WO1996011942A1

WO1996011942A1 - Therapeutic molecules

Info

Publication number: WO1996011942A1
Application number: PCT/AU1995/000683
Authority: WO
Inventors: Gordon Frood Burns
Original assignee: The University of Newcastle; Newcastle Innovation Ltd
Current assignee: The University of Newcastle; Newcastle Innovation Ltd
Priority date: 1994-10-18
Filing date: 1995-10-17
Publication date: 1996-04-25
Anticipated expiration: 1997-04-18
Also published as: AUPM885894A0

Abstract

The present invention relates generally to molecules capable of inhibiting platelet aggregation such as associated with thrombus formation. The present invention is also directed to a screening assay for such molecules. The present invention also contemplates a method of inhibiting platelet aggregation by the administration of these molecules.

Description

THERAPEUTIC MOLECULES

Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs.) for the amino acid sequences referred to in the specification are defined following the bibliography.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

The processes of platelet activation and aggregation are fundamental in the prevention of blood loss following vascular damage when the aggregated platelets form a clot that seals the wound. However, platelet clotting, or thrombus formation, can also result in undesirable pathological conditions including deep vein thrombosis, vessel occlusion and stroke and in the development of atheromatous plaques. Consequently, much research has been aimed at finding inhibitors of undesirable thrombus formation, for use after surgical intervention in general, and in particular, after angioplasty designed to remove atheromatous plaques. Such research has attempted either to "dissolve" pre-existing thrombi with agents such as heparin or tissue plasminogen activator (tPA) that break down the interplatelet linkages, or to prevent the initial platelet activation and aggregation. For therapeutic use in patients, both approaches are generally applied together. Agents used for the prevention of activation and aggregation fall into two major categories. The first category involves pharmacological drugs that inhibit some of the signalling pathways involved in platelet activation: such signalling is utilised by cell types other than platelets and this limits the usefulness of these drugs in vivo. A second approach has been specifically to target the molecules on the surface of the platelet involved in activation and or aggregation. The major cell surface glycoprotein targeted in this way is the integrin receptor αllbβ3 (also known as GPIIbllla) that appears central to most platelet aggregation, and can be functionally inactivated with .antibodies, specific peptides or their chemical analogues. Such treatments, however, are not always completely effective and it may be that some platelet aggregation can bypass this receptor.

A molecule known as thrombospondin (TSP) plays an accessory role in platelet activation and aggregation (1). TSPl is the most abundant protein component of platelet α-granules which are secreted rapidly upon platelet activation, after which, TSP binds to the surface of the activated platelet (2, 3). TSPl has been shown to promote platelet aggregation of both non-stimulated platelets and platelets stimulated with thrombin or

ADP (4, 5, 6). In addition, the monoclonal antibody C6.7, directed against the carboxy terminus of human TSPl, inhibits the secondary or secretion-dependent phase of platelet aggregation (7). Also, polyclonal antibodies directed against the amino terminal heparin binding domain of TSPl inhibit platelet aggregation (8). These results implicate a role for TSPl in mediating platelet aggregation by a mechanism involving both the amino terminal heparin binding domain and the carboxy terminal cell binding domain of TSPl . Surface bound TSPl also interacts with fibrinogen bound to the surface of the platelet via αllbβ3 (1). It has been suggested that TSPl serves to stabilise fibrinogen binding to the activated platelets surface reinforcing the strength of interplatelet interactions and thereby determining the size and reversibility of platelet aggregates; this, in turn serves to regulate clot formation (9).

Some of the TSP-derived peptides that are active in cell binding have also been tested for their capacity to inhibit platelet aggregation. The Arginine-Glycine-Aspartate (RGD) sequence, present in TSP as Arginine-Glycine-Aspartate-Alanine (RGDA), is generally tested as Arginine-Glycine-Aspartate-Serine (RGDS) since this was the canonical RGD sequence first identified as a cell-binding peptide within fibronectin (12). The RGD sequence is also present in a number of matrix glycoproteins wherein it appears to function as a cell binding domain; this sequence is also contained within fibrinogen. The peptide RGDS does inhibit platelet aggregation induced by a range of agonists and, used at low concentration by itself (without additional platelet stimulation), it can induce a conformational change in the integrin αllbβ3 and thereby initiate platelet activation (13). Since the integrin αllbβ3 binds the RGD sequence, and this peptide can inhibit fibrinogen binding by the integrin receptor, this is generally considered to be the mechanism of inhibitory action by this peptide. RGD-containing peptides and their analogues and mimetic are currently undergoing clinical trials as inhibitors of thrombus formation.

In accordance with the present invention, it has been surprisingly discovered that certain peptides derived from TSP are capable of activating platelets directly in a process that appears to exclude major involvement by αllbβ3. A major consequence of this discovery is the realisation that inhibitors of αllbβ3 function cannot be completely efficacious in inhibiting thrombus formation. The present invention provides, therefore, antagonists of TSP-mediated activation of platelets and a method for identifying such antagonists.

Accordingly, one aspect of the present invention is directed to a peptide having the characteristics of:

(i) being capable of inducing homotypic platelet aggregation; and

(ii) comprising a sequence of amino acids differing by at least one amino acid residue from the sequence set forth in SEQ ID NO:l.

This aspect of the present invention is predicated at least in part on the identification of a peptide derived from TSP which is capable of inducing homotypic aggregation of platelets even in the presence of RGD peptides or their mimetics. The peptide identified as having responsibility for platelet aggregation comprises the amino acid sequence (in single letter code):

R F Y V V M W K (SEQ ID NO:!), and is derived from the portion of TSP which binds to the carboxy terminal receptor. This peptide is also known and referred to herein as "4N1-1" (11). Accordingly, the importance of the discovery of this peptide lies in part in the ability to use it to identify antagonists that inhibit initial TSP binding to the carboxy terminal receptor which will thereby inhibit platelet aggregation in to to.

The present invention, therefore, provides a novel peptide differing in amino acid sequence and/or content from the SEQ ID NO:l peptide and which induce homotypic platelet aggregation. Modifications contemplated herein include the deletion, substitution and/or addition of at least one amino acid residue or the chemical modification of at least one amino acid residue on SEQ ID NO:l or its derivatives or homologues.

Chemical modifications of amino acids contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on the peptides or their analogues.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH,_μ amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6 -trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5 '-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal. The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4- amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.

Crosslinkers can be used, for example, to stabilise 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH2)_n spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero- bifunctional reagents which usually contain an amino-reactive moiety such as N- hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of C_α and N_α-methyl.amino acids, introduction of double bonds between C_α and Co atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

Where the sequence of SEQ ID NO:l is modified by the substitution of at least one amino acid residue for at least one other, the selection of a substitute residue may be made based on, for example, similarly in interactive groups, conformation and/or size of the residues. Alternatively, a completely unrelated amino acid residue may be employed.

The following deletions are contemplated by the present invention:

R F Y V V M W (SEQ ID NO:2) R F Y V V M K (SEQ ID NO:3) RFYVVWK (SEQIDNO-.4) R F Y V M W K (SEQ ID NO:5) RFVVMWK (SEQIDNO.6)

RYVVMWK (SEQ ID NO:7)

FYVVMWK (SEQIDNO.8)

R F Y V V M (SEQ ID NO:9)

RFYVVK (SEQIDNO:10) RFYMWK (SEQIDNO:ll)

RFVMWK (SEQIDNO:12) R V V M W K (SEQ ID NO:13) YVVMWK (SEQIDNO:14)

Deletions may include deletion in at least one amino acid residue (as exemplified above in SEQ ID NOs.2-8) or in multiple residues such as of two (e.g. in SEQ ID NOs.9-14), three or four residues provided that the peptide is still capable of inducing platelet aggregation or a form of aggregation. Particularly preferred deletion mutants are VVMWK (SEQ ID NO: 17) and VMWK (SEQ ID NO: 18).

Another aspect of the present invention contemplate the use of the peptide defined by SEQ ID NO:l or a modified form thereof as hereinbefore described to screen for .an antagonist which inhibits, reduces or otherwise interferes with platelet aggregation.

According to this aspect of the present invention, there is provided a method for identifying an antagonist of platelet aggregation, said method comprising contacting platelets with an effective .amount of the peptide defined by SEQ ID NO:l or a functional mutant, derivative or modified form thereof or a chemical equivalent thereof in the presence of a potential antagonist of platelet aggregation and measuring the extent of platelet aggregation.

In accordance with this method, the potential antagonist may be an extract of a biological source such as from coral, plants or fungi or a biological molecule or mixture of molecules such as peptides, polypeptides or proteins or carbohydrates. Alternatively, the potential antagonist may be a chemical compound whether synthetically produced or in an isolated, naturally occurring form. A single potential antagonist may be tested or mixtures of compounds may be initially tested for antagonistic properties before being separated into individual, active fractions. The potential antagonists may also be derived from the peptide defined by SEQ ID NO: 1 or from another portion of the TSP molecule.

The potential antagonists or source(s) thereof may be added first to the platelet composition followed by the aggregation inducing peptide SEQ ID NO:l or its functional mutants, derivatives or chemical equivalents. Alternatively, the components may be added substantially simultaneously or sequentially in any order.

Platelet aggregation may be detected by any convenient means such as the microtitre well plate aggregation assay of Dixit et al (10). Screening may be by macroscopic observation, spectrophotometry or by biochemical analysis such as by antibody binding or non-binding to receptors. Still another aspect of the present invention contemplates an antagonist of platelet aggregation, said antagonist being capable of interfering with the binding between the peptide defined by SEQ ID NO:l and its receptor on a platelet.

The antagonist of the present invention may be proteinaceous or non-proteinaceous, naturally occurring or chemically synthetic (including production by recombinant means). The present invention extends, therefore, to the use of the antagonists to prevent platelet aggregation and, therefore, thrombi. Accordingly, the present invention contemplates a pharmaceutical composition comprising an antagonist of platelet aggregation, said antagonist being capable of interfering with binding between the peptide defined by SEQ ID NO:l and its receptor on a platelet, said composition further comprising one or more pharmaceutically acceptable carriers and/or diluents.

The active ingredients of a pharmaceutical composition comprising one or more antagonists are contemplated herein to exhibit excellent therapeutic activity, for example, in the prevention of thrombi when administered in amount which depends on the particular case. For example, from about 0.5 ug to about 20 mg per kilogram of body weight per day may be administered. Dosage regi a may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The active compound may be administered in a convenient manner such as by the oral or intravenous (where water soluble) routes, although other routes of administration are contemplated such as by the intramuscular, subcutaneous, intranasal, intradermal or suppository route. Depending on the route of administration, the active ingredients which comprise an antagonist may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said ingredients. For example, the low lipophilicity of the antagonists will allow them to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis. In order to administer antagonists by other than parenteral administration, they will be coated by, or administered with, a material to prevent its inactivation. The active compounds may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof. As used herein "pharmaceutically acceptable carrier and/or diluent" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.

The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 μg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 μg to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

The present invention contemplates, therefore, a method for preventing, inhibiting or otherwise reducing the risk of platelet aggregation such as in thrombus formation, said method comprising the administration of an antagonist as hereinbefore described capable of interfering with binding between the peptide defined by SEQ ID NO:l and its receptor on a platelet. The recipient may be an animal such as a mammal including a human, livestock animal, laboratory test animal or companion animal. The treatment may be given alone or in conjunction with other anti-clotting treatments. The treatment may also be initiated as a routine procedure following surgery or other invasive procedure or following, for example, stroke or heart attack.

The present invention is further directed to the use of an antagonist of platelet aggregation, said antagonist capable of interfering with binding between the peptide defined by SEQ ID NO:l and its receptor on a platelet, in the manufacture of a medicament for the treatment of blood clotting disorders, such as in the prevention of thrombi.

Yet another aspect of the present invention provides an agent comprising an antagonist as hereinbefore described useful in preventing thrombi formation.

The present invention is further described by the following non-limiting Figures and/or Examples.

The peptide "7N3-1" referred to in the Examples comprises the sequence IRVVM and is disclosed by Kosfeld and Frazier (11). Peptide "4N1-2" is also disclosed by Kosfeld and Frazier (11) and is referred to herein as "SEQ ID NO:!".

In the Figures:

Figure 1 shows that SEQ ID NO:l induces homotypic aggregation of C32 melanoma cells. C32 melanoma cells were plated on vitronectin substrate in the absence of any peptide (NIL), or in the presence of CSVTCG (SEQ ID NO: 16) or SEQ ID NO:l peptide at 150 μM fnial concentration. In the box is shown a cell adhesion assay of the effect of increasing concentrations of the SEQ ID NO: 1 peptide upon the C32 melanoma cells plated on vitronectin. From the illustrations it can be seen that the melanoma cells are aggregated only in the presence of the SEQ ID NO:l peptide, and this might account for the loss of cell adhesion to vitronectin at this concentration of the peptide. Bar 50μ.

Figure 2 is a graphical representation showing induction of platelet aggregation by SEQ ID NO:l. Platelets, freshly isolated from normal human blood by centrifugation were washed one time and resuspended in tyrodes buffer at 2 x 10¹⁰/ml. To the washed platelets (500μl/cuvette) was added SEQ ID NO: 1 peptide at the concentration indicated (in μg/ml). The time of peptide addition to the cuvette is indicated with an arrow, and the tracings shown are those recorded in a standard aggregometer recorded at 2cm/min.

Figure 3 shows that a "scrambled" SEQ ID NO:l peptide, VFRWKYVM (SEQ ID NO: 15) does not induce platelet aggregation. The conditions of the assay were as described in Figure 2. The numbers after the peptide are the concentration tested (in μg/ml); the scrambled peptide shown was at lOOμg/ml.

Figure 4 shows aggregation induced by truncated SEQ ID NO:l peptides. The conditions were those described in Figure 2. All of the peptides were tested at 100 μg/ml.

Figure 5 shows that peptide 7N3-1 neither induces nor inhibits platelet aggregation. The conditions were those described in Figure 2. In the bottom trace is shown absence of aggregation with 7N3-1 peptide at 100 μg/ml. The middle tracing shows absence of aggregation induced by 7N3-1 peptide at 200μg/ml and, upon addition of SEQ ID NO:l peptide (lOOμg/ml, arrow) aggregation ensues that is comparable to that seen with the addition of SEQ ID NO:l peptide alone (top tracing).

Figure 6 shows that FYWMWK (SEQ ID NO:8) causes apparent inhibition of SEQ ID NO:l mediated platelet aggregation. The conditions used are as those described in Figure 2. In the bottom trace, SEQ ID NO:8 peptide (200 μg/ml) was added first, followed by SEQ ID NO:l peptide (lOOμg/ml, arrow). The top trace shows control SEQ ID NO:l mediated aggregation in the absence of additional peptide. Figure 7 shows that some truncated peptides do not inhibit SEQ ID NO:l mediated platelet aggregation. The conditions used were those described in Figure 2. Each of the peptides indicated was added at 200 μg/ml (or no peptide, top trace) and then SEQ ID NO:l peptide (lOOμg/ml) was added at the time indicated (arrow).

Figure 8 Heparin-binding peptide, hep 1, causes only partial inhibition of SEQ ID NO: 1 -mediated platelet aggregation. The conditions used were those described in Figure 2. The hep 1 peptide was added at the concentration shown (μg/ml) then SEQ ID NO:l (lOOμg/ml) added at the time indicated (arrow).

Figure 9 RGDS peptide partially inhibits SEQ ID NO:l induced platelet aggregation. The conditions used were those described in Figure 2. The RGDS peptide was added at the concentration shown (μg/ml) and SEQ ID NO:l peptide (lOOμg/ml) added at the time indicated (arrow).

The following single and three letter abbreviations are used for amino acid residues:

Amino Acid Three-letter One- letter Abbreviation Symbol

Alanine Ala A

Arginine Arg R

Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gin Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine He I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Any residue Xaa X EXAMPLE 1

The TSP-derived peptides were initially tested for their effects upon (melanoma) cell attachment to the matrix glycoprotein, vitronectin. As anticipated, the RGDS-containing peptide caused complete inhibition of cell attachment and spreading. The CSVTCG (SEQ ID NO.16) peptide had no effect on cell adhesion (Fig. 1) but, unexpectedly, the RFYVVMWK peptide (SEQ ID NO:l) inducted homotypic cell aggregation that prevented the attachment of the cells to vitronectin (Fig. 1). The 7N3-1 peptide (11) from the carboxy terminal region of TSP had no measurable effect on cell adhesion and, in particular, did not induce homotypic aggregation.

EXAMPLE 2

Each of the peptides in Example 1 was tested for its capacity to mediate (homotypic) platelet aggregation. Neither RGDS nor CSVTCG induced any aggregation, nor did the carboxy terminal peptide 7N3-1. The SEQ ID NO:l peptide directly stimulated the aggregation of washed human platelets in a dose-dependent manner (Fig. 2). Complete and rapid aggregation occurred at concentrations of 60 μg/ml peptide and above; at 50μg/ml, the amplitude of the response was half maximal, and at 25 μg/ml the response was minimal (Fig. 2).

A scrambled version of the SEQ ID NO:l peptide (VFRWKYVM) (SEQ ID NO: 15) had no effect upon platelet aggregation at lOOμg/ml (Fig. 3) or 200μg/ml, suggesting that the primary sequence of the amino acids is essential for inducing aggregation.

EXAMPLE 3

Progressive truncation of the SEQ ID NO:l peptide form the amino terminus was used to determine the minimum sequence of amino acids required to induce platelet aggregation. FYVVMWK (SEQ ID NO:8) did not induce aggregation as recorded by the platelet aggregometer (Fig. 3); but it was observed that the platelets formed large "fluffy" aggregates when treated with this peptide, and this is recorded as a loss in light transmission in the aggregometer, particularly at a concentration of 200 μg/ml (Fig. 3). Further truncations of SEQ ID NO:l revealed the pattern of platelet aggregation shown in Fig. 4 when the peptides were tested at 100 μg/ml. Thus, at this concentration, peptide YVVMWK (SEQ ID NO: 14) caused a loss of light transmission; VVMWK caused rapid aggregation; and peptide VMWK caused progressive, slow, microaggregation.

EXAMPLE 4

Peptides derived from the other cell-binding domains of TSP had very little inhibitory effect upon SEQ ID NO: 1 (100μg/ml)-mediated platelet aggregation. Peptide CSVTCG had no measurable effect. A long amino acid peptide representing amino acids 23-32 of TSP within the hep.arin-binding domain and termed hepl exhibited minimal inhibitory effect when tested at concentrations to 400 μg/ml (Fig. 8). The RGDS peptide also had very little effect upon this platelet response when tested at concentrations to 500μg/ml (Fig. 9).

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions .and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

REFERENCES

1. Leung, L.L.K.a.N., R.L. J. Clin. Invest. 70, 542-549 (1982).

2. Lawler, J., Slayter, H.S. & Coligan, J.E. J. Biol. Chem. 253, 8609-8616 (1978).

3. Phillips, D.R., Jennings, L.K. & Prasanna, H.R. J. Biol. Chem. 255, 11629-11632 (1980).

4. Jaffe, E.A., Leung, L.L., Nachman, R.L., et al. Nature 295, 246-248 (1982).

5. Lawler, J. Blood 67, 1197-1209 (1986).

6. Tuszynski, G.P., Rothman, V.L., Murphy, A., et al. Blood 72, 109-115 (1988).

7. Dixit, V.M., Haverstick, D.M., O'Rouke, K.M., et al. Proc. Natl. Acad. Sci. USA. 82, 3472-3476 (1985).

8. Gartner, T.K., Walz, D.A., Aiken, M., et al Biochem. Biophys. Res. Commun. 124, 290-295 (1984).

9. Leung, L.L.K. J. Clin. Invest. 74, 1764-1772 (1984).

10. Dixit, V.M., Haverstick, D.M., O'Rourke, I., et al Proc. Natl. Acad. Sci. USA. 82, 2844-3848 (1985).

11. Kosfeld, M.D. & Frazier, W.A. J. Biol. Chem. 268, 8808-8814 (1993).

12. Ruoslahti, E. & Pierschbacher, D. Cell 44, 517-518 (1986).

13. Frelinger, A.L., III, Cohen, I., Plow, E., et al J. Biol. Chem. 165 6346-6352 (1990). SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT:

(in countries other than the US) The University of Newcastle

Research Associates Limited GORDON FROOD BURNS (in US only) Gordon Frood BURNS

(ii) TITLE OF INVENTION: THERAPEUTIC MOLECULES

(iii) NUMBER OF SEQUENCES: 18

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(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT INTERNATIONAL

(B) FILING DATE: 17-OCT-1995

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: PM8858/94

(B) FILING DATE: 18-OCT-1994

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: HUGHES DR, E JOHN L

(C) REFERENCE/DOCKET NUMBER: EJH/EK

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: +61 3 9254 2777

(B) TELEFAX: +61 3 9254 2770 (2) INFORMATION FOR SEQ ID NO:1 :

<i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Arg Phe Tyr Val Val Met Trp Lys 1 5

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2

Arg Phe Tyr Val Val Met Trp 1 5

(2) INFORMATION FOR SEQ ID NO:3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Arg Phe Tyr Val Val Met Lys 1 5

(2) INFORMATION FOR SEQ ID NO: :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Arg Phe Tyr Val Val Trp Lys

1 5 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Arg Phe Tyr Val Met Trp Lys 1 5

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

( i) SEQUENCE DESCRIPTION: SEQ ID NO:6 :

Arg Phe Val Val Met Trp Lys 1 5

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7

Arg Tyr Val Val Met Trp Lys 1 5

(2) INFORMATION FOR SEQ ID NO:8 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8 :

Phe Tyr Val Val Met Trp Lys

1 5 (2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 :

Arg Phe Tyr Val Val Met

1 5

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Arg Phe Tyr Val Val Lys 1 5

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Arg Phe Tyr Met Trp Lys 1 5

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Arg Phe Val Met Trp Lys

5 (2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Arg Val Val Met Trp Lys

1 5

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Tyr Val Val Met Trp Lys

1 5

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Val Phe Arg Trp Lys Tyr Val Met 1 5

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16

Cys Ser Val Trp Cys Gly

1 5 (2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

Val Val Met Trp Lys 1 5

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Val Met Trp Lys

1

Claims

CLAIMS:

1. A peptide having the characteristics of:

(i) being capable of inducing homotypic platelet aggregation; and

2. A peptide according to claim 1 wherein the peptide defined in SEQ ID NO:l differs in .amino acid sequence by a deletion, substitution and/or addition of an amino acid.

3. A peptide according to claim 2 comprising the amino acid sequence selected from:

R F Y V V M W (SEQ ID NO:2)

R F Y V V M K (SEQ ID NO:3) RFYVVWK (SEQIDNO.4) R F Y V M W K (SEQ ID NO:5) R F V V M W K (SEQ ID NO:6) RYVVMWK (SEQIDNO:7) FYVVMWK (SEQ ID NO:8)

R F Y V V M (SEQ ID NO:9)

R F Y V V K (SEQ ID NO:10)

RFYMWK (SEQIDNO:ll)

RFVMWK (SEQIDNO:12)

RVVMWK (SEQIDNO:13)

Y V V M W K (SEQ ID NO:14)

4. A peptide according to claim 2 comprising the amino acid sequence:

V V M W K (SEQ ID NO:17).

5. A peptide according to claim 2 comprising the amino acid sequence: VM WK (SEQ ID NO: 18).

6. A method for identifying an antagonist of platelet aggregation, said method comprising contacting platelets with an effective amount of the peptide defined by SEQ ID NO:l or a functional mutant, derivative or modified form thereof or a chemical equivalent thereof in the presence of a potential antagonist of platelet aggregation and measuring the extent of platelet aggregation.

7. A method according to claim 6 wherein the agonist is derived from the peptide defined by SEQ ID NO: 1.

8. An antagonist of platelet aggregation, said antagonist being capable of interfering with the binding between the peptide defined by SEQ ID NO:l -and its receptor on a platelet.

9. A ph.armaceutical composition comprising an antagonist according to claim 20 and one or more pharmaceutically acceptable carriers and/or diluents.

10. A method for preventing, inhibiting or otherwise reducing the risk of platelet aggregation comprising administering to a subject a molecule capable of interfering with binding between the peptide defined by SEQ ID NO:l and its receptor on a platelet.

11. A method according to claim 10 wherein the subject is a human.

12. A method according to claim 10 wherein the subject is a livestock animal, laboratory test animal or a companion animal.