WO2006021942A2

WO2006021942A2 - Platelet modulating oligopeptides

Info

Publication number: WO2006021942A2
Application number: PCT/IE2005/000086
Authority: WO
Inventors: Niamh Moran; Dermot Kenny; Denis Shields
Original assignee: Royal College Of Surgeons In Ireland
Priority date: 2004-08-26
Filing date: 2005-08-25
Publication date: 2006-03-02
Also published as: GB0419053D0; WO2006021942A3

Abstract

The invention relates to the peptides shown in Sequence ID -Numbers 1 to 18 that have been found to be useful in modulating platelet activity, and modulating other types of cellular activity. The invention also relates to fragments, or analogues, of the peptides which retain the ability to modulate platelet activity. The peptides of Seq. IDs 1-18 correspond to short (10 amino acids long) cytoplasmic regions of transmembrane proteins, like CD 226, Nectin 3, ATP8A1, ABCR, ABCAl, VPPl, VPP4, CLCN7, etc..

Description

BIOACTTVE PEPTIDES

Technical Field

The invention relates to bioactive peptides and proteins. In particular, the invention relates to peptides and proteins which modulate cellular, especially platelet, activity. The invention also relates to bioactive peptides and proteins useful in treating or preventing diseases or conditions associated with thrombosis.

Background of the Invention

The availability of the human genome sequence is directing a flow of information from DNA sequence, expression and proteomic survey data to the identification of pivotal regulators of cell signalling, and of drug targets. An important bottleneck is the extent of functional validation for such panels of novel proteins. Gene silencing and animal gene knock-out models provide two approaches. A third route is to synthesise proteins or portions of proteins, either by manipulating their recombinant genes or by chemical synthesis, and to use these peptides in experimental assays or animal studies. This latter approach has the advantage that the mechanisms of action of the peptides provide models for the development of peptide or small molecule therapeutics. To date screens of short peptides have concentrated on libraries of random combinatorial peptides coupled to a functional screen, which have the advantage of screening a very large number of compounds. However, the next step of identifying the mechanism of action is often a limiting factor in further development of such peptides, since they only rarely correspond to biologically occurring linear peptide motifs. Thus, while such a screen identified the Asparagine- Glycine-Aspartic Acid (RGD) tripeptide motiffl], this had been previously been identified through biological studies as a signalling motif in fibrinogen and other proteins, and had already formed the basis for the design of small molecule RGD- mimetic drugs.

It is an object of the invention to provide a method of identifying bioactive peptides and proteins, particularly peptides and proteins which modulate platelet activity, or are involved in platelet signalling. Statements of Invention

According to the invention, there is provided a method of identifying bioactive peptides comprising the steps of:

providing a library of cell-specific, transmembrane, proteins; screening the library of proteins for peptides having the following characteristics:

between 3 and 20 amino acids in length; located in or overlapping a cytoplasmic tail and loop region of a transmembrane protein; having an N-terminal amino acid located not greater than 30 amino acids from the transmembrane region; located in a region aligned with a related region of a related human protein; containing a residue that is conserved in an orthologous protein, but different in a paralogous protein, wherein the or each residue has a Burst

After Duplication (BAD) score of 3 or greater, and the BAD scores are within the top 10% of BAD scores across all residue for the protein,

synthesising the peptides revealed by the screening step; optionally, for each peptide, synthesising a peptide from an aligned region of a paralogous protein; and assaying the synthesised peptides for bioactivity.

As used herein, the term "orthologous protein" means a protein which is equivalent to the protein containing the peptide of interest but separated by speciation (i.e. the equivalent protein in a different species).

As used herein, the term "paralogous protein" means a non-equivalent, related, protein not separated by speciation, i.e. related proteins which have arisen by duplication within the genome (i.e. related human proteins). A method of calculating a BAD score for any given residue is provided in the paper by Caffrey et al. [2].

The method of the invention is preferably a means of identifying proteins and peptides which are involved in modulating platelet activity or platelet signalling. In such cases, the library of proteins will be a library of platelet-specific, transmembrane, proteins. However, the method of the invention can likewise be utilised in identifying bioactive peptides or proteins which are involved in modulating the activity of other types of cells, such as, for example, neutrophils, megakaryocytes, inflammatory cells, cancer cells, endothelial cells, stem cells, neuronal cells, cardiac cells, bone cells, bone marrow cells, epithelial cells and hepatic cells.

Typically, the library of proteins is screened for peptides having at least 5, preferably at least 6, more preferably at least 7, more preferably at least 8, and ideally at least 9, amino acids. Suitably, the library of proteins is screened for peptides having at most 15, preferably at most 14, more preferably at most 13, more preferably at most 12, and ideally at most 11, amino acids. Ideally, the library is screened for peptides having approximately 10 amino acids.

The invention also relates to oligopeptides comprising peptides produced by the method of the invention, and additionally the use of such oligopeptides to modulate cellular, especially platelet activity. The invention also relates to the use of such oligopeptides in treating or preventing a condition or disease associated with thrombosis.

Oligopeptides

The method of identifying bioactive peptides revealed 18 short cytoplasmic regions of transmembrane platelet specific proteins which either activate or inhibit platelets. The sequences of these peptides is given in SEQUENCE ID NO 1 to 18.

Thus, the invention also relates to an isolated oligopeptide comprising: a peptide selected from the group comprising SEQUENCE ID NO 1 to 18; or a fragment or analogue of a peptide selected from the group comprising SEQUENCE ID NO 1 to 18.

As used herein, the term "oligopeptide" means an isolated amino acid sequence comprising, or consisting essentially of, a peptide selected from the group comprising SEQUENCE JD NO 1 to 18, or fragments or analogues of such peptides. However, the term should be taken to exclude the native proteins from which the peptides of any of Sequence ID No's 1 to 18 have been isolated. Details of these native proteins are provided in Table 1.

Typically, the oligopeptide will contain from 5 to 100 amino acids, suitably from 5 to 50 amino acids, preferably from 10 to 30 amino acids, more preferably from 10 to 20 amino acids, and ideally from 10 to 12 amino acids.

In a preferred embodiment, the oligopeptide, or the fragment or analogue thereof, is modified to make it cell permeable. Typically, the oligopeptide, or fragment or analogue thereof, is palmitylated by addition of a palmitylate group (H₃C-(CH₂)i₄- CO-). Methods of preparing palmitylated proteins or peptides will be well known to those skilled in the art.

A "fragment" of a peptide means a contiguous stretch of amino acid residues of at least 4 amino acids, typically at least 5 amino acids, preferably at least 6 amino acids, which retains the function of modulating platelet or other cell activity. Thus, taking SEQUENCE ID NO 1 (RRERRDLFTE) as an example, a fragment of this peptide could, for example, consist of any of the following non-exhaustive list of sequences (provided the fragment retained the function of modulating platelet or other cell activity): RRER; ERRD; DLFT; DLFTE; RRDLF; RDLFTE; and ERRDLFT.

An "analogue" of a peptide means a polypeptide modified by varying the amino acid sequence of one of the peptides of SEQUENCE ID NO's 1 to 18, or fragments thereof, e.g. by manipulation of the nucleic acid encoding the peptide or by altering the peptide itself. Such peptide analogues may involve insertion, addition, deletion and/or substitution of one or more amino acids, while providing a peptide capable of modulating platelet or other cell activity. Insertion, addition and substitution with natural and modified amino acids is envisaged.

Preferably such analogues involve the insertion, addition, deletion and/or substitution of 5 or fewer amino acids, more preferably of 4 or fewer, even more preferably of 3 or fewer, most preferably of 1 or 2 amino acids only.

Analogues also include derivatives of the above peptides, including the peptide linked to a coupling partner, e. g. an effector molecule, a label, a drug, a toxin and/or a carrier or transport molecule, or cyclised forms of the peptide. Methods of cyclising peptides will be known to those skilled in the field of protein chemistry. Techniques for coupling the peptides of the invention to both peptidyl and non- peptidyl coupling partners are well known in the art.

Analogues of and for use in the present invention further include reverse-or retro- analogues of natural peptides or their synthetic derivatives. See, for example, EP 0497 366, U.S. 5,519,115, and Merrifield et al., 1995, PNAS, 92:3449-53, the disclosures of which are herein incorporated by reference. As described in EP 0497 366, reverse peptides are produced by reversing the amino acid sequence of a naturally occurring or synthetic peptide. Reverse peptides are purported not only to retain the biological activity of the non-reversed "normal" peptide but may possess enhanced properties, including increased biological activity. (See Iwahori et al., 1997, Biol. Pharm. Bull. 20: 267-70).

Peptides (including reverse peptides, analogues and fragments thereof) of and for use in the invention may be generated wholly or partly by chemical synthesis or by expression from nucleic acid. The peptides of and for use in the present invention can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods known in the art (see, for example, 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). Cellular Activity Modulating Agents

The invention also relates to the use of an oligopeptide of the invention to modulate the activity of mammalian, especially human, cells, including megakaryocytes, inflammatory cells (inflammatory disease, i.e. RA), cancer cells (cancer), endothelial cells (vascular disease, hypertension), stem cells (direct development of pluripotent stem cells), neuronal cells (preventing cell death after stroke), cardiac cells (vascular diseases), bone cells (stimulating growth following trauma), bone marrow cells, epithelial cells (intestinal disease), hepatic cells (liver diseases), and vascular cells.

For each cell type, an example of the use of the peptides is given in brackets.

The invention also relates to a platelet activity modulating agent comprising an oligopeptide of the invention, or a fragment or analogue thereof.

The invention also relates to the use of an oligopeptide of the invention, or a fragment or analogue thereof, to modulate the activity of platelets.

The invention also relates to a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an oligopeptide according to the invention, or a fragment or analogue thereof.

The invention also relates to a method of modulating the activity of platelets in a mammalian subject comprising the step of administering to the mammal a suitable amount of an oligopeptide according to the invention, or a fragment or analogue thereof, or a pharmaceutical composition of the invention.

The invention also relates to a method of reducing or inhibiting platelet aggregation in a mammalian subject comprising the step of administering to the mammal a suitable amount of an oligopeptide according to the invention, or a fragment or analogue thereof, or a pharmaceutical composition of the invention.

The invention also relates to a method of treating or preventing a disease or condition associated with thrombosis comprising a step of administering to a subject a suitable amount of an oligopeptide according to the invention, or a fragment or analogue thereof. Diseases or conditions associated with thrombosis include Stroke, Atherosclerosis and Coronory Artery Disease, Ischemic Cerebrovascular Disease and Peripheral Vascular Disease.

Typically, the oligopeptide, fragment, or analogue is modified to allow cell permeability, typically by incorporation of a palmitylate group.

Research Tools

The invention also relates to the use of the oligopeptides of the invention, or fragments or analogues thereof, as research tools.

For example, as the peptides of SEQUENCE ID NO 1 to 18 have been found to modulate platelet activity, oligopeptides according to the invention, or fragments or analogues thereof, are used to screen a library of compounds (chemical or biological), or screen a cellular (i.e. platelet) lysate or cellular material preparation, to identify ligands which have the ability to modulate platelet activity. Means of designing such a screen will be well known to those skilled in the art. For example, the peptide of SEQUENCE ID NO 1 is modified to incorporate a HIS tag or a biotin moiety to facilitate immobilisation of the peptide to a support such as a 96 well plate. Each well in the plate is then be reacted with a different candidate compound, and then washed with a labelled moiety having a high binding affinity for the peptide bound to the support. The label may be, for example, FITC, carboxy-fluorescein or Cy-dye. Where the candidate compound binds to the peptide, reduced levels of fluorescence will be detected compared to a control.

Thus, in a further aspect, the invention provides a method of identifying ligands to the oligopeptides of the invention, which method comprises the step of contacting a putative ligand with an oligopeptide of the invention, or a fragment or analogue thereof, and determining whether the putative ligand has a binding affinity for the oligopeptide, or fragment or analogue thereof. Suitably, the putative ligand must have at least a mMolar binding affinity. The oligopeptides of the invention, or fragments or analogues thereof, are also used to screen a library of proteins to identify any proteins which interact with the oligopeptides. For example, a biotinylated version of the peptide of SEQUENCE ID NO 1 is used to screen a human foetal brain expression array to identify any proteins having a binding affinity, typically a high binding affinity, for the biotinylated peptide. Methods of performing such a screen are described in the paper by Larkin et al. [3]. Proteins identified by this screen form part of the present invention.

The invention also relates to oligopeptides of the invention, or fragments or analogues thereof, which incorporate a biotin moiety or a HIS tag. The biotin moiety may be linked to the oligopeptide through a photoactivatable group, such as, for example, benzoyl phenylalanine.

Proteins

As described above, the method of the invention revealed 18 short cytoplasmic regions of transmembrane platelet specific proteins which either activate or inhibit platelets. The sequences of these peptides is given in SEQUENCE ID NO 1 to 18. The sequence, and reference, of the native proteins containing the peptides of the invention are provided in Table 1 below. Thus, the 10 amino acid peptide of SEQUENCE ID NO. 2 is part of the Nectin 3 protein, the full sequence of which is provided in SEQUENCE ID NO 19.

Table 1

Peptide CSEO ID^') Gene/Protein Name Protein CSEO TD)

1 CD226

2 Nectin 3 19

3 ATP8A1 20

4 ABCR 21

5 ABCAl

6 VPPl 22

7 VPP4 23

8 CLCN7 24 9 ADCY4 25

10 Q8NDE1 26

10 Q8N219 27

10 Q9NSGS 28

11 EDG2

12 EDG7

13 KCNC3 29

14 KCNCl 30

15 PGRMCl 31

16 SPUF 32

17 PTGIR

18 SCN8A 33

Description of Proteins

CD226 is a platelet T-cell activation protein previously described [4,5]. The CD226- paralogous protein is the adhesion molecule Nectin 3 [6], which also acts as a herpesvivus receptor [7].

ATP8A1 is a potential phospholipid transporting ATPase IA (EC 3.6.3.1) previously described [8].

ABCR has been previously identified as a retinal specific ATP-binding cassette, with gene defects associated with retinal disease. No platelet specific role has previously been identified. The paralogous protein is encoded by the ABCl gene, which causes Tangiers disease, which is associated with platelet abnormalities [10].

VPPl is the 116 KlD subunit of the vacuolar proton pump, as determined by Swissprot database annotation (accession number Q93050; VPPl-HUMAN). While a related sequence has a role in the inhibition of endothelial cell proliferation [11], its specific role in cellular regulation is unknown. Mutations in the VPPl -paralogous protein, VPP4, are associated with tubular acidosis [12].

CLCN7 has been identified as a chloride channel protein [13]. ADCY4 is an adenylate cyclase [14], identified as the paralogue of ADCY7, which may be the predominant adenylate cyclase expressed in platelets [15].

The peptide of SEQUENCE ID NO 10 is derived from the paralogue of a platelet- associated protein of unknown function. The gene encoding the peptide has three protein sequences associated with it, with accession numbers Q8NDE1 (contains the peptide), Q8N219 (contains the peptide), and Q9NSG5 (fragment not containing the peptide).

EDG2-Human, and its paralogue EDG7, are both members of the seven transmembrane receptor lysophospholipid receptor family. Both proteins are expressed in platelets [16,17].

KNC3 and KNCl are potassium voltage-gated channel protein subunits Kv3.3 KSHIIID (KNC3) and Kv3.1/Kv4/NGK2 (KNCl). Mouse knock-out models of these genes have pronounced neurological disorders [18], but no platelet abnormalities are reported.

PGRMCl has been identified as a putative progesterone receptor [19]. The paralogous protein, SPUF, has had no functional assignments to date, and is identified by Swissprot database entry accession number Q9UMX5.

PTGER. is the prostacyclin receptor [20]. The PTGIR peptide 17 is 3 and 5 residues upstream of naturally palmitoylated cysteines, which may act to bring the region close to the membrance in the native protein [21].

SCN8A is a sodium channel subunit [22]. Mouse mutants of SCN8A exhibit neurological disorders [23-25].

Uses of Proteins

The invention also relates to a method of identifying a ligand for a protein selected from the group comprising SEQUENCE ID NO 19 TO 33, the method comprising the steps of: providing a candidate ligand; contacting a protein selected from the group comprising SEQUENCE ID NO 19 to 33 with the candidate ligand; and determining whether the candidate ligand binds to the protein. Using this method, a library of compounds may be screened to identify ligands for the proteins.

The proteins of SEQUENCE ID NO 19 TO 33, may also be used to screen a library of proteins to identify any interacting proteins. For example, a biotinylated version of the protein of SEQUENCE ID NO 19 may be used to screen a human foetal brain expression array to identify any proteins having a binding affinity, typically a high binding affinity, for the biotinylated protein. Methods of performing such a screen are described in the paper by Larkin et al [3]. Proteins identified by this screen form part of the present invention.

In a further aspect, the invention relates to the use of known agonists or antagonists of a protein selected from the group comprising SEQUENCE ID NO 19 TO 33 to modulate platelet activity. Persons skilled in the field of these proteins will be aware of agonists and antagonists of the proteins. For example, the sodium channel , SCN8A is antagonised by the naturally occuring wasp peptide beta-pompilidotoxin [26] and its derivatives [27]. Further, extracellular fragments of nectin-3 act as agonists and antagonists [28].

Combined Platelet Activation/Inhibition Assay

The invention also provides a method of assaying for modulation of platelet activity, comprising the steps of: contacting a platelet preparation with a putative platelet agonist/antagonist in a reaction vessel; assaying the mixture for platelet activation; adding a known platelet aggregating agent to the mixture; and monitoring the level of platelet aggregation.

Typically, the mixture is assayed for platelet activation by monitoring platelet aggregation. Generally, the known platelet aggregating agent is thrombin. Suitably, the assay is carried out on a multi-well plate. Detailed Description of the Invention

A fruitful approach in identifying bioactive peptides and proteins has been identified as modelling peptides directly on protein sequences [29]. Such oligopeptides derived from parent proteins can mimic the effect of the motif to either agonize or antagonize the parent protein's activity. However, some means of targeting a peptide to its site of action is desirable. Palmitoylated peptides are membrane associated, and have , been shown to modulate platelet signalling when a peptide modelled on the region inside the cytoplasmic membrane is targeted [30, 31], and this may account for the utility and robustness of such peptide models [3] . Platelet signalling is mediated through a number of identified and many unidentified receptors, many of which signal through changes in their associations with intracellular proteins.

To date such studies have been performed on a limited number of proteins, so the value of using this method for screening across many proteins was assessed. A combined bioinformatic and experimental screen of the cytoplasmic regions of transmembrane proteins (Table X in online material) was performed. The selection of peptides enriched for those that were evolutionarily conserved in comparison to orthologous sequences from other vertebrates (orthologues are the same protein in different species, separated by speciation). Secondly, we enriched for peptides with residues that, although conserved among orthologues, were strongly different from other related human proteins (termed paralogues: related proteins which have arisen by duplication within the genome). It is predicted that such residues are more likely to confer specificity within a protein family [2, 32]. The motifs analysed were 10 amino acid residues long, derived from the cytoplasmic tail and loop regions whose N-terminal side was within 50 amino acids of the transmembrane region.

The attached Figure is a flowchart illustrating the experimental design. In the Figure: X = platelet ligand; Y = universal ligand; Z = non-platelet ligand; dark grey segments are 10-mer peptide sequences of interest that are aligned between the platelet and paralogue proteins, overlapping at least one residue that exhibits apparent specificity; ticks represent peptide interaction mimicking that seen naturally for parental protein; crosses indicate peptide effect of paralogous peptide where region in parental protein would not normally interact with protein X. In order to quantify the specificity of the motifs effects acting on proteins within the protein superfamily, paralogous peptides were similarly synthesised from the aligned region of a paralogue. Hydrophobic peptides and those likely to cause problems of synthesis were excluded. These 52 decameric oligopeptides from 22 platelet proteins and their 22 paralogues were then used in aggregation and ADP- release assays of resting and thrombin-stimulated platelets. Agonists of platelet activation were defined as those that caused resting platelets to either secrete ADP or to aggregate, while antagonists were defined as those peptides that inhibited the thrombin-induced aggregation of platelet activation and ADP release. Statistical modelling compared the effects of each peptide compared to all other peptides, as well as to controls containing the medium in which each peptide was re-suspended.

129 likely transmembrane proteins were defined as being of interest in platelet biology, based on published literature, RNA array and proteomic survey studies. Of these, 91 had sufficient evolutionary information (related orthologues in other species and at least one paralogue within the human genome). From 22 of these proteins a total of 26 peptides were chosen, selecting peptides from aligned regions that included residues with apparent specificity in comparison with the paralogous protein. Peptides were synthesised chemically and a pamitylate group added to the N-terminus, permitting the targeting of the peptide to the transmembrane region[4, 8]. Peptides were then added to 96 well plate assays containing fresh platelets replicated across six donors at lOμm and 50 μm concentrations. Two assays of resting platelets tested for platelet agonists (Agg-R: level of aggregation of resting platelets; ADP-R: ADP release from resting platelets), while two assays of thrombin- activated platelets tested for platelet antagonists (Agg-TA: level of the aggregation of thrombin-activated platelets; ADP-TA: level of ADP release from thrombin-activated platelets).

Controlled comparisons of peptides are affected by insolubility, instability, and hydrophobicity, each of which may limit the activity of certain peptides, even if that peptide contains potentially active subsequences. Peptides that were strongly hydrophobic based on the Eisenberg scale [33], or were difficult to synthesise (containing dipeptide motifs DP, DC, DG, NG, or NS), were avoided. Some peptides were soluble in water, others required various concentrations of MeOH to dissolve, while more insoluble peptides required between 10% and 100% DMSO solvent. Insoluble peptides (indicated by strength of solvent required) were less likely to promote platelet activation measured by ADP release (r=0.75) and also less likely to inhibit ADP release in thrombin stimulated platelets (r=0.78), reflecting in part a direct effect of the solvent, since DMSO and water controls showed significant differences in platelet function (r=0.xx check stats on server). Accordingly, platelet activation was expressed as a proportion of the maximal activation seen for that vehicle for the same six platelet donors used for each peptide.

Peptide effects on platelet function are indicated in Table 2 below.

Experimental

1. Synthesis of Peptides

Materials

The Fmoc-protected amino acids, coupling reagents and the Rink Amide MBHA resin were purchased from Novabiochem. AU other reagents and solvents were purchased from Aldrich and used without further purification.

Peptide synthesis

The peptides were prepared by standard Solid Phase Peptide Synthesis [35,36] according to the Fmoc-tBu strategy [37,38] with HBTU/HOBt/DEEA coupling chemistry, in DMF solvent. Double coupling cycles, using a total 10-fold excess of Fmoc amino acid derivatives to resin-bound peptide, were employed. The side chain protecting groups were Acm for Cys; Boc for Lys and Trp; tBu, for Ser, Thr, and Tyr; O-tBu for Asp and GIu; Pbf, for Arg; Trt, for Asn, GIn and His. Assembly of the amino acid sequence starting from a Rink Amide MBHA resin and attachment of the N-terminal palmitic acid were carried out on a 40 mmol scale, on an automated parallel peptide synthesizer (Advanced ChemTech 396W). The peptides were deprotected and cleaved from the synthesis resin using a mixture of 81.5% trifluoroacetic Acid, 5% water, 1% triisopropylsilane, 10% thioanisole, 2.5% 1,2- ethanedithiol, at RT for 4 h. The peptides were precipitated and washed three times with diethyl ether. They were then dried, dissolved in distilled water and lyophilized. Chromatographic analysis and purification were performed on a Biocad Sprint Perfusion Chromatography Workstation (PerSeptive Biosystems) using POROS 20R2 Reversed Phase Perfusion Chromatography (4.6mmDxl00rnmL 1.7ml analytical column, 10 mmDxlOOmmL 7.9ml semi-preparative column); buffer A: 0.1% TFA in water, buffer B: 0.1% TFA in acetonitrile, linear gradient: 5 to 65% B in 30 min; flow rate: 4 ml/min (analysis) or 12 ml/min (semi-preparative); single wavelength detection at 214 nm.

The peptides were characterised by Matrix Assisted Laser Desorption Ionisation — Time Of Flight - Mass Spectrometry on a Bruker Reflex III (α-cyano-4-hydroxy- cinnamic acid matrix).

2. Platelet Activity Tests

As descibed above, two assays of resting platelets tested for platelet agonists (Agg-R: level of aggregation of resting platelets; ADP-R: ADP release from resting platelets), while two assays of thrombin-activated platelets tested for platelet antagonists (Agg- TA: level of the aggregation of thrombin-activated platelets; ADP-TA: level of ADP release from thrombin-activated platelets).

Washed platelets were prepared as described previously [31] and diluted in Buffer A (13OmM NaCl, 1OmM trisodum citrate, 9mM NaHCO₃, 6mM Dextrose, 0.9mM MgCl₂, 0.8ImM KH₂PO₄, 1.8mM CaCl₂Tris HCl pH 7.4) to a concentration of 6X10^s/ml. Peptides stock solutions (ImM) were prepared in an appropriate vehicle depending on individual solubility (H₂O, DMSO; 1% w/v or methanol; 5%w/v final concentration; See Table ??) and stored at -8O⁰C. Dual agonist-antagonist assessment was performed as follows: Platelets (80μl) and peptide pairs (lOμM and 50μM) or the relevant vehicle were added to a 96 well plate to a final volume of lOOμl and shaken at 37⁰C. Absorbance readings (405nm; 0.1 sec; Wallac Victor² 1420 spectrophotometer) were taken before addition of peptide (TO) and at subsequent 3 minute intervals. Thrombin (0.2U/ml) was added after 6 mins (T6) and an addidional 2 absorbance readings were taken at T9 and T12. A total of 6 donors (3 male and 3 female) were selected for each peptide.

For the ADP secretion assays, platelets were prepared as above but diluted to 3XlO⁸AnI and aliquoted into black and white 96 well isoplates. Peptide pairs (lOμM and 50μM) or the relevant vehicle were added and the plates shaken at 37oC. T₀ Luminescence reading at 405nm was obtained using the Wallac Victor² 1420 multi label counter from Perkin Elmer at 37⁰C. Chronolume (lOμl) was added after 3 mins and luminescence recorded to measure the peptide-induced ADP release. Parallel plates were prepared with platelets activated by 0.2U/ml thrombin. Changes in absorbance reflected antagonistic properties of peptides to inhibit thrombin-induced ADP release.

Platelet activation from all four endpoints was determined as

%Act = 100. (R - C,,) / (Q - C_n),

where R is the raw reading, C_n is the mean vehicle control reading before thrombin has been added, and C_t is the mean vehicle control after thrombin. Peptide %Act values were then compared for each peptide to all other peptides using a Mann- Whitney non-parametric test [39].

Table 2

Protein Assay Peptide p-value Activation SE Activity

VPPl ADP-R 6 0.0100 24.1% 9.62% Activator

VPP4 ADP-R 7 0.0053 35.7% 9.38% Activator

Nectin3 ADP-R 2 0.0020 35.8% 7.82% Activator

CLCN7 ADP-R 8 0.0093 31.9% 8.32% Activator

EDG7 ADP-R 12 0.0002 38.0% 5.60% Activator KCNCl ADP-R 14 0.0092 33.9% 8.78% Activator

PTGIR ADP-R 17 0.0000 59.2% 8.53% Activator

ABCR ADP-TA 4 0.0010 76.3% 4.67% Inhibitor Q8NDE1 ADP-TA lO 0.0043 79.6% 5.91% Inhibitor KCNC3 ADP-TA 13 0.0006 64.6% 11.79% Inhibitor KCNCl ADP-TA 14 0.0005 78.7% 3.56% Inhibitor

SPUF ADP-TA 16 0.0020 69.1% 12.33% Inhibitor SCN8A ADP-TA 18 0.0009 70.0% 8.79% Inhibitor

VPP4 Agg-R 7 0.0100 7.0% 4.86% Activator ADCY4 Agg-R 9 0.0004 5.6% 2.09% Activator PGRMCl Agg-R 15 0.0048 3.2% 2.21% Activator EDG2 Agg-R 11 0.0005 20.4% 15.50% Activator CD226 Agg-TA 1 0.0004 57.3% 8.99% Inhibitor KCNCl Agg-TA 14 0.0010 54.3% 10.61% Inhibitor ATP8A1 Agg-CA 3 (separate assay) Inhibitor

Activation indicates the mean % activation of the peptide across donors.

SE: Standard error of mean % activation. p-value: p-value from 2-tailed Mann-Whitney Test.

ADP-R: ADP release from resting platelets induced by peptide.

ADP-TA: inhibition by peptide of ADP release from thrombin-activated platelets.

Agg-R: aggregation of resting platelets induced by peptide.

Agg-TA: inhibition by peptide of aggregation of thrombin-activated platelets. Agg-CA: inhibition by peptide of aggregation of calcium ionophore (A23187) activated platelets.

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

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Claims

1. An isolated oligopeptide comprising the peptide of SEQUENCE ID NO

1, or a fragment or analogue thereof.

2. An isolated oligopeptide comprising the peptide of SEQUENCE ID NO

2, or a fragment or analogue thereof.

3. An isolated oligopeptide comprising the peptide of SEQUENCE ID NO

3, or a fragment or analogue thereof.

4. An isolated oligopeptide comprising the peptide of SEQUENCE ID NO

4, or a fragment or analogue thereof.

5. An isolated oligopeptide comprising the peptide of SEQUENCE ID NO

5, or a fragment or analogue thereof.

6. An isolated oligopeptide comprising the peptide of SEQUENCE ID NO

6, or a fragment or analogue thereof.

7. An isolated oligopeptide comprising the peptide of SEQUENCE ID NO

7, or a fragment or analogue thereof.

8. An isolated oligopeptide comprising the peptide of SEQUENCE ID NO

8, or a fragment or analogue thereof.

9. An isolated oligopeptide comprising the peptide of SEQUENCE ID NO 9, or a fragment or analogue thereof. 10. An isolated oligopeptide comprising the peptide of SEQUENCE ID NO

10, or a fragment or analogue thereof.

11. An isolated oligopeptide comprising the peptide of SEQUENCE ID NO

11, or a fragment or analogue thereof.

12. An isolated oligopeptide comprising the peptide of SEQUENCE ID NO

12, or a fragment or analogue thereof.

13. An isolated oligopeptide comprising the peptide of SEQUENCE ID NO

13, or a fragment or analogue thereof.

14. An isolated oligopeptide comprising the peptide of SEQUENCE DD NO

14, or a fragment or analogue thereof.

15. An isolated oligopeptide comprising the peptide of SEQUENCE ID NO

15, or a fragment or analogue thereof.

16. An isolated oligopeptide comprising the peptide of SEQUENCE ID NO

16, or a fragment or analogue thereof.

17. An isolated oligopeptide comprising the peptide of SEQUENCE ID NO

17, or a fragment or analogue thereof.

18. An isolated oligopeptide comprising the peptide of SEQUENCE ID NO

18, or a fragment or analogue thereof.

19. An isolated oligopeptide as claimed in any preceding Claim, having from 10 to 20 amino acids.

20. Use of an isolated oligopeptide as claimed in any of Claims 1 to 19 to modulate platelet activity.