Background Art Thrombosis is a critical element in morbidity and mortality associated with vascular disease. When an atheromatous plaque ruptures or the normal integrity of the vascular endothelium is disrupted the circulating multimeric protein von Willebrand factor (vWF) adheres to the subendothelium. Platelets then adhere to immobilised vWF using the Glycoprotein Ib receptor (GPIb). When platelets adhere they become activated releasing potent agonists such as thromboxane (TXA2) ADP and serotonin. These agonists amplify thrombus formation and signal to the platelet Glycoprotein IIbIIIa (aIIbß3) receptor.

The integrin aIIbß3 then undergoes a conformational change and binds its ligand, fibrinogen, and vWF, resulting in thrombus formation. The only platelet- specific drugs that are effective in treating this pathological process are aspirin, which inhibits thromboxane formation, clopidogrel (or ticlopidine), which blocks ADP and the aIIbp3 antagonists. The mechanism of action of clopridogel has only very recently been appreciated again highlighting how little is known about platelet function in thrombotic syndromes. The recent failures of oral aIIbß3 antagonists and indeed the possible increase in mortality induced by these drugs suggests that the current paradigm of platelet biology does not translate into clinical benefit. The pathway by which ligand binding to GPIb leads to aIIbß3 activation and thrombus formation, is as yet not understood.

The initial stage of formation of a thrombotic plug involves the interaction between the platelet glycoprotein Ib (GPIb) receptor and its immobilising ligand vWF (Von Willebrand Factor). The crystal structure of the GPIba-vWF-A1 complex is disclosed in Science, 2002,297, 1176-1179.

It is an object of the invention to overcome at least some of the above mentioned problems.

Statements of Invention

According to the invention, there is provided a cyclic (3-hairpin peptidomimetic comprising a reverse turn, first and second adjacent anti-parallel ß- strands flanking the reverse turn, and a hairpin stabilising motif, wherein the first (3-strand includes an amino acid sequence which is capable of forming a continuous sheet with at least a portion of a ß-3 strand from an Al domain of Von Willebrand Factor.

Typically, the first P-strand includes at least three contiguous amino acids, suitably at least four contiguous amino acids, and preferably at least five amino acids, which are capable of forming a sheet with a portion of a ß-3 strand from an Al domain of Von Willebrand Factor. The ß-3 strand from an Al domain of Von Willebrand Factor comprises the amino acid sequence S-H-A-Y. In this manner, the cyclic peptidomimetic of the invention competes with the platelet GPIb glycoprotein receptor for Von Willebrand Factor (vWF) binding sites, thereby inhibiting formation of the GPIB-vWF complex which is the initial stage in the formation of a thrombotic plug.

In this specification, the term"reverse turn" should be taken to understand an amino acid sequence of 3 or 4 amino acids which forms a reverse turn.

Suitable reverse turns are ß or y-Turns having 4 and 3 amino acids respectively. The term should not be taken to exclude reverse turns which are not based on amino acids, but which

perform the same function. Likewise, the term should be taken to include molecules comprising, or consisting of, amino acid analogues.

In this specification, the term"peptidomimetic" should be understood to mean a synthetic molecule which mimics the secondary structure of a native protein. The term should be taken to include peptides which consists entirely of amino acids, and peptides which include other moieties such as amino acid analogues, amide bond isosteres and nonpeptide structures.

Suitably, the first strand consists of an amino acid sequence which is capable of forming, along its full length, a continuous 3-sheet with the ß-3- strand from the Al domain of vWF.

A In a preferred embodiment, the amino acid sequence of the first strand comprises the sequence (K or C) -A-V. In a more preferred embodiment, the amino acid sequence of the first strand comprises (K or C)-A-V-T. Ideally, the amino acid sequence of the first ß-strand comprises (K or C)-A-V-T-S.

Preferably, the amino acid sequence of the first strand comprises (K or C)-A-V-T-S-N. Thus, the sequence of the first {3-strand may be selected from the group comprising: K-A-V-T-S-N; C-A-V-T-S-N; K-A- V-T-S; C-A-V-T-S; K-A-V-T ;'C-A-V-T ; K-A-V; C-A-V; and L-E-V-N.

In a preferred embodiment of the invention, the macrocyclic peptidomimetic includes an intramolecular di-sulphide bridge between the two adjacent ß-strands. Typically, the bridge is introduced by replacing two paired residues, on in each adjacent ß-strand, with suitable amino acids, for the formation of a covalent bond via their side chains. Ideally, the residues replaced are the central amino acids of the (3-strands or the amino acids close to the centre of the (3-strands. The bridge may be, for example, a di-sulphide bridge or a lactose bridge. Suitably, each strand flanking sequence includes a cysteine residue, wherein an intramolecular di-sulphide bridge is formed between the cysteine residues. Alternatively, one of the P- strand may contain an aspartic acid residue and the other strand contain a 2,3-diaminopropionic acid, wherein an intramolecular lactam bridge is formed between the two ß-functional groups of these two residues.

Typically, the a-turn comprises the sequence G-A1-D- V wherein Al and A2 represents any amino acid. In a preferred embodiment, Al and A2 are is selected from the group comprising: amino acids known to stabilise ß-hairpin structures and (3-turns ; and amino acids identified in gain-of-function mutations related to platelet-type von Willebrand disease. In a preferred embodiment, the (3-turn comprises the sequence G-V-D- V. Alternatively, at least one of the amino acids in the sequence G-V-D-V is substituted with a different amino acid selected from the group

comprising: D, N, E, Q, S and Y. Typically, the second residue in the ß-turn is replaced by P.

Suitably, the third residue in the turn is replaced by G.

In a preferred embodiment of the invention, the first (3-strand follows the turn in a amino (NH2)- carboxy (COOH) direction. Thus, the residue generally directly precedes the first amino acid in the first flanking sequence.

In this specification, the term"ß-hairpin stabilising motif"should be taken to mean moiety which helps constrain the cyclic peptide in a ß hairpin conformation.

Typically, the ß-turn hairpin stabilising motif is located opposite the ß-turn. Ideally, the stabilising motif is L-Pro-D-Pro. Alternatively, the stabilising motif is a dipeptide or a tripeptide such as YP, PF, PW, P-G-P, or an organic template such as monocyclic, bicyclic, fused bicyclic systems. Ideally, the ß-hairpin turn is stabilised by a stabilising motif and an intramolecular sulphide bridge linking the two adjacent ß-strands turn flanking sequences. Examples of hairpin turn stabilising motifs are disclosed in Immunomethods, 1992,1, 33-39, Synlett, 1999,4, 429-441, and J.

-Org. Chem. 2002,67, 5085-5097.

In one embodiment of the invention, the ß-turn stabilising motif may comprise a ß-turn. Generally,

each sheet consists of between 2 and 10, suitably between 2 and 8, preferably between 2 and 6, and more preferably between 2 and 4, amino acids.

Usually, each sheet will consist of the same number of amino acids as each other. However, when the cyclic peptidomimetic includes a y-turn, one of the ß-sheets may contain one amino acid more than the other.

In one embodiment of the invention, the second ß- sheet includes an amino acid sequence which is substantially, or completely, identical to the sequence of the first ß-sheet. However, in a preferred embodiment the sequence of the second ß- sheet is not identical to that of the first sheet.

Typically, the second sheet comprises the sequence W-K- (Q or C). Preferably, the second sheet comprises the sequence V-W-K- (Q or C). More preferably, the second sheet comprises the sequence Y-V-W-K- (Q or C). Ideally, the second ß- sheet comprises the sequence V-Y-V-W-K- (Q or C).

Thus, the second sheet is generally selected from the group comprising: W-K-Q; W-K-C; V-W-K-Q; V-W-K- C; Y-V-W-K-Q; Y-V-W-K-C; V-Y-V-W-K-Q; V-Y-V-W-K-C; and E-V-S-K.

In a preferred embodiment of the invention, the cyclic ß-hairpin peptidomimetic is selected from the group comprising:

EI-13 (Sequence ID No 1) X-A-V-T-S-L-D |-K-A-V-T-S-P-DP-+-= EI-16 (Sequence ID No 2) L T-LP-DP-< EI-25 (Sequence ID No 3) l-t Å~t T-P-DP- m. ___ _ - -. _ _ EII-18 (Sequence ID No 41) Ll_ç_LP Dg EI-24 (Sequence ID No 5) L EI-17 (Sequence ID No 6) EI-17 (Sequence ID No 6) -V'--LP D EI-19 (Sequence ID No 7) Lg.. rT EI-22 (Sequence ID No 8) V-K-A-V-T-G-P-D-V-K-A-V-T-G-P-D EI-22 (Sequence ID No 9) Lval-Lys-Dap-Val-LPro-DPro-Trp-Asp-Gln-Gly-Val-As1 EI-27 (Sequence ID No 10)

EI-28 (Sequence ID No 11) In the above sequences, the residues highlighted in the darkest shading form the ß (or y)-turn, i. e. the sequence G-V-D-V in sequence ID No's 1 to 6. The residues highlighted in the lightest shading form the ß-hairpin stabilising motif, i. e. LP-DP or P-G- P. The residues highlighted in the remaining tones form the first and second ß-strands, the first ß- strand being located adjacent the carboxy terminal of the ß (or y)-turn. Sequence ID No's 4 and 6 include a disulphide bridge formed between cysteine residues, one of which is located in each of the first and second ß-strand. Sequence ID No's 10 and 11 include a lactam bridge formed between an aspartic acid residue on the second (3-strand and a 2,3-diaminopropionic acid (Dap) residue on the first strand.

All molecules of Sequence ID No's 1 to 11 are cyclic molecules which mimic that portion of the platelet glycoprotein Ib (GPIb) which interacts with the P-3 strand of the Al domain of Von Willebrand Factor (vWF). Thus, the cyclic peptidomimetics of the invention compete with GPIb for binding sites on vWF, thereby inhibiting the initial stage of the formation of a thrombotic plug which involves the formation of a GPIb-vWF (A1) complex. The cyclic peptidomimetics of the invention are thus useful in

treating or preventing conditions associated with thrombosis.

Suitably, one or more of residues V227-Y-V-W-K-Q232 may be substituted with W or Y, and one or more of residues K237 -A-V-T-S-N242 may be replaced with W or Y. In one embodiment, residues V227, Y228, W230, K237, S241 or N242 may be replaced by V or I. V239 may be replaced by M or I. (Residue numbering provided above is based on the numbering of the amino- terminal domain of GPIba, residues 1 to 290, as used in the Science paper referred to above.) In sequence EI-19, one or more of residues E5 to K8 and L13 to N16 may be replaced by W, Y, V or I.

In one embodiment, the amino acid residues V6 and V15, or S7 and E14, or K8 and L13 may be substituted with the amino acid C, wherein the intramolecular disulphide bridge is formed between these cysteine residues.

In a preferred embodiment of the invention, the amino acid residues V6 and V15, or S7 and E14, or K8 and L13 may be substituted with the amino acids aspartic acid and 2, 3-diaminopropionic acid (Dap), respectively, wherein the intramolecular lactam bridge is formed between the two-functional groups of these two residues. Alternatively, the amino acid residues V6 and V15, or S7 and E14, or K8 and L13 may be substituted with the amino acids 2,3- diaminopropionic acid (Dap) and aspartic acid, respectively, wherein the intramolecular lactam

bridge is formed between the two ß-functional groups of these two residues.

The invention also relates to a cyclic P-hairpin peptidomimetic comprising a 3-turn or a 7-turn and a P-turn stabilising motif, the amino acid sequence of the peptidomimetic having a sufficient degree of homology with a sequence of V227 to N242 of platelet glycoprotein allbf33 to enable the peptidomimetic compete with the native glycoprotein for vWF binding sites, wherein the peptidomimetic is a cyclic molecule.

The invention also relates to a pharmaceutically acceptable salt of a cyclic (3-hairpin peptidomimetic according to the invention.

The invention also relates to a pharmaceutical composition comprising a cyclic ß-hairpin peptidomimetic according to the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

The invention also relates to a method of treating or preventing a condition associated with thrombosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a cyclic ß-hairpin peptidomimetic according to the invention, or a pharmaceutically acceptable salt thereof.

The invention also relates to a method'of inhibiting platelet aggregation or adhesion in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a cyclic - hairpin peptidomimetic according to the invention, or a pharmaceutically acceptable salt thereof.

The invention also relates to a use of a cyclic ß-hairpin peptidomimetic according to the invention, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prevention of a condition associated with thrombosis. Typically, the condition associated with thrombosis is selected from the group comprising: myocardial infarction; stroke; restenosis; angina; atherosclerosis; and ischemic conditions.

The invention also relates to a method of investigating signalling pathways in platelets comprising the steps of treating platelets with an agonist in the presence and absence of a cyclic - hairpin peptidomimetic according to the invention, and subsequently investigating one or more selected characteristics of the thus treated platelets. Preferably, the process steps are carried out for both male and female platelets, and the resultant characteristics compared between the male and female platelets. Suitably, the selected characteristic to be investigated is selected from the group comprising: platelet phosphoproteome; platelet proteome; platelet thromboxane production;

platelet ADP release; and platelet serotonin release. Suitably, the platelet phosphoproteome is investigated using SDS-PAGE, western blotting, and/or mass spectrophotometry.

Typically, the platelet proteome is investigated using a protein microarray. The agonist is suitably selected from the group comprising: ristocetin; thrombin; and ADP.

Typically, the cyclic (3-hairpin peptidomimetic is biotinylated or fluorescently labelled. Suitably, the peptidomimetic is conjugated to biotin or a fluorescent label through an epsilon-amino group of a lysine residue, originally present in the sequence, or added by, for example, substitution.

The invention also relates to a cyclic hairpin peptidomimetic according to the invention for use as a research tool.

Detailed Description of the Invention The invention will be more clearly understood from the following description of some embodiments thereof, given by way of example only, with reference to the accompanying Figures in which: Fig. l : EI-13 Doses Response Curves. Various concentrations of EI-13 (Sequence ID No 1) were

incubated with washed platelets and vWF and the % aggregation monitored; Figure 2: EI-13 dose response histogram-Botrocetin Induced Platelet Aggregation Various concentrations of the EI-13 peptide were incubated with washed platelets in JNL for 2mins prior to addition of 1ug/ml botrocetin (known platelet aggregation agonist). The % aggregation that occurred was monitored to investigate if EI-13 inhibited botrocetin induced platelet aggregation. Inhibition of botrocetin induced aggregation was not observed (M=male, F=female); Figure 3: Effect of EI-13 (& Control peptide) on Low Dose Thrombin Induced Platelet Aggregation: peptide EI-13 plus its vehicle, linear peptide (control peptide) were incubated at a concentration of 1000M with washed platelets in JNL for 2mins prior to addition of 0.2U/ml thrombin (known platelet aggregation agonist). The % aggregation that occurred was monitored and it was observed that EI- 13 caused slight inhibition of low dose thrombin (M=male, F=female); Figure 4: EI-13 dose response histogram-Ristocetin Induced Platelet Aggregation: various concentrations of the EI-13 peptide were incubated with washed platelets in JNL for 2mins prior to addition of 0.3mg/ml ristocetin (known platelet aggregation agonist). The % aggregation that occurred was monitored to investigate if EI-13 inhibited

ristocetin induced platelet aggregation. Ristocetin induced aggregation was reduced by 40% upon exposure to 100RM EI-13; Figure 5: Effect of EI-13 & Control peptide on Ristocetin Induced Platelet Aggregation: Peptide EI- 13 plus its vehicle, and linear peptide control were incubated at a concentration of 100uM with washed platelets in JNL for 2mins prior to addition of 0.3mg/ml ristocetin (known platelet aggregation agonist). The % aggregation that occurred was monitored and it was observed that the linear peptide was lacking the antagonist activity of the cyclic peptide; Figure 6a: EI-13 dose response histogram with male donor platelets. Effect of EI-13 & Control peptide on Ristocetin Induced Platelet Aggregation: Peptide EI-13 plus its vehicle, and linear peptide control were incubated at a concentration of 100uM with washed platelets in JNL for 2mins prior to addition of 0.3mg/ml. The % aggregation that occurred was then monitored; Figure 6b: EI-13 dose response histogram with female donor platelets. Effect of EI-13 & Control peptide on Ristocetin Induced Platelet Aggregation: Peptide EI-13 plus its vehicle, and linear peptide control were incubated at a concentration of 100pM with washed platelets in JNL for 2mins prior to addition of 0.3mg/ml. The % aggregation that occurred was then monitored;

Figure 7a: Effect of EI-13 & Control peptide on low dose thrombin Induced Platelet Aggregation with male donor platelets: Peptide EI-13 plus its vehicle, and linear peptide control were incubated at a concentration of 100uM with washed platelets in JNL for 2mins prior to addition of 0.3mg/ml. The % aggregation that occurred was then monitored; Figure 7b: Effect of EI-13 & Control peptide on low dose thrombin Induced Platelet Aggregation with female donor platelets: Peptide EI-13 plus its vehicle, and linear peptide control were incubated at a concentration of 100uM with washed platelets in JNL for 2mins prior to addition of 0.3mg/ml. The % aggregation that occurred was then monitored; and Figure 8: Effect of EI-13 & Control peptide on platelet adhesion under arterial shear i. e. 1, 500s-1.

Washed platelets were untreated or preincubated with Reopro which prevents platelet adhesion due to GPIIbIIIa binding vWF to determine average platelet adhesion due to GPlb interaction with vWF. Washed platelets were preincubated with EI-13 and its control peptide and a decrease in platelet adhesion was observed in the presence of EI-13, especially after flow.

1. Preparation of Cyclic Peptidomimetics The peptides are prepared by standard Solid Phase Peptide Synthesis according to the Fmoc-tBu strategy

with HBTU/HOBt/DIEA coupling chemistry. Assembly of the amino acid sequences is carried out on an automated peptide synthesizer (Applied Biosystems 433A).

For EI-13, the peptide chain is assembled from a Fmoc-L-Glu-Oall residue, linked on a Novasyn TGR resin. For EI-16, EI-25, EI-18, EI-24, EI-17, EI-26, EI-22 the peptide chain is assembled from a Fmoc-L- Asp-Oall residue, linked on a HMPA resin. The peptide chains are elongated by iterative deprotection (piperidine in DMF) and coupling with Fmoc protected amino acids. The side chain protecting groups were tBu, for Ser, Thr and Tyr; Trt, for Asn, Gln, His and Cys; Boc, for Lys and Trp; Pbf, for Arg; O-tBu for Asp and Glu.

Deprotection of the allylic ester with tetrakis (triphenylphosphine) palladium, N- methylmorpholine and acetic acid in chloroform, and subsequent deprotection of the N-terminal amino function with piperidine in DMF, followed by macrocyclisation on the resin using PyBOP/HOBt/DIEA coupling chemistry is performed using a Quest 210ASW (Argonaut Technologies) Organic Synthesizer. The peptide is cleaved from the synthesis resin with 82. 5% TrifluoroAcetic Acid, 5% water, 5% triisopropylsilane, 5% thioanisole, 2. 5% EDT.

Formation of the disulfide bridge in EI-17-18-26 is accomplished by air oxidation of a 0.75mM solution of the monocyclic peptide in ammonium acetate buffer (pH=8, 50mM).

For EI-27-28, a linear precursor of the beta-hairpin is prepared as described above on a Novasyn TGT resin. The sequence is elongated by iterative deprotection (piperidine in DMF) and coupling with Fmoc protected amino acids. Fmoc-L-Asp (Oall) -OH and Fmoc-Dap (Alloc) -OH are used to introduce the residues forming the lactam bridge. Deprotection of the allylic protecting groups with tetrakis (triphenylphosphine) palladium, and subsequent formation of the lactam bridge on the resin using PyBOP/HOBt/DIEA coupling chemistry is performed using a Quest 210ASW (Argonaut Technologies) Organic Synthesizer. The bridged peptide is cleaved protected from the resin and the macrocyclisation is carried out in DCM solution with HATU/HOAt/DIEA coupling chemistry at a concentration of lmg/ml. The cyclic bridged peptide is then deprotected with 82.5% TrifluoroAcetic Acid, 5% water, 5% triisopropylsilane, 5% thioanisole, 2. 5% EDT and lyophilized.

For EI-19 a linear precursor of the beta-hairpin is prepared as described above on a Novasyn TGT resin.

The sequence is elongated by iterative deprotection (piperidine in DMF) and coupling with Fmoc protected amino acids. The linear peptide is cleaved protected from the resin and the macrocyclisation is carried out in DCM solution with HATU/HOAt/DIEA coupling chemistry at a concentration of lmg/ml. The cyclic peptide is then deprotected with 82.5% TrifluoroAcetic Acid, 5% water, 5%

triisopropylsilane, 5% thioanisole, 2. 5% EDT and lyophilized.

Chromatographic analysis and purification were performed on a BioCAD SPRINT Perfusion Chromatography Workstation (PerSeptive Biosystems) using POROS 20R2 Reversed Phase Perfusion Chromatography packing for a first purification, followed by as second purification on a Jupiter column (15A, C5, 100mmd/250mmL, Phenomenex); (A mobile phase: 0.1% TFA in water; B mobile phase: 0. 1% TFA in acetonitrile), gradient: 2 to 60% B in 18 column volumes (Poros) or 5 column volumes (Jupiter); flow rate: 4 ml/mn (Poros) or 2 ml/mn (Jupiter); single wavelength detection at 214 nm.) The peptides were characterised by Matrix Assisted Laser Desorption Ionisation-Time Of Flight-Mass Spectrometry (a-cyano-4-hydroxy-cinnamic acid as a matrix).

2. Cyclic Peptidomimetics The following cyclic peptidomimetics were synthesised using the protocols described above: L G-V-D-V-K-A-V-T-S-N-P-G-P-V-Y-V-W-K-Qj EI-13 (Sequence ID No 1) L V-K-A-V-T-S-LP-DP-Y-V-W-K-Q-G-V-D EI-16 (Sequence ID No 2)

V-K-A-V-T-LP-DP-V-W-K-Q-G-V-D EI-25 (Sequence ID No 3) l LV-C-A-V-T-LP-DP-V-W-K-C-G-V-D EI-18 (Sequence ID No 4) LV-K-A-V-LP-DP-W-K-Q-G-V-D EI-24 (Sequence ID No 5) LV-C-A-V-LP-DP-W-K-C-G-V-D EI-17 (Sequence ID No 6) l L S-H-L-E-V-N-LP-DP-E-V-S-K-V-A EI-19 (Sequence ID No 7) LV-K-A-V-T-G-P-D-V-K-A-V-T-G-P-D EI-22 (Sequence ID No 8) LV-K-A-V-T-G-P-D-V-K-A-V-T-G-P-D EI-22 (Sequence ID No 9) I L Val-Lys-Dap-Val-LPro-DPro-Trp-Asp-Gln-Gly-Val-As EI-27 (Sequence ID No 10) Val-Lys-Dap-Val-Thr-LPro-DPro-Val-Trp-Asp-Gln-Gly-Val-Asp EI-28 (Sequence ID No 11)

3. Aggregation Assays Human blood was collected from healthy donors who had been aspirin free for the previous 14 days.

Blood (50ml) was drawn into a syringe containing the anticoagulant buffer ACD at a ratio of 1. 5ml ACD to every 10ml blood.

Platelet rich plasma (PRP) was obtained by centrifuging whole blood at 150g for 10 min at room temperature. The upper phase i. e. PRP was carefully transferred into a fresh tube, acidified to pH 6.5 with ACD buffer and prostaglandin Ez (PGE1) added at a final concentration of 2 WM to avoid platelet activation. Platelets were pelleted by centrifugation at 720g for 10mins. The supernatant was discarded and platelets resuspended in JNL buffer to a concentration of 200-300 x 103 platelets/Rl.

The platelet suspension was left undisturbed for 20 min at room temperature. Then, immediately prior to use CaCl2 was added to a concentration of 1.8 mM.

Freshly isolated platelets were used within 4hrs of blood draw and kept at room temperature throughout all experiments.

Aggregation Assay Aggregation assays were performed on a Platelet Aggregation Profiler, BioData Corporation, which incubated the platelets at 37°C and agitated them

via stirring at 1100rpm. Aggregation was measured by an increase in light transmission through the assay tube.

To washed-platelets in JNL buffer, vWF was added to a working concentration of 10Rg/ml, if required.

The peptide under investigation or its vehicle blank was added to the platelets and aggregation monitored for 2min. The aggregation agonist of choice was then added and the occurrence of aggregation monitored for 3mins. Agonist working concentrations were 0.3mg/ml, lRg/ml and 0.2U/ml for ristocetin, botrocetin and low dose thrombin, respectively.

4. Results EI-13 (Sequence ID No 1) was devoid of any agonist activity at working concentrations up to 10ORM (fig. l). In addition, while EI-13 was not able to reduce botrocetin induced platelet aggregation (fig. 2), it antagonised, in a dose dependent manner, low-dose thrombin (fig. 3) and ristocetin induced platelet aggregation (fig. 4).

At 100 umol concentration, EI-13 reduced low-dose thrombin induced platelet aggregation by 11%.

At 100 umol concentration, EI-13 reduced ristocetin induced platelet aggregation by 40%. This result was an average of 8 individual platelet donors (4 male and 4 female) assayed with peptide from the same stock.

This response was also confirmed by using the linear peptide containing the same sequence (EI-13 linear) as a control. The higher conformational flexibility of this peptide resulted in a partial, or total, loss of inhibitory potency for the prevention of platelet aggregation, when low-dose thrombin (fig. 3), or ristocetin (fig. 5) was used as an agonist respectively. Working concentrations up to lOOpM were assayed.

The investigation of the effects of EI-13 on platelet function revealed a striking difference in results obtained with platelets originating from donors of different gender. Indeed, inhibition of platelet aggregation was much greater in male rather than female platelet donors where aggregation was reduced by 57% and 23%, respectively (fig. 6a, b) A similar trend was also observed in low-dose thrombin induced platelet aggregation, where EI-13 reduced this process by 15% and 2. 5%, for male and female platelet donors, respectively (fig. 7a. b).

To verify that EI-13 is also active in a non-static agglutination assay and that its mechanism of action occurs through the disruption of platelet adhesion, we evaluated the cyclic peptide in a physiological environment under fluid dynamic shear, by using real-time videomicroscopy.

Washed platelets which were untreated or preincubated with reopro (10pg/ml), EI-13Linear (100uM working concentration) or EI-13 (100uM working concentration) were perfused into the flow chamber that was coated with human vWF (100ug/ml).

The chamber was then perfused with JNL buffer using a syringe pump to give an arterial shear rate of 1, 500s-l. Both during 4min of perfusion with JNL (of Flow) and after perfusion (after flow), single frame images were captured through-out the centre of the flow path and the number of adherent cells per field quantified offline (fig. 8). In the presence of EI- 13 a decrease in platelet adhesion can be clearly seen compared to platelets that were untreated, preincubated with reopro or EI-13control peptide.

This decrease in platelet adhesion is particularly substantial after flow.

5. Use of Cyclic Peptidomimetics as a Research Tool The present data has revealed a gender specific response in activation of the GPIb receptor that can be selectively investigated by using the cyclic peptidomimetics of the invention to further characterise signaling pathways in platelets in response to various agonists. These pathways are studied by looking at the platelet proteome and platelet phosphoproteome in response to different agonists in the presences and absence of specific cyclic peptidomimetics of the invention in male and female platelets.

Phosphoproteins: the alterations in phosphoproteins in platelets in response to various agonists is analyzed using SDS PAGE and western blotting. The agonists used in the experiments are ristocetin, thrombin and ADP. A complex pattern of altered phosphoproteins is characteristically seen on 1D gels in response to these agonists. These phosphoproteins can be more completely resolved on 2D gels. 1 D or 2D gels are used to identify gender specific phosphorylated protein spots in the presence of cyclic peptidomimetics and agonist.

These proteins are then identified by mass spectrometry.

Proteins: the interaction of cyclic peptidomimetics with a protein can be investigated by using the protein microarray technology. Cyclic peptidomimetics modified by conjugation with a fluorescent substrate or by biotinylation are screened on protein arrays to identify interacting proteins and receptor proteins.

Small molecules: The effects of the cyclic peptidomimetics of the invention in male and female platelets on thromboxane production, ADP and Serotonin release may also be investigated. Finally, signaling via GPIb leads to activation of the GPIIbIIIa receptor, detected by PAC1 binding, which are studied in platelets in the presence and absence of cyclic peptidomimetics in response to the agonists listed above.

The invention is not limited to the embodiments hereinbefore described which may be varied without departing from the spirit of the invention.