitive inhibition (Figure 2). The average inhibition constants (Ki) for the competitive inhibition reactions were found to be 73.6 μM for TroHCpepi and 112.5 μM for TroHCpep2 (Table 2). In similar inhibition studies, varying the concentration of prothrombin, it was observed that the K_m's were the same for different concentrations of peptides while the Vmax values were different, indicating non-competitive inhibition (Figure 3). This indicated that peptides did not bind to the substrate binding site of the FXa-FVa complex and did not compete for the interaction between prothrombin and the active complex. The average inhibition constants (Kj) for the non-competitive inhibition were calculated as 102.8 μM for TroHCpepi and 200 μM for TroHCpep2 (Table 2). These results strongly suggest that these peptides interfered with the assembly of FXa-FVa complex, most likely by binding to FVa, but did not bind to the substrate prothrombin or the active site. 1.1.6. Binding studies using surface plasmon resonance (SPR)

Real time biomolecular interaction analysis (BIA) studies were performed to obtain the affinity constants to determine the binding affinity of TroHCpepi and TroHCpep2 for FVa, FXa and prothrombin. These interactions were monitored by surface plasmon resonance (SPR) using a BIACORE instrument (Pharmacia Pte Ltd). Briefly, the system was primed with 10 mM HEPES buffer, 150 mM NaCI, pH 7.4 at 25⁰C. A CM5-sensor chip was pre-activated by amine coupling by injecting 55 μl of a mixture of equal volumes of 0.1 M N- hydrosuccinimide (NHS) and 0.4 M N-ethyl-N^!- dimethylaminopropylcarbodiimide. For immobilization on the chip, the ligands were dissolved in 10 mM HEPES, 150 mM NaCI, at a pH ~1 to 1.5 units below the isoelectric point of the ligand. Accordingly, TroHCpepi (pi = 9.7) was dissolved in 55 μl buffer of pH 8.5 and TroHCpep2 (pi = 4.5) in 55 μl buffer of pH 3.5, both to 1 mg/ml. After immobilization of ligands, excess reactive carboxyl groups on the chip were deactivated by injecting 55 μl of 1 M ethanol amine hydrochloride, pH 8.5. Then, 100 μl of a regeneration buffer, 1 M NaCI, was injected to remove ligand molecules that were not properly immobilized on the chip surface. All the above steps were carried out at a flow rate of 5 μl/min of the running buffer.

Kinetic studies were carried out on the immobilized peptides using 40 μl of six different concentrations (100 μg/ml, 75 μg/ml, 50 μg/ml, 25 μg/ml, 10 μg/ml and 1 μg/ml) of three sets of analytes (FVa, FXa and prothrombin), which were passed over each peptide at a running buffer flow rate of 35 μl/min. After each concentration of each analyte was injected, the chip was regenerated using the regeneration buffer, 1 M NaCI. Affinity constants were calculated from interaction curves using the BIAevaluation software.

Both TroHCpepi and TroHCpep2 bound specifically to FVa. The K_d for the TroHCpep1-FVa interaction was 23.8 μM, while that for the TroHCpep2-FVa interaction was 90.3 μM. As for interaction with FXa, TroHCpepi showed very weak but negligible binding, and TroHCpep2 did not show any detectable binding. Both TroHCpepi and TroHCpep2 did not bind to prothrombin. The K_d values for the TroHCpep1-FXa and TroHCpep2-FVa interaction was undetectable by the SPR. These results indicate that TroHCpepi and TroHCpep2 bind to FVa (Figure 4). Since they compete with FXa for the formation of the complex they bind to the same site as FXa (Figure 4).

1.1.7. Anticoagulant effect of peptides in human plasma Since both the peptides were effective in specifically inhibiting the FXa-FVa interaction, their anticoagulant effect on human plasma was determined. Anticoagulant activity of the peptides was determined by their effects on the recalcification time of human plasma using a fibrometer (Becton-Dickinson Microbiology Systems, Sparks, MD, USA). Freshly obtained human whole blood was centrifuged at 1500 rpm for 10 minutes, to separate blood cells from plasma. Clotting of a mixture of 100 μl human plasma, 250 μl of various concentrations peptide in 50 mM Tris-HCI buffer, pH 7.5 containing 150 mM sodium chloride, at 37°C, was initiated by the addition of 50 μl of 50 mM CaCb and the recalcification time was measured. Dose effect curves for the peptides were plotted.

Both peptides prolonged the recalcification time of citrated human plasma, though TroHCpepi was marginally more potent than TroHCpep2. The recalcification time was prolonged by 50% of the control clotting time at 50 μM concentration of TroHCpepi and 100 μM concentration of TroHCpep2 (Figure 5). The recalcification time was ~250% of the control clotting time at a peptide concentration of 500 μM. Hence the peptides are efficacious not only in a system consisting of purified factors, but also interfere in the blood coagulation cascade in plasma. They therefore have the potential to be developed into novel anticoagulants that specifically inhibit the assembly of the enzyme-cofactor prothrombinase complex. TroHCpepi was chosen for further investigation to delineate MIS.

1.1.8. Stepwise deletion of residues from amino and carboxylic acid terminal

To delineate the MIS, peptides were synthesized with stepwise deletion of residues from the amino terminal end of TroHCpepi (ETRRLLSVDKIYVHTKFVPPN) and assayed for activity. Results showed that residues ETRRLLSVD could all be removed without significantly affecting the activity of the peptide (IC₅₀ of the active peptides = 500 μM). The same process deletion was done on the carboxyl terminal. PPN was removed with the resultant peptide showing about a 50 % drop (IC₅₀ = 1000 μM) in activity. Removal of any residues beyond PPN from the carboxyl terminal resulted in a significant drop in activity. This showed that PPN at the carboxyl terminal may play a structural role of enhancing the activity while not being directly involved in the binding (Table 1 ). Previous studies have shown that the presence of prolines in the flanking regions of synthetic bioactive peptides significantly enhances their potency by limiting backbone flexibility, hence stabilizing favorable conformations (Kini and Evans, 1995a; Kini and Evans, 1995b). This explains the moderate drop in activity upon the removal of PPN. Combining these results, the MIS found contained 9 residues, KIYVHTKFV.

1.1.9. Enhancing the activity of MIS by 'proline bracket' To enhance the activity of MIS both the amino and carboxyl terminal were attached with one proline residue each to form the 'proline bracket'. The 'proline bracket' was suggested to impose conformational restrictions and stabilizing favorable conformations of the peptides. The results showed that the activity of MIS was enhanced by 3-folds to the IC50 of 300 μM by inclusion of the 'proline bracket¹ (Table 1 ). 1.1.10. Alanine scan

Alanine scan was used to identify the role of side chain of each residue of MIS. Sequential replacements of one residue at a time with alanine for all the residues spanning the MIS were done. Alanine was chosen to replace the residues as it has only a methyl group as side chain thus eliminating any group beyond the β-carbon. Unlike glycine and proline, it does not alter the main chain conformation and does not impose extreme electrostatic or steric effects. Since the replacement of alanine only disrupts the side chain structure, any difference in activity observed should be due to the loss of the original side chain. The peptide sequence V²F¹K¹I²Y³V⁴H⁵T⁶K⁷F⁸V⁹P¹⁰N¹¹ (IC₅O = 300 μM) was used as prototype in this alanine scan (underlined portion of the sequence represents the MIS for binding and prolines were added at both sides to enhance the activity) (Table 3).

K¹, I² and K⁷ of the peptide were found to possess side chains that are essential for binding. Loss of either one of the side chains resulted in a significant drop of activity as there was minimal inhibitory effect (-10 %) even at a concentration of 1000 μM. Since a single replacement of residue side chain caused such a significant loss in activity, these side chains are believed to be directly involved in the binding to their complementary residues in the receptor site. K¹ and K⁷ both possess basic side chains and are likely to be protonated in the assay. This means that the side chains are positively charged and could bind to their complementary binding residues through ionic interaction. I² possesses a bulky aliphatic side chain, which is neutral and very hydrophobic. Side chain of I² could bind to its complementary binding residue through hydrophobic interaction (Table 3).

Y³, V⁴ and F⁸ were found to possess side chains that are important but not essential for interaction. This observation is based on the results that although some activities were lost when these residues were replaced, they were to a lesser extent compared to when K¹, I² and K⁷ were replaced. These side chains might not be involved directly in binding to a specific residue at the receptor site, but their presence helps in maintaining the optimum structure for the interaction. As Y³, V⁴, and F⁸ are mostly hydrophobic (overall, Y is hydrophilic but has a hydrophobic aromatic group), the effects of these side chains are most likely brought about by hydrophobic interactions and steric effects either with each other to lock the specific binding residues in position or to interact with hydrophobic residues in the receptor site to bring the specific binding residues closer to their target. All in all, the intermediate loss of activity suggested that the role of these 3 residues were not essential (Table 3).

V⁹, H⁵ and T⁶ did not record any loss of activities when replaced with alanine. Although V⁹ proved to be essential in the C-terminal deletion scan, it had a similar activity when replaced with alanine. The side chain of V⁹ is not important in interaction but if a residue is eliminated from this position the activity is lost. The steric effect brought about by the presence of a residue is possibly needed in the backbone for interaction (Table 3).

Most interestingly, when H⁵ and T⁶ were replaced with alanine separately, the resultant peptides recorded 1200-folds (IC₅₀ = 0.25 μM) and 240-folds (IC₅O = 1.25 μM) increase in activity respectively. Based on these results the side chains of the two residues hindered the optimum binding of the peptides to receptor site. The bulky histidine side chain might introduce steric hindrance in the interaction. Threonine causes a bend in the secondary structure of proteins, and therefore replacement with alanine might have eliminated this bend and opened up the peptide structure for optimum binding (Table 3, Figure 6). These two peptides: 13VP_A7_PN (V²F¹K¹I²Y³V⁴A⁵T⁶K⁷F⁸V⁹P¹⁰N¹¹) and 13VP_A8_PN (V¹F¹K¹I²Y³V⁴H⁵A⁶K⁷F⁸V⁹P¹⁰N¹¹) are good leads for further development of anticoagulant peptides due to their high potency.

Table 1. Sequences of peptides synthesized to determine MIS, their corresponding IC₅₀/μM and percentage of inhibition at 1000 μM in the assaty of inhibition prothrombinase complex assembly.

Table 2. A comparison of inhibitory properties of TroHCpepi and TroHCpep2.

Table 3. Sequence of peptides synthesized for alanine scan, their corresponding IC₅₀/μM and percentage of inhibition at 1000 μM in the assay of inhibition prothrombinase complex assembly. References:

Camerer, E., Rottingen, J-A., Gjernes, E., Larsen, K., Skartlien, A. H., Iversen, J-G., Prydz, H. (1999). Coagulation factors Vila and Xa induce cell signaling leading to up-regulation of the egr-1 gene. J Biol Chem. 21 A, 32225-32233.

Gajdusek, C, Carbon, S., Ross, R., Nawroth, P., Stem, D. (1986). Activation of coagulation releases endothelial cell mitogens. J Cell Biol. 103, 419-28.

Gasic, G. P., Arenas, C. P., Gasic, T. B., Gasic, G. J. (1992). Coagulation factors X, Xa, and protein S as potent mitogens of cultured aortic smooth muscle cells. Proc Natl Acad Sd (USA). 89, 2317-2320.

Joseph, J. S., Chung, M. C, Jeyaseelan, K., and Kini, R.M. (1999). Amino acid sequence of Trocarin, a prothrombin activator from Tropidechis carinatυs venom: Its structural similarity to coagulation factor Xa. Blood 94, 621-631.

Kini, R. M. and Evans H. J. (1995a). A hypothetical structural role of proline residues in the flanking segments of protein-protein interaction sites. Biochem Biophys Res Commun. 212, 1115-1124.

Kini, R. M. and Evans H. J. (1995b). A novel approach to the design of potent bioactive peptides by incorporation of praline brackets: antiplatelet effects of Arg-Gly-Asp peptides. FEBS Lett. 375, 15-17.

Kini, R. M., Morita, T., Rosing, J. (2001 ). Classification and nomenclature of prothrombin activators isolated from snake venoms. Thrombos Haemostas. 85, 710-711. Ko, F. N., Yang, Y. C, Huang, S. C, Ou, J. T. (1996). Coagulation factor Xa stimulates platelet-derived growth factor release and mitogenesis in cultured vascular smooth muscle cells of rat. J CHn Invest. 98, 1493-1501.

Nicholson, A. C, Nachman, R. L., Altieri, D. C, Summers, B. D., Ruf, W., Edgington, T. S., Hajjar, D. P. (1996). Effector cell protease receptor-1 is a vascular receptor for coagulation factor Xa. J Biol Chem. 271, 28407-28413.

Papapetropoulos, A., Picardoni, P., Cirino, G., Bucci, M., Sorrentino, R., Cicala, C, Johnson, K., Zachariou, V., Sessa, W. C, Altieri, D. C. (1998). Hypotension and inflammatory cytokine gene expression triggered by factor Xa-nitric oxide signaling. Proc Natl Acad Sci (USA). 95, 4738-4742.

Senden, N. H. M., Jeunhomme, T. M. A. A., Heemskerk, J. W. M., Wagenvoord, R., van't Veer C, Hemker, H. C, Buurman, W. A. (1998). Factor Xa induces cytokine production and expression of adhesion molecules by human umbilical vein endothelial cells. J Immunol. 161 , 4318-4324.

Claims

Claims

1. An isolated peptide having an amino acid sequence of:

(a) [Σ]_nNYYYVHQNFDRVAYDYDI[U]_m ; or

(b) [Σ]_nKIX³X⁴X⁵X⁶KX⁸X⁹[U]_m wherein

X³, X⁴ and X⁸, are independently an amino acid selected from A, F, I, L, M, V, W and Y; each of X⁵, X⁶ and X⁹ is any amino acid; [Σ] is any amino acid, and n is an integer from 0 to 15; [U] is any amino acid, and m is an integer from 0 to 15.

2. The peptide of claim 1 , wherein the amino acid sequence is: [Σ]_nKIYVX⁵X⁶KFX⁹[U]_m.

3. The peptide of claims 1-2, wherein X⁵ is H or A; X⁶ is T or A; and X⁹ is V or A.

4. The peptide of claims 1-3, wherein the amino acid is: [Σ]_nKIYVX⁵TKFV[U]_m and X⁵ is H or A; [Σ]_nKIYVHX⁶KFV[U]_m and X⁶ is H or A; [Σ]_nKIYVHTKFX⁹[U]_m and X⁹ is H or A.

5. The peptide of claims 1-4, wherein the amino acid sequence is: [Σ]_nKIYVHTKFV[U]_m.

6. The peptide of claims 1-5, wherein [Z]_n is selected from ETRRLLSVD, GPLLSVD, LLSVD, SVD, D, P, PP, PPP, PPPP₁ PPPPP, VP, VPP, VPPP, VPPPP, F, FV, FVP, FVPP and [U]_m is selected from P, PP, PPP, PPPP, PPPPP, PN, PPN, PPPN, PPPPN.

7. The peptide of claims 1-6, wherein the amino acid is selected from:

SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25 and SEQ ID NO:26.

8. The peptide of claims 1-7, which is a synthetic peptide.

9. An isolated nucleic acid molecule encoding the peptide of claims 1-8.

10. A vector comprising the nucleic acid molecule of claim 9.

11.A host cell comprising the vector of claim 10 or a nucleic acid of claim 9.

12. A method of producing a peptide comprising cultivating the cell of claim 11 and expressing the peptide.

13.A composition comprising at least one peptide of claims 1-8, the nucleic acid molecule of claim 9 or the vector of claim 10.

14. The composition of claim 13, wherein the composition is a pharmaceutical composition.

15. The composition of claim 14, further comprising a pharmaceutically acceptable excipient, diluent and/or carrier.

16. The peptide of claims 1-8 for use in medicine.

17. The peptide of claim 16, for use as an anticoagulant.

18. Use of at least one peptide of claims 1-8 for the preparation of an anticoagulant.

19. A method for inhibiting the assembly of prothrombinase complex in a subject comprising administering to a subject at least one peptide of claims 1-8 or a composition of claims 14-15.

20. A method for the prevention and/or treatment of formation of intravascular blood clots in a subject comprising administering to the subject at least one peptide of claims 1-8 or a composition of claims 14-15.

21. The method of claim 20, wherein the method is for the prevention and/or treatment of ischemic events.

22. The method of claim 21 , wherein the ischemic event is coronary syndrome, myocardial infarction, stroke, venous thromboembolism and/or peripheral arterial occlusion.

23. The method of claims 20-22, wherein the subject is a mammal.

24. The method of claim 23, wherein the mammal is human.

25.A method for identifying anticoagulant peptides, comprising:

- providing at least one peptide; - determining the minimum interaction site (MIS) between the at least one peptide and FVa;

- contacting in vitro the peptide comprising the MIS with the prothrombinase complex; and - determining the ability of the peptide in inhibiting the assembly of the prothrombinase complex.

26. The method of claim 25, further comprising the step of contacting the peptide comprising the MIS to plasma, and determining the ability of the peptide to interfere with the blood coagulation cascade in plasma.

27. The method of claims 25-26, further comprising selecting the peptide comprising the MIS, modifying at least one amino acid residue inside and/or outside the MIS, and determining the ability of the modified peptide to interfere with the assembly of the prothrombinase complex and/or with the blood coagulation cascade in plasma.

28. The method of claim 27, further comprising the step of isolating or synthesising the modified peptide capable of interfering with the assembly of the prothrombinase complex and/or with the blood coagulation cascade in plasma.

29. The method of claims 25-28, wherein the provided at least one peptide comprising the MIS is obtained from fragmentation of a protein.

30. The method of claim 29, wherein the protein is trocarin.

31. The method of claims 29-30, wherein the peptide is at least one peptide of claims 1-8.