WO2007052067A2 - Von willebrand factor (vwf) binding peptides - Google Patents

Abstract

This invention relates to short triple-helical collagen peptides that are recognised by VWF A3 domain and which consist of the sequence N1RGX3X4GX5X6GX7N2, wherein; X3 is any amino acid except G, X4 is O or P, X5 is any amino acid except G or A, X6 is any amino acid except G, X7 is any amino acid except G or A, and; N1 and N2 are amino acid sequences consisting, independently, of 0 to 10 amino acid residues. Peptides described herein may be useful in the treatment of thrombosis and the diagnosis of bleeding disorders, as well as in the construction of synthetic collagens.

Description

von Willebrand factor (vWF) Binding Peptides

The present invention relates to peptides, in particular peptides based on collagen sequences, which bind the plasma protein von Willebrand factor (vWF) and are useful in modulating platelet and other cell function, including aggregation and thrombus formation, also to methods of use of the peptides and compositions comprising them.

Human collagens represent a family of 28 or more proteins each of which is characterised by the presence of a triple-helical structure, formed as three separate protein strands as left-handed helices, sometimes different gene products, wind around one another to form a right-handed super helix. This defining conformation is facilitated by a repetitive [glycine-x-x' ] _n structure, where n may be as large as 350, so that a triple helical domain (col domain) may be in excess of 1000 amino acids in length. In nature, the amino acids x and x¹ are quite often proline and hydroxyproline, which occur in- qu-ite high- proportion within col -domains and are. essential . for the formation of the triple-helical structure which is known as the polyproline helix. The collagens are widely distributed within the vertebrate organism where they perform an essential structural role. Examples of this are provided by the most abundant fibrillar collagens, types I, II and III, which occur in skin, bone, cartilage, tendon and in the vitreous humour of the eye. More subtle roles are played by the more complex non-fibrillar collagens, such as types IV and VI, which form two- and three-dimensional networks, supporting the interstitial tissues of the body and being the fundamental component of the basement membranes to which epithelial and endothelial cell layers can attach.

Circulating platelets, also called thrombocytes, within the bloodstream are the crucial cellular components of blood which regulate the clotting process. In healthy, undamaged tissues, collagens which support the blood vessel wall and its surrounding tissue are concealed by endothelial cell layers and cannot come into contact with the circulating platelets. However, should the endothelial cell layer be removed either in disease or upon tissue injury, then collagens are revealed which can interact with the cellular components of the blood as well as with proteins in blood plasma. The platelet surface contains a series of proteins known as receptors which sense the presence in the extracellular medium of specific molecules, including hormones, cytokines and other species. Collagen can bind directly to several such receptors on the platelet surface, notably integrin α2βl and Glycoprotein VI (GpVI) . See Farndale et al . for a recent review ^λ. Indirect interactions can also occur, as when von Willebrand Factor (VWF) binds to the platelet surface through the Glycoprotein Ib/V/IX complex, interacting directly with Gplbα, and also to specific sites within the collagens. This interaction has hitherto been poorly understood, and is the subject of the present work.

The platelet regulates the blood clotting process in a complex series of interacting processes. ^"First, the platelet must be captured from the circulation, a process that may vary with the shear stress experienced at the site of injury. Damage to the vessel wall may occur as a consequence of either mechanical trauma or rupture of atherosclerotic plaque in diseased blood vessel walls. The initiating event in platelet adhesion is the binding of the plasma protein von Willebrand factor (vWF) to collagen, necessary to support a transient interaction of platelets with collagen-bound VWF via the platelet receptor glycoprotein Ibα¹'²'⁸. The GPIbα-VWF interaction slows down platelets, which allows firm adhesion via other platelet adhesive receptors such as the collagen receptors α2βl and glycoprotein VI^s. The platelet must be secured at the exposed collagen surface, and the integrin α2βl is considered to be crucial for this process. Finally, the platelet must be activated, and GpVI is considered the primary activatory collagen receptor expressed on the platelet surface. There remains debate about the exact role of these individual interactions in the capture and activation of platelets. Once the platelet becomes activated, a new series of events is set in motion, with further activatory materials such as ADP and ATP being secreted as part of the platelet dense granules' content, and thromboxane A₂ being generated from endogenous arachidonic acid released from cell membrane lipids upon stimulation by various platelet agonists.

Three definable endpoints characterise the platelet activation process:

First, the activation of integrins may allow tighter binding of integrin α2βl to collagens but most importantly increases the affinity of the fibrinogen receptor integrin αllbβ3 which allows plasma fibrinogen molecules to cross-link two copies of αllbβ3 on the surfaces of adjacent platelets, the fundamental interaction in the process known as platelet aggregation, or thrombus formation.

Next, the activated platelet secretes, primarily from its α- granules, bioactive materials such as platelet-derived growth factor, which may be important in stimulating cells locally to repair the damage to the blood vessel wall and the surrounding tissue. The dense granules are also secreted and release, as well as ATP and ADP, other agents including serotonin and calcium ions, all of which activate other platelets locally and recruit them to the growing thrombus .

Finally, the platelet surface may become procoagulant, that is, the distribution of phospholipids between its inner and outer leaflets changes such that negatively charged phospholipids, phosphatidylserine and phosphatidylethanolamine, are present in greater quantities upon the outer surface of the platelet where they act as a catalytic surface for the coagulation cascade and the generation of thrombin. This thrombin causes the proteolytic cleavage of plasma fibrinogen such that fibrin is formed which polymerises and clots, trapping local red cells and leading to the occlusion of breaches in the damaged vessel wall. Thrombin is also active upon other cells nearby, and may contribute to the regulation of tissue repair.

These processes are central to the normal haemostatic response, and are well understood in the field. Each of these processes may also be important in disease, where atherosclerosis, the formation of plaques which constrict the blood vessel, progresses to the point where weak, disorganised, lipid-rich tissue may be prone to fissuring or rupture, a process which reveals underlying collagens and collagen fragments, causing the activation of platelets and the clotting process. This set of pathological events is known as atherothrombosis , and is life-threatening when it occurs in vital blood vessels such as the coronary artery, causing heart attack, or in the cerebral vasculature where it may cause stroke. Detailed reviews of these various topics can be found in recent texts ^2"4.

The recognition of collagen by the platelet receptors described above depends upon its primary sequence. The collagens must contain specific combinations of amino acids for recognition to occur. However, denatured collagens (gelatins) exist as single random coil polypeptides, which, despite sequence being preserved, will not bind to these receptors ⁵. Thus, triple-helical conformation of the collagens is also an essential pre-requisite for their recognition by platelet and other collagen receptors, and the use of synthetic triple-helical peptides comprising specific recognition motifs has allowed receptor-binding properties of the collagens to be investigated in detail ⁵'⁷. This strategy has been applied successfully to both α2βl and GpVI.

For integrin α2βl, some recognition sequences within collagens are well-known, such as the GFOGER motif which occurs in collagens I, II and IV and is a high-affinity ligand that binds well even when the integrin is in a resting state. Other similar sequences that also bind α2βl especially when the receptor is activated include GLOGER, GMOGER, GLSGER, GASGER and GAOGER 8-11. Such sequences are quite widely-distributed within the triple-helical col domains, especially of the fibrillar collagens. Related sequences that also bind integrin include GROGER found in collagen III, unusual in that it contains a positively-charged arginine rather than a hydrophobic residue in the second position of the motif (Raynal N et al . J Biol Chem. 2006 ; 281 : 3821-3831) .

For GpVI, however, no clear definition exists. Certain sequences are known to interact with GpVI, and these include the triplet GPO. The affinity of such motifs increases with the number of adjacent GPO triplets within triple-helical peptides, such that [GPO] ₂ binds detectably to GpVI, and [GPO]₄ binds almost as well as the longest such peptide hitherto described, the collagen-related peptide (CRP) which contains a [GPO]₁₀ triple-helical core ¹². CRP is widely used in ^"the field a^"s a research tool, as a selective ligand for GpVI ¹³'¹⁴ Although CRP is considered specific for GpVI, it is not known whether the GPO motifs which form about 10% of the fibrillar collagen sequence are uniquely responsible for interaction with GpVI, or whether other motifs within collagens which lack the GPO triplet can also be recognised by the receptor.

The VWF binding site(s) in collagen are unknown. Putative VWF binding sites have previously been reported in collagen type III, but the triple helical peptides containing these binding sites bound VWF with low affinity compared to full-length collagen¹⁷.

The binding site for collagen on α2βl resides within the I -domain of its α subunit, and has been well-characterised by site-directed mutagenesis and by co-crystallisation with a triple-helical peptide ^9|18. Similarly, site-directed mutagenesis of recombinant GpVI has shown that collagen binds to the hinge region of GpVI, the apex between its two immunoglobulin folds ¹⁹.

VWF bound to GpIb/V/lX, forms an important collagen-binding complex that acts as a collagen receptor, and each of its Al and A3 domains has been implicated in the recognition of collagen, although the role of Al is primarily to bind GpIb. Critical residues have previously been identified in the VWF A3 domain that are involved in the binding to collagen type III using rational site-directed mutagenesis based on the crystal structure of the complex between the VWF A3 domain and a monoclonal antibody against the VWF A3 domain, which inhibits the interaction with collagen³'¹⁵. Furthermore, Nishida et al reported mapping of the collagen-binding mode of the A3 domain by NMR analysis using transferred cross - saturation, and results of these experiments were confirmed by mutational analysis¹⁶.

The present inventors have identified short triple-helical collagen peptides -that are recognised by- VWF A3 domain and may be -useful in - the treatment of thrombosis and in the construction of synthetic collagens .

One aspect of the invention provides a peptide consisting of the sequence N₁RGX₃X₄GX₅X₅GX₇N₂ wherein;

X₃ is any amino acid except G or 0,

X₄ is O or P,

X₅ is any amino acid except G, 0, or A,

X₆ is any amino acid except G, X₇ is any amino acid except I, 0, G or A, and;

Nl and N2 are amino acid sequences consisting, independently of 0 to

10 amino acid residues. In some embodiments, X₅ may be V, I, L, or T or a conservative substitution thereof, preferably V or I .

In some embodiments, X₆ is M or NIe or a conservative substitution thereof .

In some embodiments, X₇ may be F or a conservative substitution thereof, L, Y, H or W, preferably F or a conservative substitution thereof .

In some preferred embodiments, a peptide may consist of the sequence

N₁RGX₃OGVX₆GFN₂, wherein X₃ and X₆ are, independently, any amino acid other than

G and, wherein N₁ and N₂ are amino acid sequences independently consisting of 0 to 11 amino acid residues.

In some embodiments, X₃ may be Q or E, preferably Q, and X₆ may independently be M, NIe or I, preferably M.

A suitable peptide may, for example, consist of the sequence RGX₃OGVX₆GF .

N₁ may consist of the sequence GXi or may comprise the sequence GX₁ at its C terminal, where X₁ is any amino acid other than G, preferably any amino acid other than G or 0, for example A or P. N₂ may consist of the sequence X₈ or may comprise the sequence X₈ at its N terminal, where X₈ may be any amino acid other than G, preferably P or 0. A suitable peptide may, for example, consist of the sequence GX₁RGX₃OGVX₅GFX₈

Preferably, Nl consists of the amino acid sequence GX_aX_bGX_cX_dGX_eX_fGX_1y wherein X_a to X_f are independently any amino acid other than G. For example, N₁ may consist of the sequence GROGPOGPSGP or a C terminal fragment thereof, for example P, GP, SGP, PSGP, GPSGP, OGPSGP, POGPSGP, GPOGPSGP, OGPOGPSGP or ROGPOGPSGP.

Preferably N2 consists of the amino acid sequence X₈GX₃XbGXcXdGXeXf, wherein X_a to X_f are independently any amino acid other than G. For example, N₂ may consist of the sequence OGPKGNDGAO or an N terminal fragment thereof, for example 0, OG, OGP, OGPK, OGPKG, OGPKGN, OGPKGND, OGPKGNDG or OGPKGNDGA,

A suitable peptide for use as described herein may consist of the sequence GPOGPSGPRGX₁OGVX₂GFOGPKGNDGAO .

A peptide as described herein may trimerise to form a peptidyl trimer which binds to vWF. Preferably, a peptidyl trimer binds to vWF with a Kd of less than 50OnM, less than 5OnM, less than 5nM, less than InM or less than O.lnM. For example, a peptidyl trimer may bind to vWF with a Kd of about 2.5nM, as determined by Biacore.

Preferably, a peptidyl trimer promotes or induces platelet aggregation and/or platelet activation, especially when in a peptidyl trimer.

Peptides described herein may be cross-linked by covalent bonds. Any system of covalent cross-linking may be employed and numerous suitable systems are available in the art. For example, N- and C- terminal Cys residues may be incorporated into the peptides. SPDP (Perbio Science UK Ltd) may then be used to link free Cys to free peptide N-termini. Alternatively, Lys residues may be incorporated into peptides as described herein and crosslinked using glutaraldehyde (Morton LF et al (1995) Biochem J 306: 337-344.

Another aspect of the invention relates to a peptide consisting of the sequence N₁GX₁RGX₃AGVX_SGFX₈N₂ or N₁GX₁RGX₃OGNX_SGFX₈N₂ which is capable of trimerising to form a heterotrimer which binds vWF, wherein Nl and N2 and X₁ to X₈ are as described above.

Another aspect of the invention relates to a set of peptides consisting of up to three peptides, wherein each said peptide consists of the sequence N₁GX₁X₂GX₃X₄GX₅X₆GX₇X₈N₂ wherein;

X₁ to X₈ are independently any amino acid except glycine, preferably, X₁ X₃ X₅ and X₇ are independently any amino acid except glycine or O; and;

X₂ is R in at least one of said peptides,

X₂ is R and X₄ is 0 or P, preferably 0 in at least one of said peptides, and; X₅ is V or I, preferably V, and X₇ is F, L, Y or W, preferably F, in at least one of said peptides, and N₁ and N₂ are as described above.

A set of peptides may consist of 1, 2 or 3 different peptide sequences which form a trimer comprising a vWF binding site. For example, a suitable set of peptides may consist of a peptide consisting of the sequence RGX₁AGVX₂GF and a peptide consisting of the sequence RGX₅OGNX₇GF₁ wherein X₁, X_2, X₅ and X₇ are, independently, any amino acid except G, preferably X₁, X₅ and X₇ are independently any amino acid except glycine or 0.

X₁ may be A in one, two or three of said peptides. X₂ may be R in one, two or three of said peptides, X₃ may be Q or E in one, two or three of said peptides. X₄ may be 0 or A in one, two or three of said peptides. X₅ may be V or N in one, two or three of said peptides. X₆ may be M or I in one, two or three of said peptides. X₇ may be F in one, two or three of said peptides. X₈ may be 0 in one, two or three of said peptides. The present invention also encompasses peptides that consist of sequences having one, two, three or more conservative substitutions relative to the sequences set out above. A conservative substitution is a replacement of an amino acid residue with another of similar properties, such as charge, polarity and/or hydrophobicity. For example, conservative substitutes for an amino acid within the native polypeptide sequence can be selected from other members of the class to which the amino acid belongs. Amino acids can be divided into the following four groups: (1) acidic amino acids, (2) basic amino acids, (3) neutral polar amino acids, and (4) neutral, nonpolar amino acids. Representative amino acids within these various groups include, but are not limited to, (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine,- (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine,- isoleucine, valine; proline, phenylalanine, tryptophan, and methionine. Conservative substitution tables listing functionally similar amino acids are known in the art (Altschul, S. F. 1991 Journal of Molecular Biology 219: 555-665, Crighton (1984) Proteins , W. H. Freeman and Company) . For example, a peptide consisting of a sequence having one, two, three or more conservative substitutions may show 95%, 99% or 100% sequence similarity to a sequence set out herein. Amino acid similarity may be defined with reference to the algorithm GAP (Accelerys) , or the TBLASTN program, of Altschul et al. (1990) J. MoI. Biol. 215: 403- 10.

Non-naturally occurring peptide and polypeptide fusions comprising peptides as described above are also provided as aspects of the present invention, particularly wherein the peptide is fused to one or more sequences which are not naturally fused to the peptide. Sequences which are not naturally fused to the peptide may include synthetic collagen sequences, non-collagen sequences or additional copies of the peptide sequence itself. In some embodiments, one or more heterologous amino acids may be joined or fused to the peptide structure set out herein and polypeptide or peptide of the invention may comprise a peptide as described above linked or fused to one or more heterologous amino acids .

In preferred embodiments, peptides as described above are fused to heterologous N and C terminal amino acid sequences which support the triple-helical polyproline II helix structure, for example GX_aX_b repeat sequences, where X_a and X_b are any amino acid other than G, preferably X_a is independently any amino acid except glycine or 0. Suitable triple-helical sequences include GPP and/or GPO repeats. For example, (GPP)_n, wherein n is 2-6 or more and (GPO) _nl where ni is 2-6 or more.

Peptide for use as described herein may consist of the amino acid sequence (GPP) _nNiRGXsOGVXgGFNa (GPP) _n, (GPO) _nlNiRGX₃OGVX_sGFN₂ (GPO) _nl., (GPO)₁₁₁N₁RGX₃OGVX₆GFN₂(GPP)_n, or (GPP)_nN₁RGX₃OGVX₆GFN₂(GPO)_n!.

Examples of suitable peptides include,- (GPO) ₄GFOGER (GPO) ₄GPRGX₁OGVX₂GFO (GPO) ₄ and (GPO)₃GPRGX₁OGVX₂GFO(GPO)₂, where X₁ and X₂ are as set out above.

By "heterologous" is meant not conforming with the formula of the peptide as set out herein, and not occurring in any natural collagen (e.g. type 1 or type 2 collagen) joined by a peptide bond without intervening amino acids to a peptide described herein, that is to say usually a chain of amino acids which is not found naturally joined to any peptide described herein at the position of fusion in the peptide of the invention. Usually, where heterologous amino acids are fused to the peptide, the whole contiguous sequence of amino acids does not occur within collagen, and may be 10 or more, preferably 15 or more, more preferably 20 or more, 25 or more or 30 or more amino acids with a sequence which does not occur contiguously in collagen.

Heterologous amino acids may form an additional sequence or motif, such as one or more fibrinogen fragments, other integrin ligands such as fibronectin and fragments thereof, RGD peptides and so on. Indeed, any desired additional peptide may be included in a fusion with a peptide described herein, including non-triple helical extensions of the triple helix formed by trimerising of the peptides .

Heterologous amino acids may form an additional collagen sequence or motif, for example a triple helical motif which is recognised by collagen receptors on platelets, for example GpVI and/or integrin α2βl binding motifs. Peptides comprising such motifs may activate platelets through the binding of GpVI and/or integrin α2βl, or other platelet receptor or combinations of these to the peptide. Of course, where such motifs are found in collagen, they are not naturally linked to the peptide sequence set out above.

The overall length of a peptide or polypeptide fusion comprising a peptide as described herein may be less than 100 amino acids, less than 90 amino acids, less than 80 amino acids, less than 70 amino acids, or less than 60 amino acids.

In preferred embodiments, the overall length of a peptide or polypeptide fusion may be 25 or more, 27 or more or 30 or more amino acids .

A peptide or polypeptide as described herein may be linked to a coupling partner, e.g. an effector molecule, a label, a marker, a drug, a toxin and/or a carrier or transport molecule, and/or a targeting molecule such as an antibody or binding fragment thereof or other ligand. Techniques for coupling peptides to both peptidyl and non-peptidyl coupling partners are well-known in the art.

In some embodiments, a peptide, polypeptide or peptidyl trimer may be attached or coated on to a solid surface or insoluble support. The support may be in particulate or solid form, including for example a plate, a test tube, beads, a ball, a filter, fabric, polymer or a membrane. A peptide or polypeptide may, for example, be fixed to an inert polymer, a 96-well plate, other device, apparatus or material which is used in a clinical or investigative context, for example in the diagnosis of von Willebrand disease. Methods for fixing peptides or polypeptides to insoluble supports are known to those skilled in the art.

For some applications, the support may be non-immunogenic .

In some embodiments, the support may be a protein, for example a plasma protein or a tissue protein, such as an immunoglobulin or fibronectin. In other embodiments, the support may be synthetic and may be, for example. a .biocompatible, biodegradable polymer.. Suitable polymers include polyethylene glycols, polyglycolides, polylactides . polyorthoesters, polyanhydrides , polyphosphazenes, and polyurethanes . (Gunatillake PA et al 2003, European Cells and Materials 5, 1-16 Biodegradable synthetic polymers for tissue engineering)

Another aspect of the invention provides a conjugate comprising a peptide, polypeptide or trimer as described herein attached to an inert polymer.

The inclusion of reactive groups at one end of the peptide allows chemical coupling to inert carriers such that peptides might be delivered to pathological lesions such as chronic wounds or sites of acute traumatic injury without entry into the bloodstream. Other aspects of the invention relate to peptidyl trimers which bind to vWF and modulate platelet function.

A peptidyl trimer may comprise,- three peptides consisting of the sequence GX_IX₂GX₃X₄GX₅X₆GX₇X₈ wherein;

X₁ to X₈ are independently any amino acid except glycine, more preferably, X₁ X₃ X₅ and X₇ are independently any amino acid except glycine or O, and; X₂ is R in at least one of said peptides,

X₂ is R and X₄ is O or P, preferably O in at least one of said peptides, and;

X₅ is V, I, T or L, preferably V, and X₇ is F, L, Y or W, preferably

F in at least one of said peptides.

X₁ may be A in one, two or three of said peptides of the trimer.

X₂ may be R in one, two or three of said peptides of the trimer.

X₃ may be Q or E in one, two or three of said peptides of the trimer.

X₄ may be O or A in one, two or three of said peptides of the trimer.

X₅ may be V or N in one, two or three of said peptides of the trimer.

X₅ may be M or I in one, two or three of said peptides of the trimer. X₇ may be F in one, two or three of said peptides of the trimer.

X₈ may be O in one, two or three of said peptides of the trimer.

In some embodiments, a suitable peptidyl trimer may comprise three peptides consisting of the sequence GX₁RGX₃OGVX₆GFX₈, wherein X₁, X₃, X₆ and X₈ are independently any amino acid except glycine, preferably, X₁ and X₃ are independently any amino acid except glycine or 0. In other embodiments, a peptidyl trimer may comprise one peptide consisting of the sequence GX_IX₂GX₃X₄GVX₆GFX₈, and; two peptides consisting of the sequence GX_IRGX₃OGX₅X₆GX₇X₈, or; two peptides consisting of the sequence GX₁X₂GX₃X₄GVX₆GFX₈ and one peptide consisting of the sequence GX₁RGX₃OGX₅X₆GX₇Xe wherein X₁ to X₈ are independently any amino acid except glycine, preferably, X₁ X₃ X₅ and X₇ are independently any amino acid except glycine or 0.

For example, a peptidyl trimer may comprise; two peptides consisting of the sequence GX₁RGX₃AGVX₆GFX₈ and one peptide consisting of the sequence GX₁RGX₃OGNX₆GFX₈; or, two peptides consisting of the sequence GX₁RGX₃OGNX₆GFX₈ and one peptide consisting of the sequence GX₁RGX₃AGVX₆GFX₈.

Peptides and polypeptides described herein preferably form trimers under appropriate conditions.

A peptide which forms -a peptidyl trimer may be fused to one or more sequences which are not naturally fused to the peptide, for example one or more heterologous amino acids, to form non-naturally occurring peptide and polypeptide fusions, as described above.

In some embodiments, peptides may be cross-linked within the trimer, for example using covalent bonds e.g. hexanoic acid cross-linking (such as the lysyl-lysyl amino hexanoate cross-linking) . Alternatively, a disulphide knot may be produced and selectively protected and deprotected to link three chains successively and in register.

In other embodiments, peptides may trimerise without any cross- linking, and trimers consisting of peptides as described herein may be provided without cross-linking. A peptidyl trimer may be produced by providing peptides as described herein and causing or allowing (under appropriate conditions) the peptides to associate to form a trimer.

Trimerization may be followed by isolation of trimers, e.g. for subsequent use and/or manipulation.

Peptides, particularly in trimerized form, may be useful in binding vWF and/or influencing cell adhesion to collagen, particularly adhesion of platelets. They may be used to affect activation of cells, such as platelets. This may be in a therapeutic context, e.g. to induce platelet activation and/or aggregation to prevent bleeding through injury, or in vitro for diagnostic purposes to identify dysfunction of the collagen receptor pathways or in the development of therapeutic compounds.

Peptides, particularly in trimerized form, may be useful in binding vWF in a diagnostic context, for example in the detection of von Willebrand disease.

A method of detecting vWF in a sample, in particular a blood sample, obtained from an individual may comprise; contacting a blood sample obtained from an individual with a petidyl trimer as described above, and, determining the presence or absence of binding of vWF to said trimer, wherein the binding of vWF to the trimer is indicative of the presence of vWF in the sample.

A method may be used for assessing an individual for a bleeding disorder, such as von Willebrand disease. The presence of vWF in a blood sample obtained from an individual is indicative that the individual may be suffering from a bleeding disorder. A method may be carried out in a standard format, for example by ELISA, Western blot, or immunoassay, including lateral flow assays such as immunochromatographic strips, flow-through assays, agglutination assays or solid-phase assays such as dipstick or dipstick comb assays.

Peptides may be generated wholly or partly by chemical synthesis. The peptides can be readily prepared, for example, according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available (see, for example, in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984) , in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984) ; in J. H. Jones, The Chemical Synthesis of Peptides. Oxford University Press, Oxford 1991; in Applied Biosystems 430A Users Manual, ABI Inc., Foster City, California , in G. A. Grant, (Ed.) Synthetic Peptides, A User's Guide. W. H. Freeman & Co . , New York 1992, E. Atherton and R. C. Sheppard, Solid Phase Peptide Synthesis, A Practical Approach. IRL Press 1989 and in G. B. Fields, (Ed.) Solid- Phase Peptide Synthesis (Methods in Enzymology Vol. 289). Academic Press, New York and London 1997) , or they may be prepared in solution, by the liquid phase method or by any combination of solid- phase, liquid phase and solution chemistry, e.g. by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof.

Another convenient way of producing a peptidyl molecule as described herein (peptide or polypeptide) is to express nucleic acid encoding a precursor wherein proline appears in place of the desired hydroxyproline, by use of nucleic acid in an expression system. Production of GPO-containing peptides may be achieved for example by co-expression of an appropriate hydroxylase, as has been done with lysyl residues (Nokelainen et al., 1998). For peptides containing Pro residues to be post-translationally converted by hydroxylation to Hyp (O) , prolyl-hydroxylase may be co-expressed. Myllyharju, J. et al. Biochem Soc trans 2000, 4 353-7 describes an efficient expression system for recombinant human collagens which may be useful in providing peptides as described herein. This system uses the methylotrophic yeast Pichia pastoris, with co-expression of the desired peptides chains with the alpha- and beta-subunits of prolyl 4 -hydroxylase.

Another aspect of the invention provides a nucleic acid encoding a proline precursor of a peptide or polypeptide as described herein.

Generally, nucleic acid is provided as an isolate, in isolated and/or purified form, except possibly one or more regulatory sequence (s) for expression. Nucleic acid in accordance with the present invention may be provided as part of a recombinant vector.

Nucleic acid sequences encoding a polypeptide or peptide precursor as described herein can be readily prepared by the skilled person using the information and references contained herein and techniques known in the art (for example, see Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrook and Russell, 2001, Cold Spring Harbor Laboratory Press) .

In order to obtain expression of the nucleic acid sequences, the sequences can be incorporated in a vector having one or more control sequences operably linked to the nucleic acid to control its expression. The vectors may include other sequences such as promoters or enhancers to drive the expression of the inserted nucleic acid, nucleic acid sequences so that the polypeptide or peptide is produced as a fusion and/or nucleic acid encoding secretion signals so that the polypeptide produced in the host cell is secreted from the cell. Polypeptide can then be obtained by transforming the vectors into host cells in which the vector is functional, culturing the host cells so that the polypeptide is produced and recovering the polypeptide from the host cells or the surrounding medium. Prokaryotic and eukaryotic cells are used for this purpose in the art, including strains of E. coli, yeast, and eukaryotic cells such as COS or CHO cells.

Thus, a method of making a polypeptide or peptide as described herein may comprise expression from nucleic acid encoding the polypeptide or peptide (generally nucleic acid according to the invention) . This may conveniently be achieved by growing a host cell in culture, containing such a vector, under appropriate conditions which cause or allow expression of the polypeptide. Polypeptides and peptides may also be expressed in in vitro systems, such as reticulocyte lysate. Following production by recombinant expression, proline is converted to hydroxypi-oline . As noted, this may be achieved within the expression system by provision of a prolyl -hydroxylase, or by enzymatic treatment following production.

As noted, methods of making peptides by chemical synthesis are also encompassed by the present invention.

Another aspect of the invention provides a host cell containing heterologous nucleic acid as disclosed herein. Nucleic acid may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques. The nucleic acid may be on an extra-chromosomal vector within the cell, or otherwise identifiably heterologous or foreign to the cell.

A method may include introducing the nucleic acid into a host cell. The introduction, which may (particularly for in vitro introduction) be generally referred to without limitation as "transformation", may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. As an alternative, direct injection of the nucleic acid could be employed.

Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well known in the art.

The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) under conditions for expression of the gene,, so that the encoded polypeptide (or peptide is produced. If the polypeptide is expressed coupled to an appropriate signal leader peptide it may be secreted from the cell into the culture medium. Following production by expression, a polypeptide or peptide may be isolated and/or purified from the host cell and/or culture medium, as the case may be, and subsequently used as desired, e.g. in the formulation of a composition which may include one or more additional components, such as a pharmaceutical composition which includes one or more pharmaceutically acceptable excipients, vehicles or carriers (e.g. see below).

A peptide or polypeptide as described herein may be chemically modified, for example, by addition of one or more polyethylene glycol molecules, sugars, phosphates, and/or other such molecules, where the molecule or molecules are not naturally attached to wild- type collagen proteins. Suitable chemical modifications are well known to those of skill in the art. The same type of modification may be present in the same or varying degree at several sites in the peptide or polypeptide. Also, a given the peptide or polypeptide may- contain many types of modifications.

Modifications can occur anywhere in the peptide sequence, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, _ gamma-carboxylation .of. glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. See, for instance, Proteins-Structure And Molecular Properties, 2nd Ed. , T. E. Creighton, W. H. Freeman and Company, New York (1993) and Wold, F., "Posttranslational Protein Modifications: Perspectives and Prospects," pgs . 1-12 in Posttranslational Covalent Modification Of Proteins, B. C. Johnson, Ed., Academic Press, New York (1983); Seifter et al . , Meth. Enzymol. 182:626-646 (1990) and Rattan et al . , "Protein Synthesis: Posttranslational Modifications and Aging," Ann. N. Y. Acad. Sci . 663: 48-62 (1992).

Peptides or polypeptides as described herein may be structurally modified. A structurally modified peptide is substantially similar in both three-dimensional shape and biological activity to a peptide described herein and preferably comprises a spatial arrangement of reactive chemical moieties that closely resembles the three- dimensional arrangement of active groups in the peptide sequence. Examples of structurally modified peptides include pseudo-peptides, semi-peptides and peptoids .

Peptides or polypeptides as described herein may be structurally modified to include one or more non-peptidyl bonds, for example pseudopeptide bonds. A number of suitable pseudopeptide bonds are known in the art, including retro- inverso pseudopeptide bonds

("Biologically active retroinverso analogues of thymopentin" , Sisto A. et al in Rivier, J. E. and Marshall, G. R. (eds) "Peptides, Chemistry, Structure and Biology", Escom, Leiden (1990), pp. 722- 773) and Dalposzo, et al . (1993), Int. J. Peptide Protein Res., 41:561-566), reduced isostere pseudopeptide bonds (Couder, et al .

(1993), Int. J. Peptide Protein Res., 41:181-184), ketomethylene and methylsulfide bonds.

Peptides comprising pseudopeptide bonds, may have an identical amino acid sequence to the sequence described above, except that one or more of the peptide bonds are replaced by a pseudopeptide bond. Preferably the most N-terminal peptide bond is substituted, since such a substitution will confer resistance to proteolysis by exopeptidases acting on the N-terminus. Further modifications also can be made by replacing chemical groups of the amino acids with other chemical groups of similar structure.

Peptides as described herein may be structurally modified to eliminate peptide bonds. Suitable structurally modified peptides include peptoids (Simon, et al . , 1992, Froc. Natl. Acad. Sci. USA, 89:9367-9371), which are oligomers of N-substituted glycines. The N-alkyl group of each glycine residue corresponds to the side chain of a natural amino acid. Some or all of the amino acids of a peptide may be replaced with the N-substituted glycine corresponding to the replaced amino acid.

A peptide as described herein may be structurally modified to comprise one or more D-amino acids. For example, a peptide may be an enantiomer in which one or more L-amino acid residues in the amino acid sequence of the peptide is replaced with the corresponding D- amino acid residue or a reverse-D peptide, which is a peptide consisting of D-amino acids arranged in a reverse order as compared to the L-amino acid sequence described above. (Smith C. S. et al . , Drug Development Res., 15, pp. 371-379 (1988).

Methods of producing suitable structurally modified peptides are well known in the art.

Other aspects of the invention relate to the identification of specific binding members and antibody antigen-binding domains which bind to peptides, polypeptides or peptidyl trimers as described herein, and may therefore be useful in inhibiting the binding of collagen to vWF, for example in anti-thrombotic therapy.

A method of producing an antibody may comprise: administering an immunogen comprising a peptide, polypeptide or peptidyl trimer as described herein as described herein to an animal, and; isolating from said animal an antibody which binds to said peptide, polypeptide or peptidyl trimer.

The antibody may specifically bind to the vWF binding motif of collagen and may inhibit the binding of vWF to collagen.

A molecule which binds specifically shows no significant binding to molecules other than its specific binding partner (s) . Where, for example, an antigen-binding site is specific for a particular epitope, the specific binding member carrying the antigen-binding site will be able to bind to the various molecules carrying the particular epitope. For example, an antibody antigen-binding domain specific for a peptide, polypeptide or peptidyl trimer as described herein may show no binding or substantially no binding to other regions of collagen.

The immunogen may comprise a protein carrier, such as Keyhole Limpet Haemocyanin. Other suitable carriers are well known in the art.

Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al . (1992) Nature 357 80-82).

More preferably, an antibody molecule may be a monoclonal antibody. Methods of producing monoclonal antibodies are well known in the art (see, for example, Harlow et al Antibodies : A Laboratory Manual, Cold Spring Harbor Laboratory (Cold Spring Harbor, NY, 1988) pp. 353-355) and are described in more detail below. For example, antibody-producing cells may be isolated from an immunised mammal and fused with immortalised cells to produce a population of antibody-producing hybridoma cells, which can then be screened to identify a hybridoma cell that produces an antibody which displays optimal binding characteristics.

In some embodiments, a hybridoma may be produced by a method comprising; immunising a non-human mammal with an immunogen comprising a peptide, polypeptide or peptidyl trimer as described above, producing one or more fusions of antibody producing cells from said mammal and immortalised cells to provide a population of hybridoma cells, and; screening said population to identify a hybridoma cell which produces an antibody which binds the peptide, polypeptide or peptidyl trimer.

The population of hybridoma cells is preferably screened by testing the binding of antibodies produced by cells of the population to one or more peptides, polypeptides or peptidyl trimers as described herein. Conventional techniques such as western blotting or immunoprecipitation may be used.

Hybridoma cells identified as producing antibodies which bind to the peptide, polypeptide or peptidyl trimer may be isolated and/or purified from the population.

Following isolation, the hybridoma may be expanded, maintained and/or cultured in a culture medium using methods which are well- known in the art. Antibodies produced by the hybridoma may be isolated from said culture medium. A method of producing an antibody may comprise,- culturing a hybridoma cell produced as described above in a culture medium; and, isolating from the medium an antibody as described above, for example, an antibody which binds to the peptide, polypeptide or peptidyl trimer.

Alternatively, a monoclonal antibody specific for a peptide, polypeptide or peptidyl trimer as described herein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains or other molecules comprising antibody antigen- binding domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047. The library may be immunologically naive, that is constructed from sequences obtained from an organism which has not been immunised with a peptide comprising the epitope, or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest.

A method of producing a specific binding member may comprise: contacting a peptide, polypeptide or peptidyl trimer as described above with a diverse population of antibody antigen- binding domains, and; determining the binding of members of said population to said peptide, polypeptide or peptidyl trimer.

The antibody antigen-binding domains may be comprised in antibodies or scFv, Fab, Fv, dAb, Fd or diabody molecules

An antibody antigen-binding domain may be identified in said population which binds to the peptide, polypeptide or peptidyl trimer.

Antibody antigen-binding domains may be displayed on the surface of virus particles i.e. the diverse population may be a phage display library.

The virus particle which displays the identified antibody antigen- binding domain may be isolated and/or purified and the nucleic acid encoding the antibody antigen-binding domain obtained from said particle.

The nucleic acid encoding the antibody antigen-binding domain may be sequenced and/or expressed to produce the encoded antibody antigen- binding domain that binds to the peptide, polypeptide or peptidyl trimer. An antibody antigen-binding domain produced as described above may be further tested using routine methodology to determine its specificity.

In some embodiments, the binding properties of the antibody antigen- binding domain may be further optimised using standard antibody- engineering techniques, including affinity maturation, for example by chain shuffling, and site-specific, random or combinatorial mutagenesis .

An antibody antigen-binding domain which is comprised in an antibody molecule, for example an antibody, scFv, Fab, Fv, dAb, Fd or diabody molecule, may be reformatted, for example into an IgG antibody, using standard techniques for subsequent use.

The antibody molecule or specific binding member may be tested for anti-thrombotic activity. For example, the ability of the antibody molecule or specific binding member to reduce to inhibit platelet aggregation and/ore activation may _be determined and/or the ability of the antibody molecule or specific binding member to reduce to inhibit the formation of blood clots may be determined.

An antibody molecule or specific binding member which has antithrombotic activity may be formulated into a pharmaceutical composition, for example by admixing with a pharmaceutical carrier, as described herein.

Another aspect of the invention provides a specific binding member comprising an antibody antigen-binding domain which binds to a peptide, polypeptide or peptidyl trimer as described herein.

A suitable specific binding member may be an antibody molecule, for example an antibody, scFv, Fab, Fv, dAb, Fd or diabody and may, for example, be produced by a method described above. A specific binding member may be useful in inhibiting the binding of collagen to vWF, for example in anti-thrombotic therapy.

The present invention extends in various aspects not only to peptides, polypeptides as described herein, optionally coupled to other molecules, peptidyl trimers and specific binding members but also a pharmaceutical composition, medicament, drug or other composition comprising such a peptide, polypeptide, conjugate, peptidyl trimer or specific binding member, a method comprising administration of such a composition to a patient, e.g. for a therapeutic purpose, which may include preventative treatment, use of such a peptide, polypeptide, conjugate, peptidyl trimer or specific binding member, in manufacture of a composition for administration, e.g. for a therapeutic purpose, and a method of making a pharmaceutical composition comprising admixing such a peptide, polypeptide, conjugate, peptidyl trimer or specific binding member, with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.

A pharmaceutically useful compound according to the present invention that is to be given to an individual, is preferably administered in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Liposomes, particularly cationic liposomes, may be used in carrier formulations. In other embodiments, a peptide, polypeptide or peptidyl trimer as described herein may be coupled to inert polymer support . Examples of techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed) , 1980.

The agent may be administered in a localised manner to a desired site or may be delivered in a manner in which it targets particular cells or tissues. For example, the agent may be applied topically to a wound site, for example as a pharmaceutical formulation or as a component of a wound dressing.

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

A peptide, polypeptide or peptidyl trimer or an article or device comprising a peptide, polypeptide or peptidyl trimer, including a wound dressing, may be provided in a kit, e.g. sealed in a suitable container that protects its contents from the external environment. Such a kit may include instructions for use.

Peptides, polypeptides, peptidyl trimers and specific binding members as described herein may be useful as valuable reagents in a number of laboratory and clinical settings. For example, a peptide may be useful as a reagent for research into the activation and/or aggregation of platelets. A method of activating and/or aggregating platelets may comprise treating platelets with a peptide, polypeptide or peptidyl trimer as described herein. Preferably, the platelets are treated in the presence of blood plasma. This may be in vitro.

Activity of treated platelets, i.e. platelets following contact with a trimer as described herein, may be measured or determined, for example in the presence or absence of a factor or agent, test composition or substance of interest, employing suitable control experiments as expected in the art.

The effect of a factor on platelet activation and/or aggregation may be determined by a method comprising treating platelets with a peptide trimer as described herein and determining the effect of the factor on the platelet activation and/or aggregation. Platelet activation and/or aggregation may be determined in the presence or absence of the factor or with the factor at different concentrations.

Different samples from different sources, e.g. different patients with or suspected of having a bleeding disorder, a disorder of platelet function or other disorder or disease, may be analysed and/or compared.

A peptide, polypeptide or peptidyl trimer may be useful in the diagnosis of platelet disorders, which routinely use collagen fibres extracted from animal tissues as a reagent in platelet aggregometry, or immobilised collagen preparations as in the Platelet Function

Analyzer and other instruments. A method of investigating platelet activity or function or of diagnosing a dysfunction in platelet activity in a sample, the method comprising determining activation and/or aggregation of platelets in a sample treated with a peptide, polypeptide or peptidyl trimer as described herein. For example, a peptide, polypeptide or peptidyl trimer as described herein may be contacted with a blood sample obtained from the individual and the aggregation of platelets determined.

A peptide, polypeptide or peptidyl trimer as described herein may be useful as a bioactive surface coating which acts to secure cell adhesion directly as well as to aggregate and activate platelets locally, for example leading to the production and release of other bioactive molecules. A method may for example comprise contacting platelets with a peptide or peptide trimer as described herein which is immobilised on a solid support, in the presence of blood plasma, thereby aggregating and/or activating platelets at or in the vicinity of said support.

A peptide, polypeptide or peptidyl trimer as described herein which is coupled to inert polymer support may be useful in stimulating haemostasis in the circulation and may, for example, serve as an adjunct or alternative to platelet transfusion in cases of platelet insufficiency that may result from auto-immune thrombocytopaenia or from therapeutic ablation of bone marrow as in cancer therapy, as well as from bleeding disorders from other causes, such as Glanzmann's disease. A method of stimulating haemostasis may comprise administering to an individual in need thereof a peptide or polypeptide as described herein coupled to inert polymer support.

The individual may have platelet insufficiency and may, for example, have a medical condition as set out above.

A peptide, polypeptide or peptidyl trimer as described herein which is coupled to inert polymer support may be useful in inducing thrombus formation in aortic aneurism. For example, the peptide, polypeptide or peptidyl trimer may be coated onto the outside of a stent to secure the tissue and prevent further dilation of a distended artery. A method of inducing thrombus formation in damaged vascular tissue of an individual may comprise contacting said vascular tissue with a peptide, polypeptide, or trimer as described herein coupled to inert polymer support, preferably a stent. The individual may have distended artery or other blood vessel and may, for example, have an aortic aneurism as set out above. In preferred embodiments, a petidyl trimer is coupled to the support, most preferably a triple-helical peptide with GpVI, alpha2betal and VWF- reactivity, as described above. Suitable inert supports are described in more detail above and may include proteins, PEG, or liposomes onto which the peptide is coated or attached.

A peptide, polypeptide or peptidyl trimer as described herein in a form that cannot enter the circulation, such as coupled to polymer beads, may be useful in stimulating haemostasis in acute trauma, e.g. after road traffic accident or battlefield injury, being applied topically to wounds that would otherwise cause fatal blood loss. A method of stimulating haemostasis at a wound site in an individual may comprise contacting the site with a peptide, polypeptide or peptidyl trimer as described herein coupled to an insoluble support or surface. The peptide, polypeptide or peptidyl trimer may be in the form of a pharmaceutical composition or immobilised within a wound dressing.

A peptide, polypeptide or peptidyl trimer as described herein in a form that cannot enter the circulation may be useful in stimulating haemostasis in chronic wounds such_, as ulcers, where, first, cell attachment may be enhanced, and second, the release of activated platelet granule contents may stimulate the migration of cells from the bloodstream and from nearby damaged tissues that contribute to the healing process. A method of stimulating haemostasis at a chronic wound site in an individual may comprise contacting the site with peptide, polypeptide or peptidyl trimer as described herein coupled to an insoluble support or surface.

Another aspect of the invention provides a wound dressing comprising a peptide, polypeptide or peptidyl trimer as described herein, optionally coupled to an inert support.

Such a wound dressing may be applied to a wound site to promote clotting for the treatment of traumatic or chronic wounds. A peptide, polypeptide or peptidyl trimer as described herein as described herein may also be useful in the investigation or screening of test compounds that disrupt vWF binding to collagen and inhibit platelet aggregation and activation and/or blood coagulation.

Another aspect of the invention provides method of screening for an anti-thrombotic compound comprising-. contacting a peptide, polypeptide or peptidyl trimer as described herein with a vWF polypeptide in the presence of a test compound and determining the interaction of the peptide, polypeptide or peptidyl trimer and the vWF polypeptide.

A decrease in interaction in the presence of the test compound is indicative that the test compound is an anti-thrombotic compound.

An increase in interaction in the presence of the test compound is indicative that the test compound is an pro-thrombotic compound.

A vWF polypeptide is a polypeptide which has the sequence of residues 764 to 2813 of database entry number NP_000543.1 GI: 4507907 or is a fragment or variant thereof.

A variant of vWF polypeptide retains the ability of the wild type vWF polypeptide to bind to collagen and may comprise an amino acid sequence which shares greater than about 50% sequence identity with residues 764 to 2813 of database entry number NP_000543.1 GI: 4507907, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90% or greater than about 95%.

A fragment of the full length vWF polypeptide may comprise the A3 domain of vWF . Sequence identity is commonly defined with reference to the algorithm GAP (Genetics Computer Group, Madison, WI) . GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. MoI. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444- 2448) , or the Smith-Waterman algorithm (Smith and Waterman (1981) J. MoI Biol. 147: 195-197), or the TBLASTN program, of Altschul et al . (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl . Acids Res. (1997) 25 3389-3402) may be used. Sequence identity and similarity may also be determined using Genomequest™ software (Gene- IT, Worcester MA USA) . Sequence comparisons are preferably made over the full-length of the relevant sequence described herein.

In some embodiments, the interaction of the peptide and vWF polypeptide may be determined by determining the binding of the peptide and vWF polypeptide. Methods for determining the binding of a test compound to a target polypeptide are well known in the art.

In other embodiments, the interaction of the peptide and vWF polypeptide may be determined by determining the platelet aggregation and/or activation promoting activity of the peptide. For example, the ability of the compound to inhibit platelet aggregation may be measured in vitro or in vivo.

The amount of test substance or compound which may be added to a method described herein will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.01 to 100 nM concentrations of putative inhibitor compound may be used, for example from 0.1 to 10 nM. A test compound suitable for use in the present methods may be a small chemical entity, peptide, antibody molecule or other molecule whose effect on collagen/vWF interaction is to be determined. Natural or synthetic chemical compounds may be used, or extracts of plants which contain several characterised or uncharacterised components .

Suitable test compounds may be selected from compound collections and designed compounds. Combinatorial library technology (Schultz,

JS (1996) Biotechnol . Prog. 12:729-743) provides an efficient way of testing a potentially vast number of different substances for ability to modulate peptide/vWF interaction activity.

Other suitable test compounds include molecules comprising antibody antigen binding domains. For example, libraries of antibody antigen binding domains displayed on virus particles may be screened to identify an antibody antigen binding domain which decreases or inhibits the interaction of the peptide, polypeptide or peptidyl trimer and the vWF polypeptide.

Other candidate compounds may be based on modelling the 3- dimensional structure of the vWF binding motif and using rational drug design to provide potential enhancer compounds with particular molecular shape, size and charge characteristics. Drug design is described in more detail below.

The effect of a compound identified by a method described above may be assessed in a secondary screen. For example, the effect of the compound on platelet aggregation or blood coagulation may be determined. Secondary screens may be performed in in vitro test systems or in vivo in animal models. A method as described herein may comprise identifying a test compound as an agent which inhibits the binding of vWF to collagen and therefore has anti-thrombotic activity.

The identified compound may be isolated and/or purified. In some embodiments, the compound may be prepared, synthesised and/or manufactured using conventional synthetic techniques.

Optionally, compounds identified as agents which inhibit the expression and/or activity of a using an method described herein may be modified or subjected to rational drug design techniques to optimise activity or provide other beneficial characteristics such as increased half-life or reduced side effects upon administration to an individual .

Compound produced by the screening methods and/or drug design methods described above may be formulated into a composition, such as a medicament, pharmaceutical composition or drug, with a Pharmaceutically acceptable excipient.

Controls are employed as appropriate within the routine knowledge and expectation of those skilled in the art.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure, including the following experimentation to illustrate embodiments of the invention and the accompanying figures.

All documents mentioned in this specification are incorporated herein by reference in their entirety.

The invention encompasses each and every combination and sub- combination of the features that are described above. The term "comprises" as used herein encompasses both "includes", i.e. permitting the presence of one or more additional components and "consists of" i.e. not permitting the presence of one or more additional components.

All peptide structures and sequences are indicated using the standard amino acid single letter code. "O" is hydroxyproline .

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above and tables described below.

Figure 1 shows the binding of plasma-derived VWF (1 μg/ml) to a set of 57 overlapping triple helical peptides, which together comprising the entire human collagen type III sequence, immobilized on a 96 well plate (all at 10 μg/ml) . Bound VWF was visualized with an HRP- conjugated polyclonal antibody. Shown is a representative of 3 independent experiments performed in duplicate and error bars 'ndicate standard deviation.

Figure 2 shows the binding of immobilized peptide #23 to plasma- derived VWF (1 μg/ml) in presence or absence of monoclonal antibody RU5 (1 μg/ml), or with recombinant wild-type, delta A3, or HislO23Ala VWF (all at 1 μg/ml) . Bound VWF was visualized with an HRP-conjugated polyclonal antibody. Shown is a representative of 3 independent experiments performed in duplicate. Error bars indicate standard deviation.

Figure 3 shows the binding of immobilized peptide #23 or immobilized collagen type III (100 μg/ml) to increasing concentrations of VWF.

Bound VWF was visualized with an HRP-conjugated polyclonal antibody. Shown is a representative of 3 independent experiments performed in duplicate. Error bars indicate standard deviation. Figure 4 shows the aggregation of platelets on a coverslip sprayed with Peptide #23 or vehicle (0.5 μg/cm2) and perfused with whole blood for 5 minutes at a shear rate of 300 s^"1 in presence or absence of RU5 (1 μg/ml) . Uncoated coverslips served as a control. The surface of the coverslip covered with platelets was determined by computer-assisted analysis, after visualisation of the platelets with May-Grϋnwald/Giemsa staining. Shown is the mean of three independent experiments performed in triplicate. Error bars indicate standard deviation.

Figure 5 shows the binding of plasma-derived VWF (1 μg/ml) to a set of truncated and alanine-modified peptides based on the sequence of peptide #23 immobilized on a 96-well plate. Bound VWF was visualized with an HRP-conjugated polyclonal antibody. Shown is a representative of 3 independent experiments performed in duplicate. Error bars indicate standard deviation.

Figure 6 shows the aggregation of platelets on Thermanox coverslips spray-coated with peptides based on the sequence of peptide #23 (0.5 μg/cm²) and perfused with whole blood at a shear rate of 300s^"1 for 5 minutes. The surface of the coverslip covered with platelets was determined by computer-assisted analysis, after visualisation of the platelets with May-Grϋnwald/Giemsa staining. Shown is the mean of three independent experiments performed in triplicate. Error bars indicate standard deviation.

Figure 7 shows the binding capacity for different concentrations of full-length plasma-derived VWF at equilibrium of peptide #23 and peptide GPRGQOGVMGFO immobilized onto a Biacore CM5 sensorchip via the free cysteine residue in the flanking regions of the peptides. The binding capacity of the peptides was determined for different concentrations of full-length plasma-derived VWF at equilibrium. Shown is a representative of three independent experiments. Figure 8 shows VWF binding and platelet deposition to collagen (100 μg/ml) immobilized on a 96-well plate or a thermanox coverslip and incubated with with purified VWF (1 μg/ml) or perfused with whole blood in the presence or absence of different concentrations of peptide GPRGQOGVMGFO. VWF binding and platelet deposition were evaluated as indicated above. Shown is a representative of three independent experiments.

Figure 9 shows that Platelet adhesion to Peptide 23 involves both GpVI and VWF.

Figure 10 shows that Peptide 23 causes platelet aggregation in platelet-rich plasma.

Figure 11 shows that an Integrin a2bl-specific peptide does not causes platelet aggregation in platelet-rich plasma.

-±gure 12 shows platelet aggregation induced by Peptide 23 requires plasma: washed platelets do not aggregate.

Figure 13 shows aggregation caused by Peptide 23 depends upon VWF and GpVI .

Figure 14 shows crosslinked peptide 23 elicits tyrosine phosphorylation from platelets.

Figure 15 shows solid phase binding of VWF to peptides.

Figure 16 shows peptide blocking of VWF binding to immobilised type I and type III collagen.

Table 1 shows the peptide sequences of the Toolkit. Predicted mass is shown, along with melting temperature determined by polarimetry and an estimate of peptide purity. Synthetic methodology A or B is defined in Materials and Methods section. * indicates the presence of DG sequence, where Fmoc(FmocHmb) GIy-OH was used. r indicates the use of Fmoc-PP- (GPP) 4-GPC-Tentagel R RAM at the C-terminus. GER sequences, possible integrin binding motifs, are highlighted using bold text.

Table 2 shows the amino acid sequence of truncated and alanine- modified peptide based on the sequence of peptide #23 (top sequence) . Peptide sequences are denoted by standard one-letter amino acid nomenclature, in which O represents hydroxyproline.

Table 3 shows the sequence of substituted peptides used to establish the activity of substitutions within the native collagen sequence.

Materials and Methods

Peptide synthesis

57 peptides together comprising the entire collagen type III sequence and consisting of the sequence GPC-(GPP)₅-(GXY)₃-(GPP)₅-GPC were synthesized by standard Fmoc peptide synthesis (table 1) . The - (GXY) g-part of each peptide contained native collagen sequence, with a 9 amino acid overlap on both N- and C-terminus for each sequential peptide. All peptides were shown to exist in a triple helical formation at room temperature by melting temperature analysis using polarimetry. All peptides were found to be of the correct theoretical mass using mass spectrometry.

Solid phase binding assays

Binding of VWF, purified from commercially available VWF concentrates (Haemate P, Aventis Behring, Hattersheim am Main, Germany) by size exclusion chromatography, to the peptides was performed as follows. Peptides were dissolved and diluted to 10 μg/ml in 10 mM acetic acid. In selected experiments, collagen type III (Sigma, St. Louis, MO) was dissolved in 50 mM acetic acid, dialyzed against a sodium phosphate buffer (20 mM Na₂HPO₄, pH 7.4) and diluted to 100 μg/ml. Peptides or collagen were immobilized onto 96 well plates (Immulon 2, Dynatech Laboratories Inc, Chantilly, VA, 100 μl/well) by overnight incubation at 4°C. After washing with phosphate-buffered saline (PBS, 10 mM phosphate buffer, 150 mM NaCl, pH 7.4), unoccupied binding sites were blocked with PBS containing 3% bovine serum albumin, and 0.1% tween-20. Subsequently, wells were incubated with VWF (1 μg/ml, in PBS/3% BSA/0.1% tween-20) for two hours at ambient temperature. Bound VWF was visualized by a horseradish peroxidase-conjungated polyclonal antibody (DAKO, Glostrup, Denmark, used in a 1:1000 dilution), using ortho- phenylenediamine as a substrate.

Platelet binding under flow conditions Peptides were sprayed on Thermanox coverslips using a retouching airbrush (Badgermodel 100; Badger brush, Franklin park, II) at a density of 0.5 μg/cm², whereas collagen type III was coated at a density of 6.5 μg/cm². After the spaying procedure, coverslips were olocked for 1 ^"hour at room temperature with 1% human albumin in PBS. Blood was drawn from healthy volunteers who denied ingestion of aspirin or other non-steroidal anti-inflammatory drugs for the preceding week into one-tenth volume of 3.4% sodium citrate. Blood was perfused at ambient temperature for 5 minutes over the peptide- coated coverslips using a single pass, parallel plate perfusion chamber as described previously²⁵. A constant shear rate of 300 s^"1 was used. After perfusion, coverslips were fixed and stained with May-Grunwald/Giemsa as described previously²⁵, and evaluated by computer-assisted analysis using Optimas 6.0 software (Optimas Inc, Seattle, WA) . Platelet adhesion was expressed as the percentage of the surface covered with platelets.

Surface plasmon resonance

Surface plasmon resonance analysis was performed using a Biacore

2000 system (Biacore AB, Uppsala, Sweden) . Peptides were immobilized on a CM5 sensor chip via their free cysteine residues in the flanking parts of the peptide using ligand thiol coupling as recommended by the manufacturer. A peptide with the sequence GPC- (GPP)_1O-GPC served as a control. Binding of VWF A3 domain to the channels coated with collagen peptides was corrected for binding to the GPC-(GPP)₁O-GPC coated channel. Binding experiments were performed in 25 mM HEPES, 125 mM NaCl (pH 7.4) at ambient temperature with a flow rate of 5 μl/min. VWF and A3 binding to the peptides was determined at equilibrium for different VWF and A3 concentrations. Each injection was continued for 4 minutes, after which a dissociation step of 2 minutes was performed. Regeneration of the surface was performed by subsequent application of 10 mM taurodeoxycholic acid, 100 mM Tris (pH 9.0) for 3 minutes at a flow rate of 5 μl/min.

Modeling and Docking

A model comprising residues 564 to 587 of human collagen type III (amino acid sequence GPOGPSGPRGQOGVMGFOGPKGND) and the VWF A3 domain /as constructed by the addition of specific side chains to- a model of a GPO repeat sequence in a triple helical 10-3 collagen conformation²⁴ (or 10/7 by the Emsley/Brodsky naming convention) . Subsequently, simulated annealing of side chains in torsion angle space followed by energy minimization was performed with the software package CNS²⁷ to generate an ensemble of 20 models with different side chain conformations. Two crystallographic models of the VWF-A3 domain were used as starting point for the docking calculations, namely molecule A from protein data bank entry 1AO3²⁸ and molecule B from entry IATZ¹⁹. Although these models are very similar overall, the conformation of a loop containing H1023, a residue that is critical for collagen binding¹⁵, differs significantly .

Docking of the modelled collagen peptides to the crystal structures of the VWF-A3 domain was performed with the software package HADDOCK (version 1.3)²⁹ that uses the program CNS for structure calculations. Selection of residues for the generation of ambiguous interaction restraints that guide the docking procedure by HADDOCK relied on several sources of experimental information (for an explanation of ambiguous interaction restraints and other terms used to describe the docking procedure see ref²⁹) . Selected as active residues of VWF- A3 were solvent accessible residues that have been implicated in collagen binding by mutagenesis studies³'¹⁵'¹⁵ and display intensity reduction ratios of 0.3 or higher in a transferred cross-saturation NMR experiment¹⁵. This selection includes T977, 1978, D979, L994, V997, F1013, R1016, and H1023. Nearby residues P981 and Yl017 were selected as passive residues. Active residues of collagen included R572, 0575, V577, and F580 and were selected on the basis of the results of the Ala scan presented in this study. Passive residues of collagen included all neighbouring non-glycine residues (S569, P571, Q574, M578, 0581). The protocol used for the docking calculations was essentially the same as previously published²⁹. Briefly, the docking protocol consists of three stages: (i) generation of 2000 complexes by randomisation of orientations and rigid body energy minimization, (ii) for 200 best scoring complexes semi rigid simulated annealing in torsion angle space (TAD-SA) , and (iii) final refinement in Cartesian space with explicit solvent. Solutions were clustered using a full linkage algorithm with an r.m.s.d. cut-off of 3.0 A. Throughout the TAD-SA calculations, the collagen side chains were flexible. To preserve an acceptable collagen conformation during TAD-SA the main chain dihedral angles were restrained to lie close to the idealized values of 1O₃ collagen²⁴, while distance restraints were applied to maintain the characteristic interchain hydrogen bonds of collagen.

Platelet Aggregation

Aggregation testing was carried out on a Chronolog 490 aggregometer. Each channel was blanked with 250μl of platelet poor plasma (PPP) before each run. 250μl of PRP was tested with 5μl of agonist with concentration which gave a final concentration between lOOμg/ml and O.Olμg/ml. The aggregation trace was observed for 6 minutes or until a plateau had been reached.

Static Platelet Adhesion Ninety-six-well plates (Immulon-2 from Thermo Life Sciences,

Basingstoke, UK) were coated with 100 μl of peptide (at 20 μg/ml in 0.01 M acetic acid) per well at 20°C overnight. Washed platelets were prepared from PRP as described in reference 21 except that each σentrifugation step was performed at 2600 i-pm for 8 min and the platelet pellet was resuspended in 5 ml of buffer. Platelets were then resuspended to 1.25 x 10⁸ platelets/ml in adhesion buffer (0.05 M Tris-HCl, 0.14 M NaCl, 2 mM MgCl₂, 0.1% BSA, pH 7.4) and allowed to rest for one hour at 25^CC. Rested platelets were incubated with the fibrinogen receptor antagonist GR144305F (2 μM) for 15 min, to prevent platelet-platelet interaction, as indicated. The assay was then performed as described except that ligand-coated wells were blocked with 200 μl of blocking buffer for one hour and 100 μl of platelets were added to each well. Platelet phosphatase activity was used as an index of platelet number, determined by a colorimetric procedure. These methods are well-established.

Protein Tyrosine Phosphorylation

Acid citrate dextrose (ACD) (39 mM citric acid, 75 mM tri-sodium citrate -2H₂O, 135 mM D-glucose, pH 4.5) and loading buffer (145mM NaCl, 5 mM KCl, 10 mM D-glucose, 1 mM MgSO₄, 0.5 mM EGTA, 10 mM HEPES-KOH, pH 7.36) were pre-warmed to 30⁰C. ACD (10% (v/v) ) and apyrase (0.25 U/ml final concentration) were added to PRP, followed by centrifugation (2000 rpm, 15 min) . The platelet pellet was resuspended in 5 ml of loading buffer. ACD was added as before, followed by centrifugation (2000 rpm, 10 min) . Platelets were then resuspended to 1 x 10⁹ platelets/ml in loading buffer, and allowed to rest for one hour at 30⁰C. Platelet suspensions (50 μl) were stimulated at 30 ⁰C with 5 μl of the indicated concentration of peptide agonist or the vehicle control (0.01 M acetic acid) for two minutes. Reactions were stopped by adding an equal volume of Laemmli's buffer and boiling for five minutes.

Western blotting Protein separation was carried out as described in reference 23 except for the following modifications. Membranes were blocked with 5% (w/v) BSA dissolved in Tris-buffered saline Tween (TBST) , 20 mM Tris-HCl, 140 mM NaCl, 0.1% Tween-20, pH 7.6) for 2 h at room temperature. Membranes were probed with 4G10 (1:2000 in 5% BSA-TBST, 1 h, 25°C), washed for 45 min in TBST, incubated with the secondary- antibody (1:5000 in 1% BSA-TBST, 1 h, 25⁰C) and then washed in TBST as before and subjected to enhanced chemiluminescence (ECL) .

Results A set of 57 overlapping synthetic triple helical peptides together comprising the entire collagen type III sequence was produced (table 1) . Each peptide contained 27 amino acids of native collagen sequence, flanked on both the amino- and carboxyterminus by the sequence GPC-(GPP)₅- to induce triple helical stability, and to facilitate peptide crosslinking via the free cysteine residue.

Sequential peptides had a 9 amino acid overlap on both the amino- and carboxyterminus .

Binding of plasma-derived human VWF to each peptide of the entire peptide set was studied in a solid phase binding assay. A single peptide from the set (#23) bound VWF, as shown in figure 1.

Binding of VWF to peptide #23 was analysed. The interaction could be completely blocked by a monoclonal antibody (RU5)¹⁵ directed against the A3 domain of VWF, which abrogates the VWF-collagen interaction (figure 2) . Genetically engineered variants of VWF, delta A3 VWF¹⁸ and HislO23Ala VWF¹⁵, which have severely impaired collagen interaction due to the lack of the complete A3 domain, or due to the substitution of an amino acid within the A3 domain, which was previously shown to be critical for the interaction with collagen, showed severely depressed binding to peptide #23 (figure 2) . The interaction of VWF with peptide #23 was of similar affinity as compared to full-length collagen type III, when tested in a solid phase assay (figure 3) . Peptide #23 was able to bind VWF from whole blood under conditions of flow, resulting in platelet adhesion to peptide#23 -bound VWF, and this platelet adhesion could be completely blocked by monoclonal antibody RU5 (figure 4) . Binding of VWF to peptide #23 was thus found to resemble VWF binding to collagen.

Subsequently, a set of truncated triple helical peptides based on the sequence of peptide #23 was synthesized. The sequence of one of the truncated variants formed the basis of the synthesis of an alanine scanned peptide set (Table 2) . Peptides were synthesized in which a single amino acid from the native sequence was replaced by an alanine residue. All amino acids with the exception of all glycine residues, which are critical for triple helix formation, and the exception of the first triplet, from the sequence GPSGPRGQOGVMGFO were replaced by an alanine. All variants were tested for VWF binding in a solid phase assay (figure 5) , as well as for platelet binding from whole blood under flow conditions (figure 6) . As these binding experiments showed that peptides either strongly bound VWF or platelets, or completely lacked affinity for VWF as well as for platelets, the results of these binding experiments allowed us to identify the sequence RGQOGVMGF as the minimal amino acid sequence within collagen type III required for VWF binding. In this sequence, the R, 0, V, and F residues appear critical for collagen binding, whereas replacement of the Q and M by an alanine did not affect VWF binding. The peptide GPRGQ0GVMGF0 was shown to bind VWF in a solid phase binding assay with similar affinity compared to the parent peptide #23 and to full-length collagen. Using surface plasmon resonance, a more detailed kinetic study on the VWF binding of peptide #23 and peptide GPRGQOGVMGFO was performed. A genetically engineered form of the A3 domain of VWF (the generation of which has been reported previously¹⁹) bound to the immobilized peptides with moderate affinity (Kd 1,8 μM for peptide #23, Kd 2.5 μM for GPRGQOGVMGFO) , whereas full-length, plasma- derived VWF interacted with the peptides with a much higher affinity, which is presumably attributable to the multimeric structure of VWF (Kd 2.1 nM for peptide #23, Kd 2.5 nM for GPRGQOGVMGFO, figure 7) . These data are consistent with affinity constants determined for A3 and VWF binding to full length collagen²⁰'²¹. To further confirm that the peptide GPRGQOGVMGFO contained a high affinity collagen-binding site, it was shown that the peptide added to VWF or whole blood could inhibit VWF and platelet binding under flow conditions to immobilized collagen type III. At 500 μg/ml, GPRGQOGVMGFO was able to block VWF binding to collagen type III and platelet adhesion almost completely (figure 8) . These results indicate that the sequence GPRGQOGVMGFO contains the major VWF binding site, and excludes a major role for the VWF Al domain in collagen binding.

With these experiments, we have identified RGQOGVMGF as the high- affinity VWF binding sequence in collagen type III. Using this sequence, the data obtained from the crystal structure of the VWF A3 domain, and the data obtained from our previous mutagenesis studies, in which we have identified critical residues in the VWF A3 domain with respect to the interaction with collagen, we have constructed a model of the collagen sequence GPOGPSGPRGQOGVMGFOGPKGND (residues 564 to 587) and the A3 domain of VWF. The model was constructed by a docking procedure using the published crystal structures of the A3 domain, and a model of the 24 -amino acid collagen sequence. Recent crystallographic studies of native collagen sequences capped with stabilizing GPO repeats show that helical twist varies locally as a function of amino acid sequence²²'²³. In imino acid-rich regions, in particular the GPO caps, a 7-2 helix is observed whereas imino acid- poor regions adopt a 10-3 helix. Moreover, fibre diffraction studies on intact collagen indicate that it predominantly adopts a 10-3 conformation²⁴. The collagen triple helix was therefore modelled in a 10-3 conformation.

Clustering of the 200 docking solutions using an rmsd cut-off of 3.0 A identifies two highly populated clusters (76 and 62 members) that display a reverse direction of the collagen triple helix with respect to the A3 domain. One direction is clearly preferred as judged from the calculated interaction energies of the ten best solutions that are considerably lower for cluster 1 (-319 to -222 kcal/mole) than for cluster 2 (-227 to -155 kcal/mole) . The best two solutions of cluster 1 have similar interaction energies (-319 and - 314 kcal/mole, respectively) well below that of the next best solution of this cluster (-243 kcal/mole) . In both complexes, a solvent accessible surface of about 1600 A² is buried. While both solutions have a similar orientation of the triple-helix axis, they differ in the rotation about and the translation along this axis, which results in significant differences in A3 -collagen contacts and indicates that the docking results should be interpreted with caution. Despite differences in overall binding modes, the residues of collagen shown to be essential for VWF binding in the ala-scan are engaged in prominent and similar interactions with WF-A3 in both docking solutions. Both solutions predict that collagen residue R572 forms a salt bridge with D979 of A3. 0575, located on the same collagen chain, is buried in the interface and its hydroxyl group is predicted to form a weak hydrogen bond with D979 or the nearby main chain carbonyl oxygen of 1978. A different collagen chain contributes a second salt bridge of R572 to A3 residue ElOOl. A third chain contributes hydrophobic interactions of collagen residues V577 and F580 with a hydrophobic patch formed by A3 residues 1978, F1013, Y1017 and the aliphatic region of the R1016 side chain. In one complex, F580 sits close to the side chains of F1013, Y1017, and R1016 while in the other it is translated and interacts only with the hydrophobic region of R1016. H1023, which is located in a loop that protrudes from the A3 domain, approaches the collagen triple helix side-wise and, in both complexes, hydrogen bonds through its side chain to a main chain carbonyl oxygen of the collagen peptide. The conformations of the loop containing H1023 are closest to the orientation observed in the model from protein data bank entry Iao3 and very different from that observed in protein data bank entry latz suggesting that the conformation observed in the latter structure may not be relevant for collagen binding.

A database search has identified the 9 amino acid sequence RGQOGVMGF in a single other human protein, which is collagen type II. Indeed, a triple helical peptide containing 27 amino acids of native collagen type II sequence including the sequence RGQOGVMGF (GPC- (GPP) 5-GAOGEDGROGPOGPQGARGQOGVMGFO- (GPP) ₅-GPC, amino acids 510-536) was found to bind VWF with high affinity. The relevance of the VWF- collagen type II interaction for haemostasis is, however, believed to be minor or absent, since collagen type II is predominantly found in cartilage, and not in subendothelial structures. The sequence is also highly conserved in collagen type III from a variety of species, with a 100% homology in mouse, rat, cow, and chicken. Interestingly, the sequence was also found to be present in the αl(I) chain of human collagen type I, with the exception of a single amino acid residue (RGQAGVMGF) . This 0 to A substitution was shown to lack VWF binding capacity in our homotrimeric synthetic peptide from the alanine scanned set. As, however, collagen type I is a heterotrimer, it is likely that the cc2(I) chain contributes to VWF binding in collagen type I. Interestingly, alignment of the α2(I) chain with the ccl(I) chain of human collagen type I reveals that the sequence RGQAGVMGF of the αl(I) chain aligns with the sequence RGEOGNIGF of the oc2(I) chain, providing indication that the 0 in position 4 of the VWF binding sequence, although substituted with an A in the αl(I) chain of collagen type I, is present at position 4 of the oc2(I) chain. Indeed, our modelling experiments show that the interaction of the collagen sequence with the A3 domain involves different collagen chains.

The single linear 9 amino acid sequence within collagen type III is therefore shown to be responsible for high affinity binding of VWF. The docking studies provide indication that residues of collagen shown to be important for VWF binding in the Ala- scan are not organized as a linear peptide but rather are located on different chains of the collagen triple helix. One chain contributes R572 and 0575, while another chain contributes V577 and F580 and a third chain contributes a second interaction of R572.

Platelet adhesion to Peptide 23 was measured as described above. Comparing adhesion with that to CRP, Peptide 23 was a relatively weak substrate. However, platelet adhesion to Peptide 23 was abolished by the antibody RU5 which blocks the VWF A3 domain. This adhesion was not mediated by the fibrinogen receptor, integrin αllbβ3, since the antagonist GR1440453F was without significant sffect . There was modest inhibition, in excess of 20%, of adhesion to Peptide 23 when the anti-GpVI single chain antibody 10B12 was applied to the platelets prior to addition to the adhesive surface. 10B12 fully blocked adhesion to the GpVI ligand, CRP. Data are shown in Figure 9.

To investigate further the platelet reactivity of Peptide 23, the peptide was cross-linked using the same procedure as that described above for CRP that is to use the heterobifunctional covalent reagent SDP (Perbio) according to the manufacturer's instructions to couple cysteine free thiols to the free N-terminus of the peptide. This strategy is believed to provide a random matrix of cell-recognition motifs that can cluster and so activate platelet receptors.

Aggregation was performed in platelet-rich plasma, which contains free VWF. Under these conditions, the GpVI ligand CRP-XL was a potent agonist, causing near-maximal aggregation at a concentration of lOOng/ml. In contrast, cross-linked peptide 23 was much less potent, but was able to elicit a full aggregation response at 50μg/ml. To verify that this effect was mediated by the fibrinogen receptor, integrin αllbβ3, the antagonist was used at a concentration of 5μM, at which aggregation was completely abolished. Data are shown in Figure 10. To confirm the specific activatory properties of this peptide, a control was used under identical conditions, Peptide 4, which contains a recognition motif for integrin α2βl. No aggregation was observed at up to lOOμg/ml of the cross-liked peptide. Data are shown in Figure 11.

To verify the requirement for plasma, aggregation was performed on washed platelets, and under these conditions, no aggregation was observed at a peptide concentration of up to lOOμg/ml. This is consistent with a need for VWF to support aggregation. Data are shown in Figure 12.

¹O show the involvement of VWF and its platelet receptor, Gplboc, in^' the aggregatory process, two monoclonal antibodies, RU5, which blocks the collagen-binding site in the VWF A3 domain, and 12Gl, which blocks the VWF-binding site in Gplbα, were applied to platelet-rich plasma prior to the addition of cross-linked peptide 23. Aggregation was observed with EC50 between 10 and 20μg/ml using the peptide without the inhibitory antibodies. In the presence of the antibodies, the dose curve was shifted to the right, such that an EC50 of between 30 and 50 μg/ml was obtained. A further antibody, 10B12 which blocks GpVI was used in the same manner, and again a right shift was observed, further implying the dual involvement of GpVI and VWF/Gplbα in the aggregation process stimulated by peptide 23 (figure 13) .

To examine further the activatory properties of Peptide 23, it was applied over a range of concentrations to washed platelet suspensions. After 5 minutes, the interaction was stopped by the addition of Laemmli's buffer, containing the detergent SDS and β- mercaptoethanol to denature and unfold the platelet proteins. Samples were boiled, then subjected to polyacrylamide gel electrophoresis and western blotting. The blots were probed with the antiphospho-tyrosine antibody 4G10 using standard methodology. CRP-XL was used as a control. Data are shown in Figure 14.

The control lanes (marked 0) in both blots show minor reactive protein bands at a several molecular weights, and strong bands of about 50 to 6OkDa, corresponding to the src family kinases which are phosphorylated upon tyrosine residues in their basal, inactive state. In this experiment, CRP-XL (Figure V A) caused a dose- dependent increase in the density of many protein bands, consistent with substantial activation of platelet metabolism. Peptide 23 caused a qualitatively similar increase in the intensity of the phosphotyrosine-containing protein bands, but of lower intensity than those elicited by CRP-XL (Figure 14) .

The findings above show that peptides containing the VWF-binding motif, including peptide 23 will support platelet adhesion both from flowing blood and washed platelets (in which VWF is present at much reduced levels) . Because adhesion to Peptide 23 can be fully blocked by the use of the antibody RU5, which obscures the binding site on the VWF A3 domain, adhesion is dependent on VWF.

Adhesion of washed platelets to Peptide 23 was found to be reduced but not blocked by antibody 10B12, which obscures the collagen- binding site of GpVI. This provides indication that peptide 23 also contains a GpVI -binding site. Peptide 23, when cross-linked, comprises a hybrid collagenous ligand that can activate platelets in platelet -rich plasma and cause their aggregation. Involvement of both VWF and GpIb is shown by the use of specific antibodies, indicating that the VWF-GpIb interaction, rather than the VWF-αIIbβ3 interaction, is the crucial stimulus for activation, but that aggregation can be abrogated by αllbβ3 blockade with the antagonist GR144053F as would be expected of an active process rather than simply passive adhesion. The activity of Peptide 23 in aggregation assays depends upon the presence of external VWF (in plasma) and can be attenuated by blockade of VWF A3 domain, GpIb binding site for VWF or 10B12 that blocks GpVI.

To investigate amino acid substitution within the sequence GPRGQOGVMGFO, we made the variants shown in Table 3. Peptides had the generic structure GPC [GPP] ₅- [varied sequence] - [GPP] ₅GPC-NH₂ with conservative alterations in primary sequence at positions shown to be important for binding by Alanine-scanning.

Solid phase binding of VWF to these peptides was investigated and the results are shown in Figure 15.

Substitution of Lysine for Arginine at position 3 abolished binding, as did Isoleucine for Phenylalanine at position 11. Substitution of Leucine for Valine at position 8, and of Leucine or Tyrosine for phenylalanine at position 11 showed some reduction in binding. Thus requirement for the native amino acid sequence may not be absolute. Methionine is subject to oxidation during or after synthesis, and in peptide chemistry is often replaced by the unnatural amino acid, Norleucine. The peptide containing Norleucine in place of methionine supports full VWF binding.

We investigated the ability of the non-helical peptide GPC [GAP]₅- GPRGQOGVMGFO-[GAP]₅GPC-NH₂ to support VWF binding, and found that the peptide did not support any VWF binding. The data are shown in

Figure 15, indicated by label "GPR-GFO in GAP". The triple-helical structure is not supported by the [GAP] _s extensions at each end of the primary sequence of this variant, and its inability to bind VWF is in line with the concept that the triple-helical structure of collagen is crucial for its interaction with specific ligands, including VWF and cell-surface receptors.

The parent peptide, at concentrations of 125 and 250 μg/ml, blocked VWF from binding to both immobilised collagen type I and collagen type III. These are the major fibrous collagens of the blood vessel wall. These data, shown in Figure 16, provides indication that the A3 domain of VWF is the dominant site at which VWF recognises collagen, and that its inhibition by soluble peptide is sufficient to abolish the interaction.

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(2000) .

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30. Lisman et al Blood (2006) 108 12 prepublished August 15 2006

Table 1

Table 2

Table 3

Claims

Claims :

1. A peptide consisting of the sequence N₁RGX₃X₄GX₅X₆GX₇N₂ wherein;

X₃ is any amino acid except G or 0,

X₄ is O or P,

X₅ is any amino acid except G, O or A,

X₅ is any amino acid except G, X₇ is any amino acid except G, O or A, and;

Nl and N2 are amino acid sequences consisting, independently, of 0 to 10 amino acid residues.

2. A peptide according to claim 1 wherein X₅ is V, I, L, or T or a conservative substitution thereof.

3. A peptide according to claim 2 wherein X₅ is V or I.

4. A peptide according to any one of claims 1 to 3 wherein X₇ is F, L, Y or W or a conservative substitution thereof.

5. A peptide according to any one of claims 1 to 4 wherein X₆ is M or NIe or a conservative substitution thereof.

6. A peptide according to any one of claims 1 to 4 consisting of the sequence N₁RGX₃OGVX₅GFN₂.

7. A peptide according to any one of claims 1 to 6 wherein X₃ is Q.

8. A peptide according to any one of claims 1 to 7 wherein X₆ is M or NIe.

9. A peptide according to any one of claims 1 to 8 wherein Nl consists of sequence ROGPOGPSGP or a C terminal fragment thereof.

10. A peptide according to any one of claims 1 to 9 wherein N2 consists of the sequence OGPKGNDGAO or an N terminal fragment thereof .

11. A peptide according to claim 10 consisting of the sequence GPOGPSGPRGX₃OGVX₆GFOGPKGNDGAO

12. A peptide according to any one of claims 1 to 8 consisting of the sequence RGX₃OGVX₅GF.

13. A peptide according to any one of claims 1 to 8 consisting of the sequence GXiRGX₃OGVX₆GFX₈, wherein X₁ and X₈ are, independently any amino acid except G.

14. A peptide according to claim 13 wherein X₁ is A, P or 0.

15. A peptide according to claim 13 or claim 14 wherein X₈ is P or 0.

16. A peptide consisting of the sequence GX₁RGX₃AGVX₆GFX₈ or GXiRGX₃OGNX₆GFX_8/ wherein X₁, X_3, X_g and X₈ are, independently, any amino acid except glycine.

17. A set of peptides consisting of up to three peptides wherein each said peptide consists of the sequence N₁GX₁X₂GX₃X₄GX₅X₆GX₇X₈N₂, wherein X₁ to X₈ are independently any amino acid except glycine and; X₂ is R in at least one of said peptides,

X₂ is R and X₄ is 0 or P in at least one of said peptides, and; X₅ is V or I and X₇ is F, L, Y or W in at least one of said peptides and; Nl and N2 are amino acid sequences consisting, independently of 0 to 10 amino acid residues.

18. A set of peptides according to claim 16 wherein X₂ is R and X₄ is 0 in at least one of said peptides, and; X₅ is V and X₇ is F in at least one of said peptides.

19. A set according to claim 17 or claim 18 consisting of a peptide consisting of the sequence RGX_IAGVX₂GF and a peptide consisting of the sequence RGX₅OGNX₇GF, wherein X₁, X₂, X₅ and X₇ are, independently, any amino acid.

20. A polypeptide comprising a peptide according to any one of claims 1 to 16 fused to one or more heterologous amino acids.

21. A polypeptide according to claim 20 wherein said heterologous amino acids comprise the formula (GX_aX_b)_n, wherein X_a and X_b are independently any amino acid except glycine, preferably P or 0, and n is 1 to 10.

22. A polypeptide according to claim 21 wherein said heterologous amino acids comprise the formula (GPO)₃Or (GPP)₅.

23. A polypeptide according to any one of claims 20 to 22 wherein the heterologous amino acids comprise a platelet receptor binding motif .

24. A polypeptide according to any one of claims 20 to 23 wherein the heterologous amino acids comprise a GpVI and/or integrin α2βl binding motif.

25. A peptidyl trimer comprising; three peptides consisting of the sequence GX₁X₂GX₃X₄GX₅X₆GX₇X₈ wherein X₁ to X₈ are independently any amino acid except glycine and; X₂ is R in at least one of said peptides,

X₂ is R and X₄ is O or P in at least one of said peptides, and; X₅ is V, I, T or L and X₇ is F, L, Y or W in at least one of said peptides.

26. A peptidyl trimer according to claim 25 wherein

X₂ is R and X₄ is 0 in at least one of said peptides, and; X₅ is V and X₇ is F in at least one of said peptides.

27. A peptidyl trimer according to claim 25 or claim 26 comprising three peptides consisting of the sequence GX₁RGX₃OGVX_SGFX₈, wherein X₁, X₃, X₅ and X₈ are independently any amino acid except glycine.

28. A peptidyl trimer according to claim 25 or claim 26 comprising one peptide having the sequence GX₁X₂GX₃X₄GVX₆GFXe, and; two peptides having the sequence GX₁RGX₃OGX₅X₆GX₇X₈, or; two peptides having the sequence GX₁X₂GX₃X₄GVX₆GFX₈ and one peptide having the sequence GX₁RGX₃OGX₅X₆GX₇X₈ wherein X₁ to X₈ are independently any amino acid except glycine .

29. A peptidyl trimer according to claim 25 or claim 26 comprising; two peptides having the sequence GX₁RGX₃AGVX₆GFX₈ and one peptide having the sequence GX₁RGX₃OGNX₆GFX₈; or, two peptides having the sequence GX₁RGX₃OGNX₆GFX₈ and one peptide having the sequence GX₁RGX₃AGVX₆GFX₈.

30. A peptidyl trimer according to any one of claims 25 to 29 wherein X₁ is A in one or more of said peptides.

31. A peptidyl trimer according to any one of claims 25 to 30 wherein X₂ is R in one or more of said peptides.

32. A peptidyl trimer according to any one of claims 25 to 31 wherein X₃ is Q or E in one or more of said peptides.

33. A peptidyl trimer according to one of claims 25 to 32 wherein X₃ is Q in one or more of said peptides.

34. A peptidyl trimer according to any one of claims 25 to 33 wherein X₄ is O or A in one or more of said peptides.

35. A peptidyl trimer according to any one of claims 25 to 34 wherein X₅ is V or N in one or more of said peptides.

36. A peptidyl trimer according to any one of claims 25 to 35 wherein X₆ is M or I in one or more of said peptides.

37. A peptidyl trimer according to any one of claims 25 to 36 wherein X₇ is F in one or more of said peptides

38. A peptidyl trimer according to any one of claims 25 to 37 wherein X₈ is O in one or more of said peptides.

39. A peptidyl trimer according to any one of claims 25 to 38 wherein one or more of said peptides is fused to one or more heterologous amino acids .

40. A peptidyl trimer according to any one of claims 25 to 39 comprising one or more peptides according to any one of claims 1 to

16 or one or more polypeptides according to any one of claims 20 to 24.

41. A peptidyl trimer according to any one of claims 25 to 40 wherein the peptides or polypeptides in the trimer are cross-linked to other peptides or polypeptides in the trimer.

42. A peptidyl trimer according to any one of claims 25 to 41 which is immobilised on a solid support

43. A method of making a peptidyl trimer according to any one of claims 25 to 42, the method comprising a step of bringing together under conditions for formation of a peptidyl trimer three polypeptides or peptides which form a peptidyl trimer, at least one of said three polypeptides or peptides being a polypeptide or peptide according to any one of claims 1 to 16 or 20 to 24.

44. A method according to claim 43 wherein said step is preceded by production of at least one of said three polypeptides or peptides by expressing a proline precursor peptide from encoding nucleic acid, and hydroxylating proline residues to provide hydroxyproline (O) .

45. A method according to claim 43 wherein said step is preceded by production of at least one of said three polypeptides or peptides by chemical synthesis.

46. A method according to any one of claims 43 to 45 comprising cross-linking the three polypeptides or peptides which form a peptidyl trimer.

47. A method according to claim 46 comprising immobilising the trimer on a solid support.

48. An isolated nucleic acid encoding a proline precursor of a polypeptide or peptide according to any one of claims 1 to 16 or 20 to 24.

49. A method of making a desired polypeptide or peptide according to any one of claims 1 to 16 or 20 to 24, the method comprising causing expression from nucleic acid according to claim to produce an encoded proline precursor polypeptide or peptide, and hydroxylating proline residues in the encoded precursor polypeptide or peptide to provide said desired polypeptide or peptide.

50. A method according to claim 49 further comprising forming trimers from the desired polypeptides or peptides, without cross- linking polypeptides or peptides within the trimers.

51. A method according to claim 49 further comprising forming trimers from the desired polypeptides or peptides by cross-linking polypeptides or peptides within the trimers.

52. A method of producing an antibody comprising: administering an immunogen comprising a peptide, polypeptide or trimer according to any one of claims 1 to 16 and 20 to 42 to_ an animal, and; isolating from said animal an antibody which binds to said a peptide, polypeptide or trimer.

53. A method of producing a hybridoma comprising; immunising a non-human mammal with an immunogen peptide, polypeptide or trimer according to any one of claims 1 to 16 and 20 to 42, producing one or more fusions of antibody producing cells from said mammal and immortalised cells to provide a population of hybridoma cells, and; screening said population to identify a hybridoma cell which produces an antibody which binds the peptide, polypeptide or trimer.

54. A method of producing an antibody comprising; culturing a hybridoma cell produced by a method according to claim 53 in a culture medium; and, isolating from the medium an antibody as described above, for example, an antibody which binds to the GPIIIa peptide

55. A method of producing an antibody molecule comprising: contacting a peptide, polypeptide or trimer according to any one of claims 1 to 16 and 20 to 42 with a diverse population of antibody antigen-binding domains and; determining the binding of members of said population to said peptide.

56. A method according to claim 55 wherein the antibody antigen- binding domains are displayed on the surface of virus particles.

57. A method according to claim 55 or claim 56 wherein the antibody antigen-binding domains are comprised in antibodies or scFv, Fab, Fv, dAb, Fd or diabody molecules.

58. A method according to any one of claims 55 to 56 comprising identifying an antibody antigen-binding domain in said population that binds to the peptide.

59. A specific binding member which specifically binds to a peptide, polypeptide or trimer according to any one of claims 1 to 16 and 20 to 42.

60. A method of activating platelets and/or inducing platelet aggregation, comprising treating platelets with a peptidyl trimer according to any one of claims 25 to 42.

61. A method according to claim 60 wherein platelets are treated in vitro.

62. A method according to claim 60 or claim 61 wherein said platelets are treated in the presence of vWF.

63. A method according to claim 62 wherein said platelets are treated in the presence of plasma.

64. A method of investigating platelet activity or function or of diagnosing a dysfunction in platelet activity in a sample, the method comprising determining activation or aggregation of platelets in a sample treated with a peptidyl trimer according to any one of claims 25 to 42.

65. A method of determining the presence of vWF in a sample obtained from an individual comprising; contacting the sample with a petidyl trimer according to any one of claims 25 to 42 and, determining the binding of vWF to said trimer, wherein the presence of vWF bound to the trimer is indicative of^" the presence of vWF in the sample.

66. A method according to claim 65 wherein the presence of vWF in the sample is indicative that the individual is suffering from a bleeding disorder.

67. A method of inducing thrombus formation in damaged vascular tissue in an individual comprising contacting said tissue with a peptidyl trimer according to claim 42.

68. A method according to claim 67 wherein the peptidyl trimer is immobilised on the surface of a stent.

69. A method according to claim 67 or claim 68 wherein the individual has an aortic aneurism.

70. A peptidyl trimer according to any one of claims 25 to 42, a peptide according to any one of claims 1 to 16, a polypeptide according to any one of claims 20 to 24, or a specific binding member according to claim 59 for use in a method of treatment of the human or animal body by therapy.

71. A peptidyl trimer, peptide, polypeptide or a specific binding member according to claim 70 for use in a method of treatment to activate platelets and/or induce platelet aggregation in the body.

72. Use of peptidyl trimer according to any one of claims 25 to 42, a peptide according to any one of claims 1 to 16, a polypeptide according to any one of claims 20 to 24, or a specific binding member according to claim 59, in the manufacture of a medicament for promoting platelet activation and/or aggregation in the body, and/or treating a disorder of platelet activation and/or aggregation.

73. A pharmaceutical .composition comprising a peptidyl trimer according to any one of claims 25 to 42, a peptide according to any one of claims 1 to 16, a polypeptide according to any one of claims 20 to 24, or a specific binding member according to claim 59.

74. A pharmaceutical composition according to claim 73 for topical application.

75. A wound dressing comprising a peptidyl trimer according to any one of claims 25 to 42, a peptide according to any one of claims 1 to 16, a polypeptide according to any one of claims 20 to 24, or a specific binding member according to claim 59.

76. A kit for detecting the presence of vWP in a sample comprising; a peptidyl trimer according to any one of claims 25 to 42 immobilised to a solid surface, and detection reagents for determining binding of vWF to said trimer.

77. A kit according to claim 76 wherein the detection reagents comprise a first antibody that binds to vWF.

78. A kit according to claim 77 wherein the detection reagents comprise a second antibody that binds to the first antibody.

79. A kit according to claim 77 or claim 78 wherein the first antibody or the second antibody are labelled with a detectable label.

80. A kit according to any one of claims 76 to 80 wherein the peptidyl trimer is immobilised in a lateral flow device.

81. A method of screening for a compound which modulates platelet activation and/or platelet aggregation comprising: contacting a peptidyl trimer according to any one of claims 25 to 42 with a vWF polypeptide in the presence of a test compound and, determining the interaction of the peptidyl trimer and the vWF polypeptide, wherein a decrease in interaction in the presence of the test compound is indicative that the test compound modulates platelet activation and/or platelet aggregation.

82. A method according to claim 81 wherein the interaction of the peptidyl trimer and vWF polypeptide are determined by determining the binding of the peptidyl trimer to the vWF polypeptide.

83. A method according to claim 81 wherein the interaction of the peptidyl trimer and vWF polypeptide is determined by determining the ability of said peptidyl trimer to promote platelet activation and/or platelet aggregation.

84. A method of determining the effect of a factor or substance on platelet activation and/or platelet aggregation, the method comprising treating platelets with a peptidyl trimer according to any one of claims 25 to 42 and determining the effect of the factor or substance on the platelet activation or aggregation.

85. A method according to claim 84 wherein platelet activation or aggregation is determined in the presence or absence of the factor or substance or with the factor or substance at different concentrations.