WO2001094411A1 - Peptides modulating protease activated receptors and methods of using same - Google Patents

Abstract

The present invention provides PAR4 modulating polypeptides, PAR1 modulating polypeptides, polypeptides that modulate both PAR1 and PAR4, polynucleotides encoding these polypeptides, and methods using these modulating polypeptides. The PAR1 and PAR4 modulating peptides may be used to modulate platelet activation. Further, the invention provides methods for screening agents which may modulate PAR1 or PAR4 or platelet or any combination thereof. The invention also provides methods to specifically modulate PAR4-mediated activation through modulating the Gq signaling pathway.

Description

PEPTIDES MODULATING PROTEASE ACTIVATED RECEPTORS AND METHODS OF USING SAME

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH This work was supported in part by grants from the National Institutes of Health (HL 44907 and HL59202). The government may have certain rights in the invention.

TECHNICAL FIELD This invention relates to the field of molecular biology, more particularly to polypeptides which modulate protease-activated receptors PARI and/or PAR4.

BACKGROUND ART

Platelet activation is critical for normal hemostasis and plays key roles in tissue remodeling, injury and inflammatory stimuli. Platelet-dependent arterial thrombosis underlies most myocardial infarctions. Insufficient activation of platelets causes disorders such as hemophilia.

Thrombin is the most potent activator of platelets. Davey and Luscher (1967) Nature 216:857-858; Berndt and Phillips (1981) In Platelets in biology and pathology (Gordon, ed. Elsevier Amsterdam, the Netherlands) 43-74. Thrombin, a coagulation protease generated at sites of vascular injury, activates platelets, leukocytes, and mesenchymal cells. Vu et al. (1991a) Cell 64:1057-1068. Activation of platelets by thrombin is thought to be critical for hemostasis and thrombosis. In animal models, thrombin inhibitors block platelet-dependent thrombosis, which is the cause of most heart attacks and strokes in humans. Available data in humans suggest that thrombosis in arteries can be blocked by inhibitors of platelet function and by thrombin inhibitors. Thus it is likely that thrombin's actions on platelets contribute to the formation of clots that cause heart attack and stroke. Thrombin's other actions on vascular endothelial cells and smooth muscle cells, leukocytes, and fibroblasts may mediate inflammatory and prohferative responses to injury, as occur in normal wound healing and a variety of diseases (atherosclerosis, restenosis, pulmonary inflammation (ARDS), glomerulosclerosis, etc.). A thorough understanding of how thrombin activates cells is an important goal.

Characterization ofthe receptors that mediate thrombin's actions on platelets is therefore necessary for understanding hemostasis and thrombosis. Moreover, such receptors are potential targets for novel antiplatelet therapies.

Receptors are cell-associated proteins that bind to a bioactive molecule (i.e., a ligand) and mediate the effect ofthe ligand on the cell. Membrane-bound receptors are characterized by a multi-domain structure (also sometimes referred to as a "multi-peptide", wherein subunit binding and signal transduction can be functions of separate subunits) comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell. This interaction in turn leads to an alteration in the metabolism ofthe cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. Thrombin signaling is mediated at least in part by a family of G-protein-coupled protease-activated receptors (PARs), for which PARI is the prototype. Vu et al. (1991a); USP 5,256,766; Rasmussen et al. (1991) FEBS Lett. 288:123-128. Thrombin activates PARI by binding to and cleaving PARI amino-terminal exodomain at the R41/S42 peptide bond. The cleavage serves to unmask a new receptor amino terminus beginning with the sequence SFLLRN. This new amino terminus then serves as a tethered peptide ligand, binding intramolecularly to the body ofthe receptor to effect transmembrane signaling. Vu et al. (1991a); Vu et al. (1991b) Nature 353:674-677; Chen et al. (1994) J Biol. Chem. 269:16041-16045. PARs are thus in essence peptide receptors that carry their own ligands, which remain silent until unmasked by site-specific receptor cleavage. The role of PARs in platelet activation is evolving rapidly. Four distinct PARs are now known. PARI, PAR3, and PAR4 can be activated by thrombin. Vu et al. (1991a); Kahn et al. (1998) Nature 394:690-694; Ishihara et al. (1997) Nature 386:502-506; Xu et al. (1998) Proc. Natl. Acad. Sci. USA 95:6642-6646; WO 99/50415; Dery and Bunnett (1999) Biochem. Soc. Trans. 27:246-254. PAR2 is activated by trypsin and trypsin-like enzymes. Nystedt et al. (1994) Proc. Natl. Acad. Sci. USA 91 :9208-9212. PARI mRNA and protein were detected in human platelets. Vu et al. (1991a); Hung et al. (1992a) J. Clin. Invest. 89:444-450; Brass et al. (1992) J. Biol. Chem. 267:13795-13798; Molino et al. (1997) J. Biol. Chem. 272:6011-6017. PARI -blocking antibodies inhibited human platelet activation triggered by low but not high concentrations of thrombin. Hung et al. (1992a); Brass et al. (1992). These data suggested a role for

PARI in activation of human platelets by thrombin but held open the possibility that other receptors might contribute. PARI appeared to play no role in mouse platelet activation. PARI expression was difficult to detect in rodent platelets, and PARI -activating peptides did not activate rodent platelets. Moreover, platelets from PARI -deficient mice responded like wild-type platelets to thrombin. PAR3 is expressed in mouse platelets but could not be detected in human platelets. Ishihara et al.; Kahn et al. (1999) J. Clin. Invest. 103:879-887. Inhibition of PAR3 function with antibodies that bound to PAR3's hirudin-like domain or by gene knockout prevented mouse platelet activation triggered by low but not high concentrations of thrombin. Kahn et al. (1998); Ishihara et al. (1998) Blood 91:4152-4157. These results established that P AR3 is necessary for normal thrombin signaling in mouse platelets but also pointed to the existence of another platelet thrombin receptor. Such a receptor, PAR4, was recently identified. Kahn et al. (1998); Xu et al. (1998); WO99/43809; WO99/50415. See also WO 98 31810 A. PAR4 appears to function in both mouse and human platelets. Kahn et al. (1998); Xu et al. (1998); WO 99/50415; Nystedt et al. (1994). Thus in both mouse and human, platelets utilize two thrombin receptors. A

"high-affinity" thrombin receptor (PARI in human, PAR3 in mouse) is necessary for responses to low concentrations of thrombin, whereas a "low-affinity" receptor (PAR4 in both species) mediates responses at higher concentrations of thrombin.

Pharmacological studies of human platelets suggest that PARI and PAR4 account for thrombin activation of platelets. Activation of either PARI and PAR4 triggered platelet activation. Inhibition of PARI function alone — whether by blocking antibody, antagonist, or desensitization — inhibited platelet responses at 1 nM thrombin but only slowed responses at 30 nM thrombin. Inhibition of PAR4 function alone with a blocking antibody had no effect at either concentration. Strikingly, combined inhibition of PARI and PAR4 signaling profoundly inhibited platelet responses even at high concentrations of thrombin.

Kahn et al. (1999). The synthetic peptide SFLLRN, which mimics the first six amino acids ofthe new PARI amino terminus unmasked by receptor cleavage, functions as an agonist for PARI and activates the receptor independently of thrombin and proteolysis. Vu et al. (1991a); Vassallo et al. (1992) J. Bio. Chem. 267:6081-6085; Scarborough et al. (1992) J. Bio. Chem. 26'/ ^r : 13146- 13149. Such peptides have been used as pharmacological probes of

PARI function in various cell types, as well as a starting point for PARI antagonist development. Andrade-Gordon et al. (1999) Proc. Natl. Acad. Sci. USA 96:12257-12262. The cognate PT-P6' peptides of other PARs have been useful as agonists for probing the role of these receptors in various cellular responses. Hung et al. (1992b) J. Cell Biol. 116:827-832; Nystedt et al.(1994) Proc. Natl. Acad. Sci. USA 91 :9208-9212; Kahn et al.

(1998).

PAR4 is expressed in human platelets along with PARI. Kahn et al. (1998); Xu et al. (1998); Kahn et al. (1999); WO99/50415. PAR4 is activated when thrombin cleaves its amino terminal exodomain at the R47/G48 peptide bond to unmask the tethered ligand GYPGQV. Kahn et al. (1998); Xu et al. (1998); WO99/50415. The synthetic peptide

GYPGQV functions as an agonist for PAR4, but a concentration of 200-500 μM is required for activity. This lack of potency severely limits the utility of this peptide for probing PAR4 function in culture systems and virtually precludes its use in vivo. Structure-function relationships for the PAR4 tethered ligand have not been explored. The identification ofthe receptors that mediate platelet activation by thrombin raises important questions regarding strategies for the development of antithrombotic therapies. Kahn et al. (1999); Bernatowicz et al. (1996) J Med. Chem. 39:4879-4887. The observation that PARI inhibition blocked platelet responses to low concentrations of thrombin and slowed responses to high concentrations raises the question of whether PARI inhibition alone might be sufficient for an antithrombotic effect. Kahn et al. (1999); Cook et al. (1995) Circulation 91:2961-2971. Alternatively, it may be necessary to block both PARI and PAR4 to prevent or arrest thrombosis in vivo. However, agents to carry out such function to modulate PARI and PAR4 simultaneously are nonexistent in the art.

In order to elucidate clearly the in vivo roles of PARs and to develop therapeutically useful PAR-targeted drugs, there is a need to synthesize potent PAR modulators that are specific for one or more receptor subtypes. In view ofthe alarming prevalence of life-threatening diseases such as heart attack, stroke, atherosclerosis, resternosis, pulmonary inflammation (ARDS), and glomerulosclerosis, and given the role of platelet activation in hemostasis, thrombosis, and normal wound healing, there is a pressing need for developing more effective therapeutic agents and methods to modulate PAR function. There is also a pressing need for a single agent and/or method that modulates both PARI and PAR4 functions.

All publications and patent applications cited herein are hereby incorporated by reference in their entirety.

DISCLOSURE OF THE INVENTION

The invention provides peptide compositions that modulate PAR4, peptide compositions that modulate PARI, peptide compositions that modulate both PARI and PAR4, as well as methods for using these compositions, such as methods for modulating PARI and or PAR4 activity and in methods for identifying agents that modulate PARI and/or PAR4 activity, such as screening assays.

Accordingly, in one aspect, the invention provides peptides AYPGKF, SYPGKF, TYPGKF, GFPGKF, G(F)PGKF, GYPAKF, GYP(Cha)KF, GYPLKF, GYPIKF, S(F)(Cha)(Cha)(homoR)K, S(F)(Cha)(Cha)RK, GYPGRF, GYPG(homoR)F, GYPG(Orn)F, GYPGKY, GYPGKW, GYPGKK, SYPAKF, SYAGKF, SYPGRF, and SYPG(homoR)F. In some embodiments, said peptides present higher potency than the native PAR4 or PARI tethered ligand peptides to modulate activity (based on thrombin mediated activation). Other peptides are provided in Table 1.

In another aspect, the invention provides polynucleotides (including isolated naturally-occurring and non-naturally occurring polynucleotides) encoding any ofthe peptide embodiments of this invention. The polynucleotides may be isolated, chemically synthesized, in cloning or expression vectors, and/or in suitable host cells.

In another aspect, the invention provides compositions comprising any ofthe peptide and polynucleotide embodiments described herein. In some embodiments, the compositions also contain a pharmaceutically acceptable excipient. In some embodiments, an effective amount of a peptide is contained within a pharmaceutical composition, wherein an effective amount is an amount sufficient to modulate PARI and/or PAR4 activity and/or platelet activity.

In another aspect, the present invention provides methods for identifying an agent that modulates PAR4 activity, said method comprising (a) contacting a suitable host cell comprising PAR4 function with said agent to be tested and a PAR4 activating peptide selected from the group consisting of AYPGKF, SYPGKF, TYPGKF, GFPGKF and G(F)PGKF, GYPAKF, GYPGRF, GYPG(homoR)F, GYPG(Orn)F, GYPGKY, GYPGKW, GYPGKK, SYPAKF, SYAGKF, SYPGRF, SYPG(homoR)F, wherein (homoR) is homoarginine and (Orn) is omithme; and (b) analyzing at least one characteristic which is associated with PAR4 function in said host cell, wherein an agent is identified by its ability to modulate at least one such characteristic as compared to contacting the suitable host cell with said PAR4 activating peptide without the agent.

In yet another aspect, the present invention provides a method for identifying an agent that modulates PARI activity, said method comprising (a) contacting a suitable host cell comprising PARI function with said agent to be tested and a PARI activating peptide selected from the group consisting of GYPAKF, GYP(Cha)KF, GYPLKF, GYPIKF, S(F)(Cha)(Cha)(homoR)K, and S(F)(Cha)(Cha)RK, GFPGKF, and G(F)PGKF, wherein (Cha) is cyclohexylalanine, (F) is parafluoro-phenylalanine, and (homoR) is homoarginine; and (b) analyzing at least one characteristic which is associated with PARI function in said host cell, wherein an agent is identified by its ability to modulate at least one such characteristic as compared to contacting the suitable host cell with said PARI activating peptide without said agent.

In an additional aspect, the present invention provides a method for identifying an agent that modulates both PARI and PAR4 activity, said method comprising, (a) contacting a suitable host cell comprising PARI function with said agent to be tested and a PARI activating peptide selected from the group consisting of GYPAKF, GYP(Cha)KF, GYPLKF, GYPIKF, S(F)(Cha)(Cha)(homoR)K, S(F)(Cha)(Cha)RK, GFPGKF and G(F)PGKF wherein (Cha) is cyclohexylalanine, (F) is parafluoro-phenylalanine, and (homoR) is homoarginine, and analyzing at least one characteristic which is associated with PARI function in said host cell, wherein an agent is identified by its ability to modulate at least one such characteristic as compared to contacting the suitable host cell with said PARI activating peptide without the agent; (b) contacting a suitable host cell comprising PAR4 function with said agent to be tested and a PAR4 activating peptide selected from the group consisting of GYPGRF, GYPG(homoR)F, GYPG(Orn)F, GYPGKY, GYPGKW, GYPGKK, SYPAKF, SYAGKF, SYPGRF, SYPG(homoR)F, AYPGKF, SYPGKF, TYPGKF, GFPGKF, and G(F)PGKF, wherein (homoR) is homoarginine and (Orn) is ornithine, and analyzing at least one characteristic which is associated with PAR4 function in said host cell, wherein an agent is identified by its ability to modulate at least one such characteristic as compared to contacting the suitable host cell with said PAR4 activating peptide without the agent; and (c) selecting an agent that modulates at least one characteristic associated with PARI function and at least one characteristic associated with PAR4 function, wherein the steps (a) and (b) are performed in any order or simultaneously.

In some embodiments, the host cell has native PAR4 and/or PARI function. In other embodiments, the host cell lacks native PAR4 and/or PARI function and comprises a recombinant polynucleotide encoding PAR4 and/or PARI or a functional fragment thereof, wherein PAR4 and/or PARI function is restored in said host cell. In another embodiment, the host cell is a platelet cell. In other embodiments, the characteristic wliich is associated with PAR4 and/or PARI function is phosphoinositide hydrolysis, intracellular calcium mobilization, platelet shape change, platelet ATP secretion or platelet aggregation.

In further aspects, the present invention provides methods for identifying an agent that antagonizes PAR4 activity, said method comprising, (a) contacting a suitable host cell comprising PAR4 function with said agent to be tested and a PAR4 activating peptide selected from the group consisting of GYPGRF, GYPG(homoR)F, GYPG(Orn)F, GYPGKY, GYPGKW, GYPGKK, SYPAKF, SYAGKF, SYPGRF, SYPG(homoR)F, AYPGKF, SYPGKF, TYPGKF, GFPGKF, and G(F)PGKF, wherein (homoR) is homoarginine and (Orn) is ornithine, and (c) analyzing at least one characteristic associated with the inhibition of PAR4 activation in said host cell, wherein an agent is identified by its ability to modify at least one such characteristic as compared to contacting the suitable host cell with said PAR4 activating peptide without the agent.

In an additional aspects, the present invention provides methods for identifying an agent that antagonizes PARI activity, said method comprising (a) contacting a suitable host cell comprising PARI function with said agent to be tested and a PARI activating peptide selected from the group consisting of GYPAKF, GYP(Cha)KF, GYPLKF, GYPIKF, S(F)(Cha)(Cha)(homoR)K, S(F)(Cha)(Cha)RK, GFPGKF and G(F)PGKF wherein (Cha) is cyclohexylalanine, (F) is parafluoro-phenylalanine, and (homoR) is homoarginine; and (b) analyzing at least one characteristic associated with the inhibition of PARI activation in said host cell, wherein an agent is identified by its ability to modify at least one such characteristic as compared to contacting the suitable host cell with said PARI activating peptide without the agent.

In additional aspects, the present invention provides methods for identifying an antagonist of both PARI and PAR4 activity, said method comprising (a) contacting a suitable host cell comprising PARI function with said agent to be tested and a PARI activating peptide selected from the group consisting of GYPAKF, GYP(Cha)KF, GYPLKF, GYPIKF, S(F)(Cha)(Cha)(homoR)K, S(F)(Cha)(Cha)RK, GFPGKF and G(F)PGKF wherein (Cha) is cyclohexylalanine, (F) is parafluoro-phenylalanine, and (homoR) is homoarginine and analyzing at least one characteristic associated with the inhibition of PARI activation in said host cell, wherein an agent is identified by its ability to modify at least one such characteristic as compared to contacting the suitable host cell with said PARI activating peptide without the agent; (b) contacting a suitable host cell comprising PAR4 function with said agent to be tested and a PAR4 activating peptide selected from the group consisting of GYPGRF, GYPG(homoR)F, GYPG(Orn)F, GYPGKY, GYPGKW, GYPGKK, SYPAKF, SYAGKF, SYPGRF, SYPG(homoR)F, AYPGKF, SYPGKF, TYPGKF, GFPGKF, and G(F)PGKF, wherein (homoR) is homoarginine and (Orn) is ornithine and analyzing at least one characteristic associated with the inhibition of PAR4 activation in said host cell, wherein an agent is identified by its ability to modify at least one such characteristic as compared to contacting the suitable host cell with said PAR4 activating peptide without the agent; and (c) selecting an agent that modifies at least one characteristic associated with the inhibition of PARI activation and at least one characteristic associated with the inhibition of PAR4 activation, wherein the steps (a) and (b) are performed in any order or simultaneously.

In some embodiments, the host cell has native PAR4 and/or PARI function. In other embodiments, the host cell lacks native PAR4 and/or PARI function and comprises a recombinant polynucleotide encoding PAR4 and/or PARI or a functional fragment thereof, ₍

wherein PAR4 and/or PARI function is restored in said host cell. In other embodiments, the host cell is a platelet cell.

In other embodiments, the characteristic associated with PAR4 and or PARI function is phosphoinositide hydrolysis, intracellular calcium mobilization, platelet shape change, platelet ATP secretion or platelet aggregation.

In other aspects, the present invention provides kits comprising an isolated peptide selected from the group consisting of GYPAKF, GYP(Cha)KF, GYPLKF, GYPIKF, S(F)(Cha)(Cha)(homoR)K, and S(F)(Cha)(Cha)RK, wherein (Cha) is cyclohexylalanine, (F) is parafluoro-phenylalanine, and (homoR) is homoarginine wherein the isolated peptide activates PARI . In additional aspects, the present invention provides kits comprising an isolated peptide selected from the group consisting of GYPGRF, GYPG(homoR)F, GYPG(Orn)F, GYPGKY, GYPGKW, GYPGKK, SYPAKF, SYAGKF, SYPGRF, SYPG(homoR)F, wherein (homoR) is homoarginine and (Orn) is ornithine, wherein the isolated peptide activates PAR4. In another aspect, the invention provides methods for modulating PARI , PAR4 and/or platelet activity comprising the administration of peptides disclosed herein. Accordingly, the present invention provides method for modulating PARI activity in an individual, comprising administering an effective amount of an agent comprising a peptide selected from the group consisting of GYPAKF, GYP(Cha)KF, GYPLKF, GYPIKF, S(F)(Cha)(Cha)(homoR)K, S(F)(Cha)(Cha)RK, GFPGKF and G(F)PGKF to said individual, wherein (Cha) is cyclohexylalanine, (F) is parafluoro-phenylalanine, and (homoR) is homoarginine. In another aspect, the present invention provides methods for modulating PAR4 activity in an individual, comprising administering an effective amount of an agent comprising a peptide selected from the group consisting of GYPGRF, GYPG(homoR)F, GYPG(Orn)F, GYPGKY, GYPGKW, GYPGKK, SYPAKF, SYAGKF,

SYPGRF, SYPG(homoR)F, AYPGKF, SYPGKF, TYPGKF, GFPGKF, and G(F)PGKF to said individual, wherein (homoR) is homoarginine and (Orn) is ornithine. In another embodiment, the present invention provides a method for modulating both PARI and PAR4 activity in an individual, comprising administering an effective amount of an agent comprising peptides ofthe present invention disclosed herein that activate both PARI and PAR4. In yet other embodiments, the present invention provides methods for modulating platelet activation in an individual, comprising administering an effective amount of an agent comprising a peptides ofthe present invention disclosed herein to said individual.

In another embodiment, the invention provides methods of inhibiting inappropriate clotting in an individual comprising administering an amount of an antagonist (which could be for example, a peptide disclosed herein that does not show receptor activating capability) effective to inhibit inappropriate clotting, such as in the treatment of disorders such as myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, and other blood system thromboses. Any PARI and/or PAR4 antagonist/inhibitors as discovered by screening methods disclosed herein would also be suitable.

In another embodiment, the invention provides methods of activating blood clotting in an individual comprising administering an amount of a PARI and/or PAR4 activating peptide effective to activate clotting, such as in the treatment of disorders involving insufficient clotting. The dual action of peptide compositions disclosed herein on both PARI and PAR4 may increase the activation of platelets. Alternatively, a sample may be taken from an individual (e.g. a blood sample), treated ex vivo with a peptide composition ofthe invention that activates blood clotting, and returned to the individual.

In another aspect, the invention provides methods for selectively modulating PAR4- activated platelet activity and function through specifically modulating protein Gq activity and/or function.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph depicting intracellular calcium mobilization in response to α- thrombin (30 nM) and synthetic agonist peptides AYPGKF peptide (500 μM), SYPGKF peptide (500 μM), or SFLLRN (100 μM) in PARI or PAR4 expressing cells.

Figure 2 is a graph depicting phosphoinositide hydrolysis in response to PAR4 activation. KOLF-PAR4 cells were treated with either α-thrombin (30 nM) (filled square), or the indicated concentrations of GYPGKF (open squares), AYPGKF (open triangles), or SYPGKF (open circles) in the presence of LiCl (20 mM) for 1 hour at 37°C. Values from untreated cells (ranging from 500-1,200 cpm in different experiments) were subtracted as background. Figure 3 is a graph depicting intracellular calcium mobilization of KOLF-PAR4 in response to increased concentrations of AYPGKF. Concentrations (from bottom curve to top curve) were 1, 5, 10, 100, and 500 μM.

Figures 4A and 4B are graphs depicting the effect of thrombin on forskolin- stimulated adenylyl cyclase activity in KOLF-PARl cells (Figure 4A) and KOLF-PAR4 cells (Figure 4B). 3H-adenine-loaded KOLF-PARl (Figure 4A) or KOLF-PAR4 (Figure 4B) were loaded with 3H-adenine as described in Example 3, and treated with 50 μM forskolin in the presence ofthe indicated concentrations of α-thrombin for 30 min at 37°C. Prior to agonist treatment, cells were either left untreated (closed circles) or treated (open circles) with pertussis toxin (100 ng/ml) for 5 hours at 37°C. Data presented are 3H-cAMP as a fraction of 3H-cAMP + 3H-ATP (mean ± S.E.; n=3).

Figure 5 are graphs depicting phosphoinositide hydrolysis in response to α- thrombin or agonist peptides in the presence of increasing concentrations of pertussis toxin. KOLF-PAR4 or KOLF-PARl were incubated with the indicated concentration of pertussis toxin for 5 hours at 37°C, then either left untreated (open triangles), treated with α- thrombin (30 nM) (open circles), or treated with the receptor agonist peptides (open squares) GYPGKF (500 μM) for PAR4-expressing cells or SFLLRN (100 μM) for PAR1- expressing cells. Agonist treatment was for 1 hour at 37°C in the presence of LiCl (20 mM). Total (3H)-inositol phosphates released (mean ± S.E.; n=3) was quantitated as described in Example 1.

Figure 6 are graphs depicting aggregation and ATP secretion in human platelets in response to PARI and PAR4 agonist peptides. AYPGKF, GYPGKF, and SFLLRN were added to human platelet-rich plasma at the indicated concentrations (μM) and aggregation (left) and secretion (right) followed in a lumiaggregometer. Figure 7 is a bar graph depicting calcium mobilization in human platelets following

PAR4 desensitization. Washed human platelets were loaded with FURA-2/AM and agonist-triggered increases in cytosolic calcium measured fluorometrically as described in

Example 1. Platelets were either left untreated prior to measurement (gray bars) or were pretreated with the PARI -antagonist peptide BMS200261 (100 μM) for 5 min and AYPGKF (500 μM) for 30 min at room temperature (white bars). These platelets were then stimulated with a saturating concentration of agonist as indicated: α-thrombin (30 nM), SFLLRN (100 μM), AYPGKF (500 μM), or U46619 (10 μM).

Figure 8 are graphs depicting mouse platelet secretion and aggregation in platelet- rich plasma. Platelet-rich plasma was prepared from wild-type C57BL6 mice, and agonist- triggered ATP secretion (right) and aggregation (left) were measured as a function of time in response to indicated concentrations (in μM) of AYPGKF, GYPGKF, or the thromboxanc receptor agonist U46619. Note that the native PAR4 agonist GYPGKF triggered minimal aggregation (bottom left) and no detectable secretion even at 500 μM.

MODES FOR CARRYING OUT THE INVENTION

We have discovered and synthesized novel peptides that function as potent activators, i.e., agonists, of protease-activated receptor 4 (PAR4), protease-activated receptor 1 (PARI), and both PAR4 and PARI.

Significantly, some peptides described herein are more potent than the native tethered ligand peptides GYPGKF in mouse and GYPGQV in human with respect to activation of PAR4. For example, the peptide AYPGKF activated and desensitized PAR4 in platelets and, like thrombin, triggered phosphoinositide hydrolysis but not inhibition of adenylyl cyclase in PAR4-expressing cells. When combined with PARI inhibition, desensitization of PAR4 signaling by prolonged incubation with AYPGKF effectively blocked platelet activation by thrombin. This observation supports the model that PARI and PAR4 together account for most if not all platelet activation by thrombin. Prior to our discovery ofthe extent ofthe activity of these peptides, potent agonists were virtually nonexistent for PAR4, which hindered both the research of PAR4 function in culture systems and in vivo therapy of platelet-activation related disorders and diseases. Accordingly, the peptides are useful tools for probing PAR4 function and signaling in culture systems and in platelets, and therefore useful for the development of therapeutic treatments of platelet- activation related disorders and diseases.

We have also discovered that certain peptides that function as potent activators, i.e., agonists, of both PARI and PAR4 which account for virtually all activation of human platelets. Thus, modulating both PARI and PAR4 with a single agent is desirable. However, such agents were non-existent prior to our discovery. Our discovery of identifying a single agonist for both PARI and PAR4 raises the possibility that a single antagonist for both receptors might be developed using, for example, the screening assays described herein. These peptides which modulate both PARI and PAR4 are useful tools for probing both PARI and PAR4 functions and signaling in culture systems and in platelets, and therefore for developing therapeutic treatments of platelet-activation related disorders and diseases.

We discovered several stmcture-function relationships of PARI and PAR4 agonists. First, the PAR4 tethered ligand' s amino terminal protonated amino group appears to be important for agonist activity with respect to PAR4, consistent with the notion that the proteolytic switch that activates the tethered ligand involves not only removal of sequence amino to the scissile bond between R47 and G48 but also generation of a new protonated amino group at the nitrogen that was part of that peptide bond. This suggests that the proteolytic switch in PARs utilizes a common mechanism. Second, the basis for agonist specificity for PAR4 vs. PARI was in part attributable to the tyrosine at position 2 in GYPGKF and AYPGKF. Several peptides in which this tyrosine was substituted by phenylalanine or para-fluorophenylalanine were capable of acting at both PARI and PAR4.

We have also discovered that PAR4 couples to Gq but not to G[. In contrast, the prototype of PARs, PARI, couples to G-proteins ofthe Gi, Gq and Gi2/13 subfamilies. Elevated cAMP levels inhibit platelet activation, hence PARl's ability to activate G[ and inhibit adenylyl cyclase (in addition to activating Gq) may contribute to its effectiveness in activating human platelets. Conversely, PAR4's lack of ability to activate Gj may be a factor in its apparent lesser role in thrombin-triggered platelet activation. Our discovery differentiates PAR4 signaling from PARI signaling, and allows to specifically modulate PAR4-mediated platelet activation via the PAR4-Gq signaling pathway. Accordingly, the invention provides a method for specifically activating PAR4 mediated signaling pathway, said method comprising administering an effective amount of a protein Gq signaling pathway modulator wliich does not modulate Gi activity.

Collectively, these results further understanding of platelet activation and thrombin signaling and provide valuable tools. A single peptide that activates both PARI and PAR4 can be used to promote platelet activation in contexts in wliich enhancement of clotting is desirable, such as wound healing.

Further, the peptides disclosed herein can be used in screening assays to identify agents which modulate PARI and/or PAR4 function. A single agent that modulates, particularly inhibits, both PARI and PAR4 can be used as a drug which would be useful in preventing or treating conditions caused by thrombosis or involving platelet activation by thrombin. These conditions include but are not limited to myocardial infarction and unstable angina, stroke and transient ischemic attacks, pulmonary embolism and deep venous thrombosis, platelet activation in extracorporeal circulation, diffuse intravascular coagulation, and thromboembolism secondary to atrial arehythmia or ventricular dysfunction. General Techniques

The practice ofthe present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill ofthe art. Such techniques are explained fully in the literature, such as: Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ. Gait, ed., 19 4); Animal Cell Culture (R.I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Wei & C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller & M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Platelets, A Practical Approach (Watson and Authi eds., 1996).

Definitions

"Protease-activated receptor 4", "PAR4", "PAR4 receptor" and the like, used interchangeably herein, refer to all or part of a vertebrate cell surface protein which is specifically activated by thrombin or a thrombin agonist or cathepsin G thereby activating PAR4 mediated signaling events (effector functions, e.g., phosphoinositide hydrolysis, Ca²⁺ efflux, platelet aggregation). PAR4 is characterized as having the properties (including the agonist activating and the antagonist inhibiting properties) associated with PAR4 function (such as effector functions, as described herein) known in the art, described herein and in US Application No. 09/032,397 and 09/360,482; Kahn et al. (1998); Xu et al. (1998); Kahn et al. (1999); WO 99/43809; WO 99/50415; WO 98 31810 A;

PCT/US99/19158. PAR4 may refer to a naturally occurring form ofthe receptor and/or a recombinantly produced form ofthe receptor (or, when suitable, a fragment thereof). In addition, the term may include variants of PAR4 that retain at least one activity and/or property of naturally-occurring PAR4. "Protease-activated receptor 1 ", "PARI ", "PARI receptor" and the like, used interchangeably herein, refer to all or part of a vertebrate cell surface protein which is specifically activated by thrombin or a thrombin agonist thereby activating PAR-1 mediated signaling events (effector functions, e.g., phosphoinositide hydrolysis, Ca²⁺ efflux, platelet aggregation). PARI is characterized as having the properties (including the agonist activating and the antagonist inhibiting properties) known in the art, described herein and in Vu et al. (1991a). See also PCT/US92/01312 and U.A. 5,256,766. PARI may refer to a naturally occurring form ofthe receptor and/or a recombinantly produced form ofthe receptor (or, when suitable, a fragment thereof). In addition, the term may include variants of PARI that retain at least one activity and/or property of naturally- occurring PARI.

A "functionally preserved" variant or "functionally equivalent" variant of PAR4 (or polynucleotide encoding PAR4) or PARI (or polynucleotide encoding PARI) is a PAR4, or PARI, sequence which retains at least one aspect of PAR4 or PARI function, respectively. Functionally preserved variants may arise, for example, by conservative and/or non-conservative amino acid substitutions, amino acid analogs, and deletions. The function that is preserved depends upon the relevant function being considered. For example, if a PAR4, or PARI, polypeptide is considered for its ability to bind to a particular entity (such as a thrombin or other serine protease), then the ability of a variant sequence to bind to that entity is the relevant function being considered. PAR4 or PARI "function" refers to any activity or characteristic associated with expression of PAR4 or PARI, respectively, including one or more effector activities. These activities and characteristics include, but are not limited to, binding other proteins (particularly serine proteases); regulation (whether induction or repression) of certain genes; and particular phenotypic characteristics of activation, such as calcium efflux, phosphoinositide hydrolysis, and platelet aggregation. These activities and characteristics will be described in more detail below. Because PAR4 (or PARI) exerts control over a number of other genes, it is understood that the term "PAR4 (or PARI) function" encompasses results and characteristics that stem from PAR4 (or PARI) activation which include affecting gene expression of any gene(s) that is regulated by PAR4 (or PARI) gene product or an active fragment thereof. For example, if gene A is repressed by expression and/or activation of PAR4 (or PARI), then lack of expression of gene A is a function of

PAR4 (or PARI). Conversely, expression of gene A indicates a compromise of PAR4 (or PARI) function. The terms "function" and "activity" are used interchangeably herein.

As used herein, a characteristic which is associated with a "compromise of PAR4 (or PARI) function" or a characteristic which is associated with "inhibition of PAR4 (or PARI) activity" or "inhibition of activation" is a characteristic which is associated with a decrease in PAR4 (or PARI) function. This decrease may range from partial to total loss, or knockout, of PAR4 (or PARI) function. Compromise of PAR4 (or PARI) function can occur as a result of an effect at any point along any pathway in which PAR4 (or PARI) exerts control, from transcription ofthe PAR4 (or PARI) gene, to PAR4 (or PARI) expression (i.e., transcription and/or translation), to affecting any activity associated with

PAR4 (or PARI) activation or deactivation.

As used herein, a characteristic which is associated with a "modulation of PAR4 (or PARI) function" is a characteristic which is associated with an alteration or change in PAR4 (or PARI) function. The modulation can be an increase or decrease. Decrease may range from partial to total loss, or knockout, of PAR4 (or PARI) function. Modulation of

PAR4 (or PARI) function can occur as a result of an effect at any point along any pathway in which PAR4 (or PARI) exerts control, from transcription ofthe PAR4 (or PARI) gene, to PAR4 (or PARI) expression (i.e., transcription and or translation), to affecting regulation of any gene(s) under PAR4 (or PARI) control, to activity associated with regulation of these gene(s). Other PAR4 (or PARI) functions are described above and herein. For example, the measurable effector functions such as intracellular calcium mobilization, phosphoinositide hydrolysis, and change in cell morphology are a result of intermediate steps, activities, and/or cascades.

"PAR4 (or PARI) activation" refers to a state in which PAR4 (or PARI) is able to cause one or more PAR4 (or PARl)-mediated effector functions, which are known in the art and described herein.

As used herein, "PARI and/or PAR4" refers to PARI, PAR4, or both PARI and PAR4.

As used herein, "PARI, PAR4 and/or platelet" refers to PARI, PAR4, platelet, both PARI and PAR4, or both PARI and platelet, or both PAR4 and platelet, or PARI, PAR4 and platelet.

"Platelets" are miniature cells without a nucleus that circulate in the blood and help to mediate blood clotting at sites of damage. They also release various factors that stimulate healing. One ofthe major functions of platelets is to initiate blood clotting. Some characteristics of "platelet function" include but are not limited to platelet aggregation, ATP secretion and platelet cell shape change.

The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, it may be interrupted by non-amino acids, and it may be assembled into a complex of more than one polypeptide chain. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfϊde bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that, because the polypeptides of this invention are based upon an antibody, the polypeptides can occur as single chains or associated chains.

A polypeptide "fragment" (also called a "region") of PAR4 (or a "PAR4 fragment" or "PAR4 region") or of PARI (or a "PARI fragment" or "PARI region") is a polypeptide comprising an amino acid sequence of PAR4 (or PARI) that has at least about any ofthe following lengths of contiguous amino acids of a sequence of PAR4 (or PARI): 5, 10, 15 25 30, 40, 50, 80. A PAR4 (or PARI) fragment may be characterized as having any ofthe following functions: (a) ability to bind another protein, particularly a serine protease; (b) ability to elicit a humoral and/or cellular immune response; (c) ability to regulate (i.e., repress or induce) another gene in the pathway regulated by PAR4 (or PARI); or (d) ability to elicit a characteristic associated with PAR4 (or PARI) activation.

A "fusion polypeptide" is a polypeptide comprising regions in a different position than occurs in nature. The regions may normally exist in separate proteins and are brought together in the fusion polypeptide, or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. A fusion polypeptide may also arise from polymeric forms, whether linear or branched, of PARI and/or PAR4 modulating peptides.

The terms "polynucleotide" and "nucleic acid", used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include a single-, double- or triple-stranded DNA, genomic DNA, cDN A, RNA, DNA-RN A hybrid, or a polymer comprising purine and pyrimidine bases, or other natural, chemically, biochemically modified, non-natural or derivatized nucleotide bases. The backbone ofthe polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone ofthe polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate- phosphodiester oligomer. A phosphorothiate linkage can be used in place of a phosphodiester linkage. In addition, a double-stranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.

The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Preferably, the polynucleotide is DNA. As used herein, "DNA" includes not only bases A, T, C, and G, but also includes any of their analogs or modified forms of these bases, such as methylated nucleotides, internucleotide modifications such as uncharged linkages and thioates, use of sugar analogs, and modified and/or alternative backbone structures, such as polyamides. Although conventional sugars and bases will be used in applying the method ofthe invention, substitution of analogous forms of sugars, purines and pyrimidines can be advantageous in designing a final product, as can alternative backbone structures like a polyamide backbone.

A nucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the polypeptide or a fragment thereof. For purposes of this invention, and to avoid cumbersome referrals to complementary strands, the anti-sense (or complementary) strand of such a polynucleotide is also said to encode the sequence; that is, a polynucleotide sequence that "encodes" a polypeptide includes both the conventional coding strand and the complementary sequence (or strand).

"Naturally occurring" or "native" refers to an endogenous polynucleotide or polypeptide sequence, i.e., one found in nature. The term includes alleles and allelic forms ofthe encoded protein, as well as full-length as processed polynucleotides and polypeptides. Processing can occur in one or more steps, and these terms encompass all stages of processing. Conversely, a "non-naturally occurring" sequence refers to all other sequences, i.e., ones which do not occur in nature, such as recombinant sequences.

"Recombinant," as applied to a polynucleotide or gene, means that the polynucleotide is the product of various combinations of cloning, restriction and/or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature.

The term "degenerate nucleotide sequence" denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp). "Transformation" or "transfection" refers to the insertion of an exogenous polynucleotide into a host cell, irrespective ofthe method used for the insertion, for example, lipofection, transduction, infection or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome.

An "isolated" or "purified" polynucleotide, polypeptide, antibody or cell is one that is substantially free ofthe materials with which it is associated in nature. By substantially free is meant at least 50%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% free ofthe materials with which it is associated in nature. As used herein, an "isolated" polynucleotide or polypeptide also refers to recombinant polynucleotides or polypeptides, which, by virtue of origin or manipulation: (1) are not associated with all or a portion of a polynucleotide or polypeptide with which it is associated in nature, (2) are linked to a polynucleotide or polypeptide other than that to which it is linked in nature, or (3) does not occur in nature, or (4) in the case of polypeptides arise from expression of recombinant polynucleotides.

A "vector" is a self-replicating nucleic acid molecule that transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily for insertion of a nucleic acid molecule into a cell, replication of vectors that function primarily for the replication of nucleic acid, and expression vectors that function for transcription and/or translation ofthe DNA or RNA. Also included are vectors that provide more than one ofthe above functions.

"Expression vectors" are defined as polynucleotides which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide(s). An "expression system" usually connotes a suitable host cell comprised of an expression vector that can function to yield a desired expression product.

A "cell line" or "cell culture" denotes eukaryotic cells, derived from higher, multicellular organisms, grown or maintained in vitro. It is understood that the descendants of a cell may not be completely identical (either morphologically, genotypically, or phenotypically) to the parent cell. Cells described as "uncultured" are obtained directly from a living organism, and are generally maintained for a limited amount of time away from the organism (i.e., not long enough or under conditions for the cells to undergo substantial replication).

A "host cell" includes an individual cell or cell culture which can be or has been a recipient for vector(s) or for incorporation of nucleic acid molecules and/or proteins. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic of total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of this invention.

As used herein, "expression" includes transcription and/or translation. A "biological sample" encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom, and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides. The term "biological sample" encompasses a clinical sample, and also includes cells in culture, cell supematants, cell lysates, serum, plasma, biological fluid, and tissue samples.

A "reagent" polynucleotide, polypeptide, or antibody, is a substance provided for a reaction, the substance having some known and desirable parameters for the reaction. A reaction mixture may also contain a "target", such as a polynucleotide, antibody, polypeptide, or assembly of polypeptides that the reagent is capable of reacting with. For example, in some types of diagnostic tests, the presence and/or amount ofthe target in a sample is determined by adding a reagent, allowing the reagent and target to react, and measuring the amount of reaction product (if any). In the context of clinical management, a "target" may also be a cell, collection of cells, tissue, or organ that is the object of an administered substance, such as a pharmaceutical compound.

As used herein, the term "agent" means a biological or chemical compound such as a simple or complex organic or inorganic molecule, a peptide, a protein or an oligonucleotide. A vast array of compounds can be synthesized, for example oligomers, such as oligopeptides and oligonucleotides, and synthetic organic compounds based on various core structures, and these are also included in the term "agent". In addition, various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. Compounds can be tested singly or in combination with one another. An agent that "inhibits or suppresses PAR4 activation" or an agent that "antagonizes PAR4 activation" is an agent that reduces the extent of PAR4 activation mediated by thrombin or a thrombin mimic or cathepsin G (i.e., the extent of activation of PAR4 in the presence of agent and thrombin or a thrombin mimic or cathepsin G is reduced when compared to the extent of activation in the presence of thrombin or a thrombin mimic or cathepsin G without presence of agent). An agent that "inhibits or suppresses PARI activation'Or an agent that "antagonizes PARI activation" is an agent that reduces the extent of PARI activation mediated by thrombin or a thrombin mimic (i.e., the extent of activation of PARI in the presence of agent and thrombin or a thrombin mimic is reduced when compared to the extent of activation in the presence of thrombin or a thrombin mimic without presence of agent). The inhibition or suppression of PAR4 (or PARI) activation may be partial or total. Methods of indicating PAR4 (or PARI) activation are known in the art and are described herein. Preferably, the inhibition is specific for PAR4, i.e., the effect is greater with respect to PAR4 than with respect to PARI . Or preferably, the inhibition is specific for PARI, i.e., the effect is greater with respect to PARI than with respect to PAR4. Assays for determining specificity are known in the art. Examples of agents which inhibit PAR4 activation include, but are not limited to, antibodies that block PAR4 cleavage by thrombin or a thrombin mimic or cathepsin G; agents which bind PAR4 and block tethered ligand binding and/or transmembrane signaling; agents which are thrombin or thrombin mimic or cathepsin G inhibitors. Examples of agents which inhibit PARI activation include, but are not limited to, antibodies that block PARI cleavage by thrombin or a thrombin mimic; agents which bind PARI and block tethered ligand binding and/or transmembrane signaling; agents which are thrombin or thrombin mimics.

An agent that "inhibits or suppresses platelet activation" is an agent that reduces the extent of platelet activation mediated by thrombin or a thrombin mimic or cathepsin G (i.e., the extent of platelet activation in the presence of agent and thrombin or a thrombin mimic or cathepsin G is reduced when compared to the extent of activation in the presence of thrombin or a thrombin mimic or cathepsin G without presence of agent). The inhibition or suppression of platelet activation may be partial or total. Methods of indicating platelet activation are known in the art and are described herein. Preferably, the inhibition is specific for PAR4, i.e., the effect is greater with respect to PAR4 than with respect to PARI . Or preferably, the inhibition is specific for PARI, i.e., the effect is greater with respect to PARI than with respect to PAR4. Examples include, but are not limited to, agents which inhibit activation of thrombin or a thrombin mimic or cathepsin G. For instance, dipeptidyl peptidase I is necessary for generating active cathepsin G. Pham et al. (1999) Proc. Natl. Acad. Sci. USA 96:8627-8632. An inhibitor of this peptidase (or an inhibitor that suppresses binding of this peptidase to cathepsin G) could suppress or prevent cathepsin G from becoming biologically active (biologically available), thereby suppressing or preventing neutrophil-dependent platelet activation via PAR4.

An agent or substance is said to be "selective" or "specific" if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances. For instance, an antibody "specifically binds" to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances.

As used herein, "specifically activates" means an agent, such as thrombin, a thrombin analog, a PAR agonist or other agents including chemicals, polypeptides or antibodies, which activates protease-activated receptor, receptor polypeptide or a fragment or analog thereof to initiate PAR-mediated biological events as described herein, but which does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally includes a protease-activated receptor polypeptide. Preferably, the agent activates the biological activity in vivo or in vitro ofthe protein to which it binds.

The characteristic which is associated with the alteration of PARI, PAR4 and/or platelet may include, for example, the changes in activity, function, conformation, shape, aggregation of PARI, PAR4 and/or platelet.

As used herein, "specifically inhibits" means an agent, such as a thrombin analog, a PAR antagonist or other agents, including chemicals, polypeptides, antibodies, which inhibits activation of protease-activated receptor, receptor polypeptide or a fragment or analog thereof, such as by inhibiting thrombin or by blocking activation of PAR by thrombin or other PAR activator. Preferably, the agent inhibits the biological activity in vivo or in vitro ofthe protein to which it binds.

An agent that "mimics" thrombin or cathepsin G or a "mimic" or "mimetic" of thrombin or cathepsin G is a substance which binds and activates PARI or PAR4. Further, and preferably, a mimic of cathepsin G fails to significantly stimulate PARI activation.

A "PAR4-expressing cell" is a cell which produces PAR4, preferably in a form such that PAR4 is able to bind to thrombin or a thrombin mimic or cathepsin G (in the absence of any agents which inhibit thrombin or a thrombin mimic or cathepsin G/PAR4 interaction) and initiate one or more PAR4-mediated effector functions. Examples of PAR4-expressing cells (which may be recombinant or naturally-occurring) are known in the art and described herein.

A "PARI -expressing cell" is a cell which produces PARI, preferably in a form such that PARI is able to bind to thrombin or a thrombin mimic (in the absence of any agents which inhibit thrombin or a thrombin mimic) and initiate one or more PARI -mediated effector functions. Examples of PARI -expressing cells (which may be recombinant or naturally-occurring) are known in the art and described herein.

An agent or substance (or composition comprising an agent or substance) that increases an activity or other measurable phenotypic characteristic preferably increases that activity or other measurable phenotype by at least about any ofthe following as compared to control conditions: 1.5 fold, 2-fold, 5-fold, or 10-fold increase.

An agent or substance (or composition comprising an agent or substance) that decreases or reduces an activity or other measurable phenotypic characteristic preferably decreases that activity or phenotypic characteristic to about the following percentages or less than about any ofthe following percentages as compared to control conditions: 80%, 50% , 25%, or 10%.

A "PAR4 agonist" refers to a molecule which mimics a particular activity, such as a ligand activity, or affects an interaction between a ligand and PAR4 or between thrombin or a thrombin mimic or cathepsin G and PAR4 thereby activating PAR4 and triggering the biological events which normally result from the interaction (e.g., phosphoinositide hydrolysis, Ca²⁺ efflux, and platelet aggregation). A "PARI agonist" refers to a molecule which mimics a particular activity, such as a ligand activity, or affects an interaction between a ligand and PARI or between thrombin or a thrombin mimic and PARI thereby activating PARI and triggering the biological events which normally result from the interaction (e.g., phosphoinositide hydrolysis, Ca²⁺ efflux, and platelet aggregation). Preferably, an agonist initiates an increase in receptor activity relative to control assays in the absence of activator or candidate agonist. An agonist may possess the same, less, or greater activity than a naturally-occurring cathepsin G-mediated PAR4 activation or thrombin-mediated PAR4 (or PARI) activation.

A "PAR4 antagonist" refers a molecule which blocks or suppresses activation of PAR4 activation as mediated by thrombin or a thrombin mimic or cathepsin G, thereby suppressing the biological events resulting from such an interaction (e.g. , phosphoinositide hydrolysis, Ca²⁺ efflux, and platelet ATP secretion, or platelet aggregation). An antagonist may bind to and thereby block the activation of PAR4, as long as this inhibition is specifically due to thrombin or a thrombin mimic or cathepsin G. In another aspect, an antagonist may bind to thrombin or a thrombin mimic or cathepsin G and thereby block the activation of PAR4.

A "PARI antagonist" refers a molecule which blocks or suppresses activation of PARI activation as mediated by thrombin or a thrombin mimic, thereby suppressing the biological events resulting from such an interaction (e.g., phosphoinositide hydrolysis, Ca²⁺ efflux, and platelet ATP secretion, or platelet aggregation). An antagonist may bind to and thereby block the activation of PARI, as long as this inhibition is specifically due to thrombin or a thrombin mimic. In another aspect, an antagonist may bind to thrombin or a thrombin mimic and thereby block the activation of PARI .

"Modulating" a characteristic, activity or interaction includes increase or decrease ofthe characteristic, activity or interaction as compared to control conditions.

"Stimulating" PAR4 (or PARI) activation means an increase in the level of PAR4 (or PARI) activation. Such an increase may be as compared to no activation or as compared to a previously lower level of activation.

"Stimulating" PAR4 (or PARl)-mediated platelet activation means an increase in the level of platelet activation. Such an increase may be as compared to no activation, or may be an increase as compared to a previously lower level of activation. Methods of indicating platelet activation are known in the art and are described herein.

The term "complement/anti-complement pair" denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti- complement pair. Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation ofthe complement/anti- complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of <10⁹ M"¹.

A "reporter gene" is a polynucleotide sequence that encodes for a detectable product ("reporter"). The reporter gene may encode all or a portion of a detectable product. Examples of reporter genes are known in the art, and include genes whose products give rise to luminescence, such as luciferase, aequorian, β-galactosidase, chloramphenicol acetyl transferase (CAT), as well as genes whose produces provide a basis for selection, such as an antibiotic resistance gene.

"Operably linked" or "operatively linked" refers to a juxtaposition, wherein the components so described are in a relationship permitting them to function in their intended manner. A transcriptional regulatory sequence or element (TRE) is operably linked to a coding sequence is the TRE promotes or allows transcription ofthe coding sequence. An operably linked TRE is generally joined in cis with the coding sequence, but it is not necessarily contiguous with or directly adjacent to it.

The term "secretory signal sequence" denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a second polypeptide, directs the second polypeptide through a secretory pathway of a cell in which it is synthesized.

The second polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.

The term "biological activity" of PAR refers to the ability ofthe PAR to bind thrombin or an agonist and signal the appropriate cascade of biological events (e.g., phosphoinositide hydrolysis, Ca²⁺ efflux, and platelet aggregation, and the like). The term "substantial increase" refers to an increase in activity or other measurable phenotypic characteristic that is at least approximately a 2-fold increase over control level (where control assays are performed in the absence of activator), preferably at least approximately a 5-fold increase, more preferably at least approximately a 10-fold increase in activity over a control assay.

The term "substantial decrease" or "substantial reduction" refers to a decrease or reduction in activity or other measurable phenotypic characteristic that is approximately 80% or the control level, preferably reduced to approximately 50% ofthe control level, or more preferably reduced to approximately 10% or less ofthe control level. A "modulator" refers to an agent that is either an agonist or an antagonist.

Accordingly, an agent "modulates" the activity of PARI or PAR4 or platelet by either increasing/activating or decreasing/inhibiting the activity of PARI or PAR4 or platelet. In one embodiment, a modulator is a "modulating peptide".

The terms "antagonist assay", "antagonist screening" and the like, refer to a method of screening a candidate compound for its ability to antagonize interaction between a naturally-occurring activating ligand or an agonist and the PAR, either PARI, PAR 4 or both.

The terms "treatment", "treating", "treat" and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, particular a human, and includes:

(a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it;

(b) inhibiting the disease symptom, i.e., arresting its development; or

(c) relieving the disease symptom, i.e., causing regression ofthe disease.

An "effective amount" is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of a PARI and/or PAR4 modulating polypeptide(s) is an amount sufficient to modulate PARI and/or PAR4 function. In terms of treatment, an "effective amount" of a PARI and/or PAR4 modulating polypeptide(s) is an amount sufficient to palliate, ameliorate, stabilize, reverse, slow or delay progression of a PARI and/or PAR4-associated disease state (i.e., a state in which PARI and/or PAR4 indicate potential or actual pathology). Detection and measurement of indicators of efficacy are generally based on measurement of PARI and/or PAR4 and/or clinical symptoms associated with the disease state, such as heart attack, stroke, atherosclerosis, resternosis, pulmonary inflammation (ARDS), and glomerulosclerosis, and with the disorders in such as, but not limited to, hemostasis, thrombosis, and normal wound healing. An "individual" is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, primates, rodents and pets.

"A," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a DNA sequence" includes mixtures and large numbers of such sequences, reference to "an assay" includes assays ofthe same general type, and reference to "the method" includes one or more methods or steps ofthe type described herein.

Conpositions ofthe Invention

PAR-activating polypeptides Compositions ofthe invention encompass PAR4 modulating peptides, PARI modulating peptides, and peptides modulating both PARI and PAR4 simultaneously. Table 1 provides examples of PAR activating compounds.

The peptides have a variety of uses, including their use as an agent to screen pharmaceutical candidates (both in vitro and in vivo), their use in rational (i.e., structure- based) drug design, as well as possible therapeutic uses. For example, a PAR activating peptide could be used to stimulate or enhance platelet activation including clotting. As another example, a PARI and/or PAR4 modulating peptide that binds competitively to PARI and/or PAR4 could compromise PARI and/or PAR4 function as a competitive inhibitor and thus exert regulatory/modulating, preferably therapeutic, activity. The PARI and/or PAR4 modulating peptides may also be used to identify proteins, especially those of human origin that bind (or interact physically) with PARI and/or PAR4 which could thus themselves be drug targets. The peptides described herein are also useful as reagents for probing aspects of PAR function.

Various peptides ofthe invention are shown in Table 1 (see also Table 2, which indicates structure and function). In some embodiments, the invention provides PAR4 activating peptides GYPGRF, GYPG(homoR)F, GYPG(Orn)F, GYPGKY, GYPGKW,

GYPGKK, SYPAKF, SYAGKF, SYPGRF, SYPG(homoR)F, AYPGKF, SYPGKF, TYPGKF, GFPGKF, and G(F)PGKF, wherein (homoR) is homoarginine and (Orn) is ornithine; PARI activating peptides GYPAKF, GYP(Cha)KF, GYPLKF, GYPIKF, S(F)(Cha)(Cha)(homoR)K, and S(F)(Cha)(Cha)RK, GFPGKF, and G(F)PGKF, wherein (Cha) is cyclohexylalanine, (F) is parafluoro-phenylalanine, and (homoR) is homoarginine and peptides GFPGKF, G(F)PGKF and GYPAKF that activate both PARI and PAR4.

Table 1

Peptide SEQ ID No. Activate PAR4? Activate of PARI?

GYPGKF Yes No SFLLRN No Yes

AYPGKF Yes No SYPGKF Yes No TYPGKF Yes/No No MprYPGKF No No

GFPGKF Yes Yes

G(F)PGKF Yes Yes

GLPGKF No No

GYAGKF No No

GYGGKF No No

GYLGKF No No

GYIGKF No No

GYKGKF No No

GYRGKF No No

GY(Cha)GKF No No

GYPAKF Yes/No Yes/No GYP(Cha)KF No Yes GYPLKF No Yes GYPIKF No Yes

GYPGRF Yes No

GYPG(homoR)F Yes No

GYPG(Orn)F Yes No

GYPGKY Yes No GYPGKW Yes No GYPGKK Yes/No No GYPGK(Orn) No No

SYPAKF Yes No SYAGKF Yes/No No SYPGRF Yes No SYPG(homoR)F Yes No S(F)(Cha)(Cha)(homoR)K No Yes

S(F)(Cha)(Cha)RK No Yes

(Cha), cyclohexylalanine; (F), parafluoro-phenylalanine; (homoR), homoarginine; (Orn), ornithine; Mpr, mercaptoproprionic acid; other letters represent the single letter amino acid code.

The invention encompasses modifications to the PARI and/or PAR4 modulating peptides as disclosed herein. Such modifications may produce functionally equivalent modulators ofthe PARI and/or PAR4 modulating peptides (which do not have significantly altered properties) as well as variants wliich have enhanced or decreased activity, provided that these sequences are different from that of PARI and/or PAR4. Modification of polypeptides is routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids which do not significantly deleteriously change the functional activity, or use of chemical analogs. Amino acid residues which can be conservatively substituted for one another include but are not limited to: glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; serine/threonine; lysine/arginine; and phenylalanine/tryosine. These polypeptides also include glycosylated and nonglycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation. Preferably, the amino acid substitutions would be conservative, i.e., the substituted amino acid would possess similar chemical properties as that ofthe original amino acid. Such conservative substitutions are known in the art, and examples have been provided above. Amino acid modifications can range from changing or modifying one or more amino acids to complete redesign of a region, such as the variable region. Changes in the variable region can alter binding affinity and/or specificity. Other methods of modification include using coupling techniques known in the art, including, but not limited to, enzymatic means, oxidative substitution and chelation. Modifications can be used, for example, for attachment of labels for immunoassay, such as the attachment of radioactive moieties for radioimmunoassay. Modified PARI and/or PAR4 modulating peptides are made using established procedures in the art and can be screened using standard assays known in the art.

The invention also encompasses fusion proteins comprising one or more PARI and/or PAR4 modulating peptides. For purposes of this invention, a PARI and/or PAR4 modulating peptide fusion protein contains one or more PARI and/or PAR4 modulating peptides and another amino acid sequence to which it is not attached in the native molecule, for example, a heterologous sequence or a homologous sequence from another region. Useful heterologous sequences include, but are not limited to, sequences that provide for secretion from a host cell, enhance immunological reactivity, or facilitate the coupling of the polypeptide to an immunoassay support or a vaccine carrier. For instance, a PARI and/or PAR4 modulating peptide can be fused with a bioresponse modifier. Examples of bioresponse modifiers include, but are not limited to, cytokines or lymphokines such as GM-CSF, interleukin-2 (IL-2), interleukin 4 (IL-4), and γ-interferon. Accordingly, the invention includes PARI and/or PAR4 modulating peptide fusion polypeptides that contain GM-CSF or IL-2. Another useful heterologous sequence is one which facilitates purification. Examples of such sequences are known in the art and include those encoding epitopes such as Myc, HA (derived from influenza virus hemagglutinin), His-6, or FLAG. Other heterologous sequences that facilitate purification are derived from proteins such as glutathione S-transferase (GST), maltose-binding protein (MBP), or the Fc portion of immunoglobulin.

The invention also encompasses polymeric forms of PARI and/or PAR4 modulating peptides. As used herein, a polymeric form of a PARI and/or PAR4 modulating peptide contains a plurality (i.e., more than one) of PARI and or PAR4 modulating peptides. In one embodiment, linear polymers of PARI and/or PAR4 modulating peptides are provided. These PARI and/or PAR4 modulating peptide linear polymers may be conjugated to carrier. These linear polymers can comprise multiple copies of a single PARI and/or PAR4 modulating peptide, or combinations of different PARI and/or PAR4 modulating peptides, and can have tandem PARI and/or PAR4 modulating peptides, or PARI and/or PAR4 modulating peptides separated by other amino acid sequences. These linear polymers can be made using standard recombinant methods well known in the art. In another embodiment, multiple antigen peptides (MAPS) are provided. MAPS have a small immunologically inert core having radically branching lysine dendrites, onto which a number of PARI and/or PAR4 modulating peptides can be anchored (i.e., covalently attached). Posnett et al. (1988) J Biol. Chem. 263:1719-1725; Tam (1989) Meth. Enz. 168:7-15. The result is a large macromolecule having a high molar ratio of PARI and/or PAR4 modulating peptides to core. MAPS are useful, efficient immunogens as well as useful antigens for assays such as ELISA, in addition to potent activating peptides. PARI and/or PAR4 modulating peptide MAPS can be made synthetically. In a typical MAPS system, a core matrix is made up of three levels of lysine and eight amino acids for anchoring PARI and/or PAR4 modulating peptides. The MAPS may be synthesized by any method known in the art, for example, a solid-phase method, for example, Merrifield (1963) J. Am. Chem. Soc. 85:2149.

In another embodiment, PARI and/or PAR4 modulating peptides can be conjugated, or complexed with, with carrier or label. For example, in instances where the PARI and/or PAR4 modulating peptide is correctly configured so as to provide a binding site, but is too small to be effective for modulating PARI and/or PAR4 in vivo, the polypeptide may be linked to a suitable carrier. A number of techniques for obtaining such linkage are known in the art and need not be described in detail herein, such as chemical cross-linking methods. Most preferably, such combinations are formed by cross-linking a natural or recombinant polypeptide PAR antagonists to carriers that have been synthesized with a cross-linking moiety, such as dinitrofluorobenzene, at its NH2 terminus. Alternatively, the carrier peptide may be conjugated to a natural or recombinant PARI and PAR4 antagonist by the use of agents such as glutaraldehyde, dimethyladipimidate, or any other bifunctional cross-linkers known in the art. The conjugated antagonist preferably involves a 1 : 1 stoichiometry with the carrier peptide. Any carrier can be used which does not itself induce the production of antibodies harmful to the host. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins; polysaccharides, such as latex functionalized sepharose, agarose, cellulose, cellulose beads and the like; polymeric amino acids, such as polyglutamic acid, polylysine, and the like; amino acid copolymers; and inactive virus particles or attenuated bacteria, such as Salmonella.

Especially useful protein substrates are serum albumins, keyhole limpet hemacyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and other proteins well known to those of skill in the art. Labels are known in the art and are described herein.

Preparation of polypeptides

The polypeptides of this invention can be made by procedures known in the art. The polypeptides can be produced by recombinant methods (i.e., single or fusion polypeptides) or by chemical synthesis. Polypeptides, especially shorter polypeptides up to about 50 amino acids, are conveniently made by chemical synthesis. Methods of chemical synthesis are known in the art and are commercially available. For example, a polypeptide could be produced by an automated polypeptide synthesizer employing the solid phase method. Polypeptides can also be made by chemical synthesis using techniques known in the art.

Polypeptides can also be made by expression systems, using recombinant methods. For any PARI and/or PAR4 modulating peptides, one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information known in the art. The availability of polynucleotides encoding polypeptides permits the construction of expression vectors encoding the modulating polypeptide, functionally equivalent fragments thereof, or recombinant forms. A polynucleotide encoding the desired polypeptide, whether in fused or mature form, and whether or not containing a signal sequence to permit secretion, may be ligated into expression vectors suitable for any convenient host. Both eukaryotic and prokaryotic host systems can be used. The polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use. Purification or isolation ofthe polypeptides expressed in host systems can be accomplished by any method known in the art. For example, cDNA encoding a polypeptide intact or a fragment thereof can be operatively linked to a suitable promoter, inserted into an expression vector, and transfected into a suitable host cell. The host cell is then cultured under conditions that allow transcription and translation to occur, and the desired polypeptide is recovered. Other controlling transcription or translation segments, such as signal sequences that direct the polypeptide to a specific cell compartment (i.e., for secretion), can also be used. Examples of prokaryotic host cells are known in the art and include, for example, E. coli and B. subitilis. Examples of eukaryotic host cells are known in the art and include yeast, avian, insect, plant, and animal cells such as COS7, HeLa, CHO and other mammalian cells. When using an expression system to produce PARI and/or PAR4 modulating peptides, it is often preferable to construct a fusion protein that facilitates purification. Examples of components for these fusion proteins include, but are not limited to myc, HA, FLAG, His-6, glutathione S-transferase, maltose binding protein or the Fc portion of immunoglobulin. These methods are known in the art. See, for example, Redd et al. (1997) J Biol. Chem. 272:11193-11197. Techniques known in the art may be employed to remove unwanted amino acids from fusions, such as His-6. For example, carboxypeptidase A may be used to eliminate carboxyterminal amino acids. Carboxypeptidase A stops at amino acids proline or arginine. For convenience of purification, solid phase carboxypeptidase A (Sigma) may be used.

In some systems, especially some recombinant systems, for embodiments which contain an extra cysteine, or a cysteine which is to be reduced (in order to, for example, conjugate the polypeptide to a platform molecule), the initial product may comprise low molecular weight mixed disulfides, in which the extra, reactive cysteine is covalently linked to other, relatively low molecular weight moiety or moieties. In these instances, selective reduction ofthe extra cysteine is desired. Such selective reduction may be accomplished by using a solid phase reductant agent, such as DTT, on a solid support, such as acrylamide (such as REDUCTACRYL by CalBiochem, San Diego).

Preferably, especially if the polypeptide is to be conjugated to a platform, chemical synthesis is used. Chemical synthesis permits modification ofthe N or C terminus, wliich facilitates conjugation to a platform molecule. Preferably, especially if used for diagnostic or therapeutic purposes, the polypeptides are at least partially purified or isolated from other cellular constituents. Preferably, the polypeptides are at least 50% pure. In this context, purity is calculated as a weight percent ofthe total protein content ofthe preparation. More preferably, the proteins are 50-75% pure. More highly purified polypeptides may also be obtained and are encompassed by the present invention. For clinical use, the polypeptides are preferably highly purified, at least about 80% pure, and free of pyrogens and other contaminants. Methods of protein purification are known in the art and are not described in detail herein.

Polynucleotides encoding PAR-activating peptides The invention also provides polynucleotides which encode any ofthe polypeptides described herein. Synthesis of these DNA molecules may be achieved by methods well known in the art. For example, the recombinant DNA molecules may be isolated from a human hematopoetic cDNA library. The synthesis of cDNA libraries and the choice of vector into which the cDNA molecules may be cloned are conventional techniques, see Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, second edition. A wide variety of methods may be used in locating and identifying cDNA sequences corresponding to the compositions ofthe present invention.

The DNA molecules of this invention may be synthesized from nucleotides by chemical means using an synthesizer. Such nucleic acids may be designed based on identified amino acid sequence ofthe agonists or antagonists. Standard methods may be applied to synthesize a gene encoding such a peptide. For example, the complete amino acid sequence may be used to construct a back-translated gene. A DNA oligomer containing a nucleotide sequence capable of coding for the desired polypeptide may be synthesized in a single step. Alternatively, several smaller oligonucleotides coding for portions ofthe agonist and antagonists may be synthesized and subsequently ligated together. Preferably, such gene is synthesized as 10-20 separate oligonucleotides which are subsequently linked together. The individual oligonucleotides contain 5' or 3' overhangs for complementary assembly.

Following synthesis ofthe nucleic acid and cleavage ofthe desired vector, assembly (if any) of a desired polynucleotide may be achieved in one or more steps by techniques well known in the art. Once assembled, the polynucleotide is characterized, for example, by sequences which are recognized by restriction endonucleases, including unique restriction sites for direct assembly into a cloning or an expression vector; preferential codons based upon the host expression system to be used, and a sequence which, when transcribed, produces an mRNA with minimal secondary structure. Proper assembly may be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active antagonist or agonist in a platelet aggregation assay.

Cloning and expression vectors comprising a polynucleotide that encodes PAR-activating peptides

The present invention further includes a variety of vectors (i.e., cloning and expression vectors) having cloned therein polynucleotide(s) that encode PAR-activating peptide(s). These vectors can be used for expression of recombinant polypeptides as well as a source of polynucleotides that encode PAR-activating peptide(s). Cloning vectors can be used to obtain replicate copies ofthe polynucleotides they contain, or as a means of storing the polynucleotides in a depository for future recovery. Expression vectors (and host cells containing these expression vectors) can be used to obtain polypeptides produced from the polynucleotides they contain. They may also be used where it is desirable to express PAR-activating polypeptides in an individual. Suitable cloning and expression vectors include any known in the art, e.g., those for use in bacterial, mammalian, yeast and insect expression systems. Specific vectors and suitable host cells are known in the art and need not be described in detail herein. For example, see Gacesa and Ramji, Vectors, John Wiley & Sons (1994).

Cloning and expression vectors typically contain a selectable marker (for example, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector), although such a marker gene can be carried on another polynucleotide sequence co-introduced into the host cell. Only those host cells into which a selectable gene has been introduced will survive and/or grow under selective conditions. Typical selection genes encode protein(s) that (a) confer resistance to antibiotics or other toxins substances, e.g., ampicillin, neomycin, methotiexate, etc.; (b) complement auxotrophic deficiencies; or (c) supply critical nutrients not available from complex media. The choice ofthe proper marker gene will depend on the host cell, and appropriate genes for different hosts are known in the art. Cloning and expression vectors also typically contain a replication system recognized by the host.

Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors will generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUCl 8, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mρl8, mpl9, pBR322, ρMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen. The Examples provided herein also provide examples of cloning vectors. Expression vectors generally are replicable polynucleotide constructs that contain a polynucleotide encoding a PAR-activating polypeptide of interest. The polynucleotide encoding the PAR-activating polypeptide is operatively linked to suitable transcriptional controlling elements, such as promoters, enhancers and terminators. For expression (i.e., translation), one or more translational controlling elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons. These controlling elements (transcriptional and translational) may be derived from PAR polynucleotides (i.e., the PAR gene), or they may be heterologous (i.e., derived from other genes and/or other organisms). A polynucleotide sequence encoding a signal peptide can also be included to allow a PAR-activating polypeptide to cross and/or lodge in cell membranes or be secreted from the cell. A number of expression vectors suitable for expression in eukaryotic cells including yeast, avian, and mammalian cells are known in the art.

The Examples provided herein contain a number of examples of expression vectors for yeast systems, particularly S. cerevisiae and C. albicans. For instance, pRD53 can be used for Gal-induced expression in S. cerevisiae. Other common vectors, such as YEpl3 and the Sikorski series pRS303-306, 313-316, 423-426 can also be used. Vectors pDBV52 and pDBV53 are suitable for expression in C. albicans.

To determine whether plasmids containing polynucleotides are capable of expression, cells can be transfected with the plasmids. Expression resulting in a polypeptide(s) is then determined by RIA, ELISA, immunofluorescence of fixed cells, or western blotting of cell lysate using an antibody as a probe. Alternatively, expression of smaller polypeptides can be detected, for example, by constructing the plasmid so that the resultant polypeptide is labeled recombinantly, such as with an enzymatic label. Further characterization ofthe expressed polypeptide can be achieved by purification ofthe polypeptide using techniques known in the art.

The vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (where the vector is an infectious agent, such as vaccinia virus). The choice of means of introducing vectors or PAR-activating polynucleotides will often depend on the host cell. Host cells transformed with polynucleotides that encode PAR-activating peptides Another embodiment of this invention are host cells transformed with (i.e., comprising) polynucleotides that encode PAR-activating peptide(s) and/or vectors having polynucleotide(s) sequences that encode PAR-activating peptide(s), as described above. Both prokaryotic and eukaryotic host cells may be used. Prokaryotic hosts include bacterial cells, for example E. coli, B. subtilis and mycobacteria. E. coli cells are particularly useful for producing PAR-activating polypeptides. Komachi et al. (1994) Genes Dev. 8: 2857-2867. Among eukaryotic hosts are yeast, insect, avian, plant and mammalian cells. Host systems are known in the art and need not be described in detail herein. Examples of fungi (including yeast) host cells are S. cerevisiae, Kluyveromyces lactis (K. lactis), species of Candida including C. albicans and C. glabrata, C. maltosa, C. utilis, C. stellatoidea, C. parapsilosis, C. tropicalus, Neurospora crassas, Aspergillus nidulans, Schizosaccharomyces pombe (S. pombe), Pichia pastoris, and Yarowia lipolytica. The host cells of this invention can be used, inter alia, as repositories of polynucleotides that encode PAR-activating peptide(s) and/or vehicles for production of polynucleotides that encode PAR-activating peptide(s) and/or polypeptides.

Compositions and kits comprising the polypeptides and polynucleotide ofthe invention The present invention provides peptide compositions comprising PARI and/or

PAR4 activating peptides (including all polypeptide embodiments described above, such as fusions, polymeric polypeptides, and conjugates), as well as compositions comprising any ofthe polynucleotide embodiments described herein. These compositions are especially useful for administration to those individuals who may benefit from modulation of platelet activation. When these compositions are to be used pharmaceutically, they are combined with a pharmaceutically acceptable excipient. The compositions are also useful as reagents in detection systems. When these compositions are to be used as reagents, they are combined with a buffer.

Generally, the compositions ofthe invention for use in modulating platelet activation comprise an effective amount of a PARI and/or PAR4 activating peptide(s), preferably in a pharmaceutically acceptable excipient, and may be in various formulations. As is well known in the art, a pharmaceutically acceptable excipient is a relatively inert substance that facilitates administration of a pharmacologically effective substance. For example, an excipient can give form or consistency, or act as a diluent. Suitable excipients include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers.

Excipients as well as formulations for parenteral and nonperenteral drug delivery are set forth in Remington 's Pharmaceutical Sciences 19th Ed. Mack Publishing (1995).

Compositions capable of eliciting the modulating activity on platelet activation in an individual when administered in an effective amount. In this context, an "effective amount" is an amount sufficient to elicit such modulating activity, and an effective amount may be administered in one or more administrations.

Generally, these compositions are formulated for administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Accordingly, these compositions are preferably combined with pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, and the like. Generally, the conjugate will normally constitute about 0.01%o to 10% by weight ofthe formulation due to practical, empirical considerations such as solubility and osmolarity. The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history. Generally, a dose of about 1 μg to about 100 mg/kg body weight, preferably about 100 μg to about 10 mg/kg body weight, will be given weekly.

Empirical considerations, such as the half life, generally will contribute to determination of the dosage. Other appropriate dosing schedules may be as frequent as daily or 3 doses per week, or one dose per week, or one dose every two to four weeks, or one dose on a monthly or less frequent schedule depending on the individual or the disease state. Repetitive administrations, may be required to achieve and/or maintain a state of platelet activation or inactivation. Such repetitive administrations generally involve treatments of about 1 μg to about 10 mg/kg body weight or higher every 30 to 60 days. Alternatively, sustained continuous release formulations ofthe compositions may be indicated for some pathologies. Various formulations and devices for achieving sustained release are known in the art. Other formulations include suitable delivery forms known in the art including, but not limited to, carriers such as liposomes. Mahato et al. (1997) Pharm. Res. 14:853-859. Liposomal preparations include, but are not limited to, cytofectins, multilamellar vesicles and unilamellar vesicles. In some embodiments, more than one PARI and/or PAR4 activating peptide(s) may be present in a composition. Such compositions may contain at least one, at least two, at least three, at least four, at least five different PARI and/or PAR4 activating peptide(s). Such "cocktails", as they are often denoted in the art, may be particularly useful in treating a broader range of population of individuals. They may also be useful in being more effective than using only one (or fewer than are contained in the cocktail) PARI and/or

PAR4 activating peptide(s).

The compositions may be administered alone or in conjunction with other forms of agents that serve to enhance and/or complement the effectiveness of a PARI and/or PAR4 activating peptide(s). Generally, efficacy of administering any of these compositions is adjudged by measuring any change in the clinical parameters of platelet activation, such as platelet secretion and aggregation. However, measurement of any parameter that is thought or has been shown to be associated with the condition being treated is suitable.

With respect to those compositions which may be used as reagents (such as in detection assays), these compositions generally comprise an amount of a PARI and/or

PAR4 modulating peptide(s) (i.e., one or more polypeptide) sufficient to effect detection. These amounts are readily determined empirically. These compositions may further comprise a substance, such as a buffer, to effect detection. These compositions may also optionally be complexed to a detection matrix, such as solid phase (e.g., in an immunoaffinity column).

These compositions may also contain a variety of other conventional antiplatelet or anti-thrombin or anti-cathepsin G compounds in addition to a naturally purified, recombinant or synthetic polypeptide inhibitor of platelet activation ofthe invention. The most widely used antiplatelet agent is aspirin, a cyclooxygenase inhibitor. Although aspirin blocks ADP- and collagen-induced platelet aggregation, it fails to prevent cyclooxygenase- independent platelet aggregation initiated by agonists, such as thrombin. Altemative anti- thrombin compounds are hirudin derivative.

The composition ofthe invention containing additional anti-platelet activation compounds may be a single dosage form, wherein a polypeptide inhibitor of platelet activation ofthe invention may be chemically conjugated to a conventional polypeptide platelet inhibitor or to a conventional anti-thrombin or anti-cathepsin G polypeptide. Alternatively, a single dosage form which contains the polypeptide inhibitor of platelet activation and the other polypeptide in the same composition, but as separate compounds. The composition may also contain multiple dosage forms, wherein the PARI and PAR4 inhibitors and the other polypeptide that inhibits platelet activation are administered separately, but concurrently, or wherein the two forms are administered sequentially.

Compositions containing polynucleotide vectors described herein are encompassed by this invention. The invention also provides compositions comprising a vector(s) containing an polynucleotide as well as compositions comprising a host cell, such as a mammalian host cell or an invertebrate host cell, containing an polynucleotide, as described herein. Examples of mammalian host cells include human host cells; examples of invertebrate host cells include frog oocytes. The invention also provides compositions comprising a PAR-activating peptide. When these compositions are to be used pharmaceutically, they are combined with a pharmaceutically acceptable excipient. Such excipients are well known in the art. Suitable excipients include, but are not limited to, stabilizing agents, wetting and emulsifying agents, salts, and buffers. If used in kits or for other experimental manipulations, these compositions generally comprise a suitable substance, such as a buffer or water. These compositions generally comprise an effective amount ofthe vector(s) and/or activating polypeptide (s) and/or modulator(s). An effective amount can vary depending on which context the vector or the modulator is to be used. For example, if an artificial chromosome vector is to be used as an expression or cloning system, an effective amount is an amount sufficient to allow transformation into a suitable host cell. If a vector is the basis for a screening assay, an effective amount is an amount sufficient to allow testing and detection. If a PAR-activating peptide is to be used to activate PAR, an effective amount is an amount sufficient to allow activating the PAR. The invention also provides kits containing (i.e., comprising) one or more vectors and/or activating polypeptides and/or modulators described herein. A PAR-activating polypeptide(s) may be a component of a kit for activating coagulation (clotting). Such a kit would enable the activation of PARI, PAR4, and/or platelet in the thrombosis pathway. The kits would also be useful for detection and/or screening with respect to PARI and/or

PAR4 as well as platelet activity. The kits of this invention are in suitable packaging, and may optionally provide additional components that are useful in the procedure in which the vector(s) will be used. These optional components include, but are not limited to, buffers, capture reagents, developing reagents, labels, reactivating surfaces, means for detection, control samples, instructions and interpretive information.

The kits may further comprise instructions for administration to an individual in order to effect, or stimulate, PARI and/or PAR4 activation in an individual. The instructions can be for any ofthe following: effecting PARI and/or PAR4 activation; stimulation of PARI and/or PAR4-mediated platelet activation; and/or treatment of a condition for which PARI and/or P AR4 activation or stimulation of PARI and/or PAR4- mediated platelet activation is indicated.

In some embodiments, the kits further comprise an agent which interferes with thrombin or cathepsin G/PAR4 (or PARI) interaction (or a composition comprising one or more such agents) in suitable packaging. In some embodiments, the agent is other than a PAR4 (or PARI ) antagonist.

In some embodiments, a kit comprises instructions for administering the composition to an individual to effect inhibition of PAR4 (or PARI) activation, inhibition of PAR4 (or PARl)-mediated platelet activation, and/or treatment of a condition for which inhibition of P AR4 (or P AR1 ) activation or inhibition of PAR4 (or PARI )-mediated platelet activation is indicated.

Administration and assessment

In methods which involve administration to an individual, a peptide (or composition comprising a peptide) may be administrated to the individual using any convenient means capable of resulting in the desired target protein activity modulation. Thus, the peptide(s) (or composition comprising the peptide) can be incorporated into a variety of formulations for therapeutic administration. Compositions can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparation in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, transdermal patches, suppositories, injections, inhalants, and aerosols. As such, administration can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, etc., administration.

In pharmaceutical dosage forms, compositions may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting. For oral preparation, compositions can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules. Examples of additives are conventional additives, such as lactose, mannitol, corn starch or potato starch; binders, such as corn starch, potato starch or sodium carboxymethylcellulose; lubricants, such as talc or magnesium stearate; and if desired, diluents, buffering agents, moistening agents, preservatives and flavoring agents.

Compositions can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol. If desired, conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives may also be added. The concentration of therapeutically active compound in the formulation may vary from about 0.5-100 wt.%.

Compositions can be utilized in aerosol formulation to be administered via inhalation and can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, compositions can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases, and can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature. Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit (e.g., a teaspoonful, tablespoonful, tablet or suppository) contains a predetermined amount ofthe composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

The compounds are added to a host in a physiologically acceptable carrier. The dosage for compounds suppressing thrombin response is elected so that the PARI and/or PAR4 activity is increased or reduced by about 10 to 80%, respectively, more preferably about 20 to 70%, respectively and even more preferably about 25-50%), respectively. The dosage for compounds inhibiting the activity of PARI and PAR4 is elected so that the ability of platelets to respond to thrombin is reduced by about 20 to 80%, preferably about 40 to 50%, respectively.

Agents for use in the methods ofthe invention may be any compound displaying requisite activity. For example, they may be small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate compounds comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and may generally include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two ofthe functional chemical groups. The candidate compounds are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivative, structural analogs or combinations thereof.

These compositions may be formulated in a physiologically acceptable carrier, at a dosage suitable to achieve the desired result. The dosage for compounds suppressing cathepsin G-mediated PAR4 activation is such that PAR4 activation is reduced by at least about any ofthe following: 40%, 50%, 75%, 80%, 90%, 95%. In other embodiments, the decrease in degree of PAR4 activation includes, but is not limited to, about any ofthe following ranges: about 50% to 100%; 50% to 90%; 60% to 90%; 75% to 85%; 75% to 90%; 80% to 100%; 80% to 95%; 80% to 90%.

Platelet activation may be induced by a number of biological phenomenon, including injury, response to certain compounds, etc. Compositions are generally administered daily, although they may be administered less often, such as bi-weekly, weekly or monthly. With respect to inhibition of platelet activation, compositions are administered in an amount to provide at least about 50%, more preferably at least about 75%, even more preferably at least about 80%, even more preferably at least about 90% decrease in platelet activation. In other embodiments, the decrease in degree of platelet activation includes, but is not limited to, about any ofthe following ranges: about 50% to 100%; 50% to 90%; 60% to 90%; 75% to 85%; 75% to 90%; 80% to 100%; 80% to 95%; 80% to 90%. The amount may vary with the general health ofthe patient, the response of the patient to the drug, whether the composition is used by itself or in combination with other drugs, and the like. Daily administrations may be one or more times, usually not more than about four times, particularly depending upon the level of drug which is administered.

Administration ofthe compositions is particularly useful in the treatment of diseases such as myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, and other blood thromboses such as microvascular thrombosis and reperfusion injury. Inhibition of platelet activation in such disorders may allow localized treatment at the site ofthe clotting, thus eliminating some ofthe more unpleasant side effects of systemic treatment, e.g., hemorrhage.

Generally, but not necessarily, the composition(s) will be administered acutely (i.e., upon presentation ofthe clinical indication), by I.V.

Methods using PARI and/or PAR4 activating peptides and polynucleotides encoding the peptides

The polypeptides and polynucleotides of this invention have a variety of uses. They can be used, for example, as an agent to screen pharmaceutical candidates (both in vitro and in vivo), for rational (i.e., structure-based) drug design, as well as possible therapeutic uses as described above. Uses in pharmaceutical development will be described in more detail below. The PARI and/or PAR4 modulating peptides may also be used to identifying proteins, especially those that bind (or interact physically) with PARI and/or PAR4 and could thus themselves be drug targets. PARI and/or PAR4 modulating peptides may also be used to detect the presence of an antibody that binds to these polypeptide(s) or fragment(s) thereof. They may also be used to raise antibodies in a suitable host, which may be rabbit, mouse, rat, goat, or human, as non-inclusive examples. Such an antibody may also bind receptor and be clinically significant. It is possible that such antibodies, when present in humans, may confer some degree of activation or inhibition of PARI and/or PAR4. It is also possible that these antibodies may provide a therapeutic function inhibiting or stimulating platelet activation. The polypeptides of this invention thus may well prove to be useful in pharmaceutical applications, such as in therapeutic and/or prophylactic vaccines.

Methods of modulating PARI and/or PAR4 activity

The invention provides methods for modulating PARI and/or PAR4 activity (also referred to as modulating PARI and/or PAR4) by administering PARI and/or PAR4 modulating peptides. Administration of PARI and/or PAR4 modulating peptides has potential therapeutic uses. For example, if full-length PARI and/or PAR4 exerts action by binding to another protein, a PARI and/or PAR4 modulating peptide that binds competitively to PARI and/or PAR4 could compromise PARI and/or PAR4 function as a competitive inhibitor and thus exert regulatory/modulating activity. Table 1 provides peptides ofthe invention that activate PARI and/or PAR4. In particular, PAR4 activating peptides include GYPGRF, GYPG(homoR)F, GYPG(Orn)F, GYPGKY, GYPGKW, GYPGKK, SYPAKF, SYAGKF, SYPGRF, SYPG(homoR)F, AYPGKF, SYPGKF, TYPGKF, GFPGKF, and G(F)PGKF, wherein (homoR) is homoarginine and (Orn) is ornithine. PARI activating peptides include GYPAKF, GYP(Cha)KF, GYPLKF, GYPIKF, S(F)(Cha)(Cha)(homoR)K, and S(F)(Cha)(Cha)RK,

GFPGKF, and G(F)PGKF, wherein (Cha) is cyclohexylalanine, (F) is parafluoro- phenylalanine, and (homoR) is homoarginine. Peptides which activate both PARI and PAR4 include GFPGKF, G(F)PGKF and GYPAKF. Assays to measure and/or indicate activity of PARI and/or PAR4 are described herein and include for example, examining platelet shape change and platelet aggregation. Methods using PARI and/or PAR4 modulating peptides: modulate platelets The invention provides a method for modulating platelet function, preferably human platelet function through modulating PAR4 by administering potent PAR4- modulating peptides. The invention provides another method for modulating platelets, for example human platelets, through modulating both PARI and PAR4 simultaneously by administering the modulating peptide(s) that modulate both PARI and PAR4. PARI and

PAR4 are receptors that mediate human platelet activation by thrombin, a process critical in hemostasis and thrombosis. Activating either receptor can trigger platelet shape change, calcium signaling, secretion and aggregation, and inhibiting both receptors dramatically inhibits platelet activation by thrombin. Kahn et al. (1998); Kahn et al. (1999).

Accordingly, a single agent that modulates both PARI and PAR4 is highly desirable as a drug for modulating thrombin-triggered platelet activation. The invention provides such peptides that modulate both PARI and PAR4 activities.

Methods using PARI and/or PAR4 activating peptides: Desensitizing platelets The invention provides a method for desensitizing human platelets through desensitizing PAR4 by administering PAR4-activating peptides.

The invention provides another method for desensitizing human platelets through desensitizing both PARI and PAR4 simultaneously by administering the activating peptides that activate both PARI and PAR4.

Desensitization with PAR-specific activating peptides is useful for probing the role of specific receptors in differentiated cells. For examples, see Example 4.

Methods using PARI and/or PAR4 modulating peptides: Targeting other detectable and/or therapeutic agents to platelets

Suitable detectable molecules may be directly or indirectly attached to the polypeptides, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like. Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or vttrium-90 (either directly attached to the polypeptide, or indirectly attached through means of a chelating moiety, for instance). Polypeptides may also be conjugated to cytotoxic drugs, such as adriamycin. For indirect attachment of a detectable to cytotoxic molecule, the detectable or cytotoxic molecule can be conjugated with a member of a complementary/anti-complementary pair, where the other member is bound to the polypeptide. For these purposes, biotin/streptavidin is an exemplary complementary/anti- complementary pair.

In another embodiment, polypeptide-toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues).

Alternatively, if the polypeptide has multiple functional domains (i.e., an activation domain or a ligand binding domain, plus a targeting domain), a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest. In instances where the domain only fusion protein includes a complementary molecule, the anti-complementary molecule can by conjugated to a detectable or cytotoxic molecule. Such domain- complementary molecule fusion proteins thus represent a generic targeting vehicle for cell/tissue-specific delivery of generic anti-complementary-detectable/cytotoxic molecule conjugates. In another embodiment, PARI and/or PAR4 modulating peptide-cytokine fusion proteins can be used for enhancing in vivo killing of target tissues (for example, blood and bone marrow cancers), if the PARI and/or PAR4 modulating peptide targets the hyperproliferative blood or bone marrow cell (see, generally, Hornick et al. (1997) Blood 89:4437-4447). This reference described fusion proteins that enable targeting of a cytokine to a desired site of action, thereby providing an elevated local concentration of cytokine.

Suitable PARI and/or PAR4 modulating peptide target an undesirable cell or tissue (i.e., a tumor or a leukemia), and the fused cytokine mediated improved target cell lysis by effector cells. Suitable cytokines for this purpose include interleukin-2 and granulocyte- macrophage colony-stimulating factor (GM-CSF), for instance. In yet another embodiment, if the PARI and/or PAR4 modulating peptide targets vascular cells or tissues, such polypeptide may be conjugated with a radionuclide, and particularly with a beta-emitting radionuclide, to reduce restenosis. Such therapeutic approach poses less danger to clinicians who administer the radioactive therapy. For instance, iridium- 192 impregnated ribbons placed into stented vessels of patients until the required radiation does was delivered showed decreased tissue growth in the vessel and greater luminal diameter than the control group, which received placebo ribbons. Further, revascularisation and stent thrombosis were significantly lower in the treatment group. Similar results are predicated with targeting of a bioactive conjugate containing a radionuclide, as described herein.

The bioactive polypeptide conjugates described herein can be delivered intravenously, intraarterially or intraductally, or may be introduced locally at the intended site of action. These principles also apply to contexts in which a polynucleotide encoding a PAR-activating peptide is administered.

Screening assays for PARI and/or PAR4 modulating polypeptides The present invention encompasses methods of identifying agents that may modulate PARI and/or PAR4-mediated platelet activation based on their ability to modulate a characteristic associated with PARI and/or PAR4. These methods may be practiced in a variety of embodiments.

The methods described herein are in vitro and in vivo screening assays. In the in vitro embodiments, an agent is tested for its ability to modulate function of PARI and/or PAR4. In the in vivo embodiments, living cells having PARI and/or PAR4 function are used for testing agents. For purposes of this invention, an agent may be identified on the basis of only partial loss of PARI and or PAR4 function, although characteristics associated with total loss of PARI and/or PAR4 function may be preferable for antagonist. An agent may also be identified by its ability to enhance thrombin mediated PARI function and/or thrombin or cathepsin G-mediated PAR4 function. Accordingly, the screening methods ofthe invention encompass methods of identifying agonists which increase or elicit activation as well as methods of identifying antagonists which inhibit activity.

As indicated in the definition of "agent" above, agents which may be used in the screening methods described herein encompass numerous chemical classes. Candidate compounds can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological compounds may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. In addition, any polynucleotide could be used as an agent, the synthesis of which may be achieved by methods well known in the art. For example, the recombinant DNA molecules may be isolated from a human hematopoetic cDNA library. The synthesis of cDNA libraries and the choice of vector into wliich the cDNA molecules may be cloned are conventional techniques. With respect to the screening methods described herein, it is understood that (intact)

PAR4 (or PARI), functional fragments of PAR4 (or PARI), and/or functional equivalents (including functional equivalent fragments) of PAR4 (or PARI) may be used, as appropriate. For example, if thrombin or cathepsin G binds to a region(s) of PAR4 (or PARI) which may be isolated from intact PAR4 (or PARI), this region(s) may be used for certain studies, such as binding studies. Preparation of polypeptide fragments (and polynucleotides encoding polypeptide fragments) as well as preparation and testing of functionally equivalent variants uses techniques standard in the art. A functionally equivalent variant may be constructed, for example, by employing conservative amino acid substitutions. Accordingly, as the definitions of "PAR4" and "PARI" make clear, reference to "PAR4" or "PARI" in this section and throughout this application applies to any ofthe above PAR4 (or PARI) embodiments.

In vitro screening methods

In in vitro screening methods of this invention, an agent is screened in an in vitro system, in which an agent is tested for its ability to bind PARI and /or PAR4, and/or its ability to modulate PARI or PAR4 or platelet or any combination thereof. For an assay for an agent that binds to PARI and/or PAR4 or PARI and/or PAR4 polypeptides, PARI and/or PAR4 is first recombinantly expressed in a prokaryotic or eukaryotic expression system as a native or as a fusion protein in which the full length PARI and/or PAR4 or fragment of PARI and/or PAR4 is conjugated with a well- characterized epitope or protein. Recombinant PARI and/or PAR4 is then purified by, for instance, immunoprecipitation using anti-PARl and/or PAR4 antibodies or anti-epitope antibodies or by binding to immobilized ligand ofthe conjugate. An affinity column made of PARI and/or PAR4 or PARI and/or PAR4 fusion protein is then used to screen a mixture of compounds which have been appropriately labeled. Suitable labels include, but are not limited to flurochromes, radioisotopes, enzymes and chemiluminescent compounds.

The unbound and bound compounds can be separated by washes using various conditions (e.g. high salt, detergent ) that are routinely employed by those skilled in the art. Lechner and Carbon (1991) Cell 64:717-725. Similar methods can be used for screening for an agent(s) that competes for binding to PARI and/or PAR4. Competitive assays are known in the art, and generally involve measuring degree of binding in the presence of increasing amounts ofthe putative competitor. In addition to affinity chromatography, there are other techniques such as solution based binding systems. Non-specific binding to the affinity column can be minimized by pre-clearing the compound mixture using an affinity column containing merely the conjugate or the epitope. A similar method can be used for screening for agents that competes for binding to PARI and/or PAR4 polypeptides. In addition to affinity chromatography, there are other techniques such as measuring the change of melting temperature or the fluorescence anisotropy of a protein which will change upon binding another molecule. For example, a BIAcore assay using a sensor chip (supplied by Pharmacia Biosensor, Stitt et al. (1995) Cell 80: 661-670) that is covalently coupled to native PARI and/or PAR4 or PARI and/or PAR4-fusion proteins, may be performed to determine the PARI and/or PAR4 binding activity of different agents.

It is also understood that the in vitro screening methods of this invention include structural, or rational, drug design, in which the amino acid sequence, three-dimensional atomic structure or other property (or properties) of PARI and/or PAR4 (or PARI and/or PAR4 polypeptide) provides a basis for designing an agent which is expected to bind to PARI and/or PAR4 (or PARI and/or PAR4 polypeptide). Generally, the design and/or choice of agents in this context is governed by several parameters, such as the perceived function ofthe PARI and/or PAR4 (or PARI and/or PAR4 polypeptide) target, its three- dimensional structure (if known or surmised), and other aspects of rational drug design. Techniques of combinatorial chemistry can also be used to generate numerous permutations of candidate agents. For purposes of this invention, an agent designed and/or obtained by rational drug designed may also be tested in the in vivo assays described below. As detailed in Example 1, alanine or serine substitution at position one of GYPGKF yielded a gain-of-function of PAR4 activation. Substitution of phenylalanine or parafluorophenylalamne for tyrosine at position two of GYPGKF yielded a peptide that can activate both PARI and PAR4. Based on such structure-function data for PARI and/or

PAR4 activating peptide epitopes, additional PARI and/or PAR4 modulating peptides can be rationally designed based on the peptide-mimetic design concept. Andrade-Gordon et al. (1999).

In vivo screening methods

In in vivo screening assays, a living cell containing a functioning PARI and/or PAR4 gene, or a living cell containing a polynucleotide construct comprising a PARI and/or PAR4 encoding sequence are exposed to an agent. In contrast (as described above), conventional drug screening assays have typically measured the effect of a test agent on an isolated component, such as an enzyme or other functional protein.

The in vivo screening assays described herein have several advantages over conventional drug screening assays: 1) if an agent must enter a cell to achieve a desired therapeutic effect, an in vivo assay can give an indication as to whether the agent can enter a cell; 2) an in vivo screening assay can identify agents that, in the state in which they are added to the assay system are ineffective to elicit at least one characteristic which is associated with compromise of PARI and/or PAR4 function, but that are modified by cellular components once inside a cell in such a way that they become effective agents; 3) most importantly, an in vivo assay system allows identification of agents affecting any component of a pathway that ultimately results in characteristics that are associated with PARI and/or PAR4 function. In general, screening is performed by adding an agent to a sample of appropriate cells, and monitoring the effect. The experiment preferably includes a control sample which does not receive the candidate agent. The treated and untreated cells are then compared by any suitable phenotypic criteria, including but not limited to intracellular calcium mobilization, phosphoinositide hydrolysis, change in platelet cell shape

(morphology) and platelet activation.

For the cell-based screening assays described herein, it is understood that thrombin or cathepsin G-mediated modulation of PAR4 (or PARI) is measured, i.e., the modulation must be specific interference, mimicry, and/or enhancement of thrombin or cathepsin G- mediated PAR4 (or PARI) activation. This specificity may be determined using experimental methods and designs standard in the art. For example, a reaction is conducted using a PAR4 (or PARI) expressing cell (whether naturally-occurring or recombinant) in the presence of agent and thrombin; a parallel experiment is conducted using a PAR4 (or PARI) expressing cell in the presence of agent without thrombin. As another example, a reaction is conducted using a PAR4 (or PARI) expressing cell(s) in the presence of agent; an effect, if any, is compared to conditions in the presence of varying amounts of thrombin (or, conversely, in the presence of varying amounts of agent while in the presence of thrombin). As another example, a reaction is conducted using a PAR4 expressing cell (whether naturally-occurring or recombinant) in the presence of agent and cathepsin G; a parallel experiment is conducted using a P AR4 expressing cell in the presence of agent without cathepsin G. As yet another example, a reaction is conducted using a PAR4 expressing cell(s) in the presence of agent; an effect, if any, is compared to conditions in the presence of varying amounts of cathepsin G (or, conversely, in the presence of varying amounts of agent while in the presence of cathespin G). Competition-based assays to establish specificity are known in the art as well as described herein. For example, an assay can be performed with and without thrombin using PAR4 (or PARI) expressing cells and cells that do not express PAR4 (or PARI). As another example, an assay could be performed in the presence of thrombin, and PAR4 (or PARI) cleavage could be monitored using, for instance, an antibody that binds to the PAR4 (or PARI) cleavage site (i.e., an antibody which binds to the amino terminal exodomain amino cleavage site). As an example, an assay can be performed with and without cathepsin G using PAR4 expressing cells and cells that do not express PAR4. As another example, an assay could be performed in the presence of cathepsin G, and PAR4 cleavage could be monitored using, for instance, an antibody that binds to the PAR4 cleavage site (i.e., an antibody which binds to the amino terminal exodomain amino cleavage site). In one embodiment, an agent is identified by its ability to modulate a characteristic associated with PARI and/or PAR4 function in a suitable host cell. In some embodiments, the modulation is an increase or activation of thrombin or cathepsin G-mediated PAR4 (or PARI) activation (i.e., the screening methods identify agonists). In other embodiments, the modulation is a decrease (which can be partial to total loss) of thrombin or cathepsin G- mediated PAR4 (or PARI) activation (i.e., the screening methods identify antagonists).

For characterizing a PARI and/or PAR4 modulating peptide for its ability to modulate platelet function, standard assays exist in the art. For instance, the ability of a PARI and/or PAR4 modulating peptide to modulate PARI and/or PAR4 activity can be determined by testing phosphoinositide hydrolysis and intracellular calcium mobilization as described in Example 4. The ability of a PARI and/or PAR4 modulating peptide to modulate platelet activation can be determined by observing platelet shape change or by platelet secretion or aggregation assay as described in Example 4.

Accordingly, in one embodiment, the invention provides methods for identifying an agent that may modulate PARI and/or PAR4 comprising the following steps: (a) contacting a suitable host cell comprising PARI and/or PAR4 function with said agent to be tested and a PARI and/or PAR4 activating peptide; and (b) analyzing at least one characteristic which is associated with PARI and/or PAR4 function in said host cell, wherein an agent is identified by its ability to modulate at least one such characteristic as compared to contacting the suitable host cell with said PARI and/or PAR4 activating peptide without the agent. The present invention also encompasses agents that modulate PARI and/or

PAR4 identified by the methods.

For these methods, the host cell may be any cell in wliich PARI and/or PAR4 function has been demonstrated. PARI and/or PAR4 function may arise due to naturally- occurring PARI and/or PAR4 encoding sequences in the host cell or due to recombinant expression (i.e., expression of recombinant PARI and/or PAR4 sequence). Thus, examples of host cells include, but are not limited to, platelet cells, or other suitable host cells, preferably mammalian cells, expressing recombinant PARI and/or PAR4. Examples of suitable host cells include, but are not limited to, Xenopus oocytes, COS7 cells, mouse fibroblasts, Ratl cells, HEK 293 cells, CHO cells, CV1 cells, L cells, and HeLa cells. In another embodiment, these methods further comprise the step of introducing a polynucleotide encoding PARI and/or PAR4 (or a functional fragment thereof) into a suitable host cell that otherwise lacks PARI and/or PAR4 function. The host cell used for these methods initially lacks PARI and/or PAR4 function (i.e., lacks PARI and/or PAR4 function before introduction of polynucleotide encoding PARI and/or PAR4). Lacking PARI and/or PAR4 function may be partial to total. A suitable host cell in this context is any host cell in which recombinant PARI and/or PAR4 complements a defect of host cell PARI and/or PAR4 function. For example, PARI -deficient mouse lung fibroblasts (KOLFs, see Trejo et al. (1996) J Biol. Chem. 271:21536-21541; Connolly et al. (1996) Nature 381:516-519) provide a new and convenient basis for screening. Devising host cells that lack PARI and/or PAR4 (or its homologue) function may be achieved in a variety of ways, including, but not limited to, genetic manipulation such as deletion mutagenesis, recombinant substitution of a functional portion ofthe gene, frameshift mutations, conventional or classical genetic techniques pertaining to mutant isolation, or alterations of the regulatory domains. In addition, host cells derived from certain type of tissues, such as fibroblasts lack PAR4 function. Determination of whether a cell lacks PAR4 function is well within the skill ofthe art.

Determining whether recombinant PARI and/or PAR4 product can substitute for the host cell's PARI and/or PAR4 (or homologue) gene product is within the skill ofthe art. For example, the host cell's PARI and/or PAR4 (or homologue) function may be deleted by, for instance, recombinant methods. A polynucleotide encoding PARI and/or PAR4 or a functional fragment thereof, is then introduced into the cell, depending on the particular host cell used, by using any ofthe many methods known in the art, including but not limited to electroporation, CaCl₂ precipitation, and lipofectamine treatment ofthe host cells. Polynucleotides introduced into a suitable host cell(s) are polynucleotide constructs comprising a polynucleotide encoding PARI and/or PAR4 or a functional fragment thereof. These constructs contain elements (i.e., functional sequences) which, upon introduction of the construct, allow expression (i.e., transcription, translation, and post-translational modifications, if any) of PARI and/or PAR4 amino acid sequence in the host cell. Exemplary methods and procedures for generating such host cells either transiently or stably expressing recombinant PARI and/or PAR4 are described in Kahn et al. (1999); Connolly et al. (1996), and are generally known to the practitioners in the art. Restoring PARI and/or PAR4 (or its homologue) function in the host cell(s) may be determined by analyzing the host cell(s) for various detectable parameters associated with PARI and/or PAR4 function (i.e., wild type). Such parameters include, but are not limited to, response to thrombin or activating peptides. Examples of determining complementarity of recombinant PARI and/or PAR4 for PARI and/or PAR4 function are described in Example 1.

For genes that are de-repressed upon loss of PARI and/or PAR4 function, loss of PARI and/or PAR4 function may be measured using a reporter system, in which a reporter gene sequence is operatively linked to the PARI and/or PAR4-repressed gene of interest. As used herein, the term "reporter gene" means a gene that encodes a gene product that can be identified (i.e., a reporter protein). Reporter genes include, but are not limited to, alkaline phosphatase, chloramphenicol acetyl transferase, β-galactosidase, luciferase and green fluorescence protein. Identification methods for the products of reporter genes include, but are not limited to, enzymatic assays and fluorimetric assays. Reporter genes and assays to detect their products are well known in the art and are described, for example in Current Protocols in Molecular Biology, eds. Ausubel et al., Greene Publishing and

Wiley-Interscience: New York (1987) and periodic updates. Reporter genes, reporter gene assays and reagent kits are also readily available from commercial sources (Strategene, Invitrogen and etc.).

The screening methods described above represent primary screens, designed to detect any agent that may exhibit modulation activity on PARI or PAR4 or platelet or any combination thereof. The skilled artisan will recognize that secondary tests will likely be necessary in order to evaluate an agent further. For example, a secondary screen may comprise testing the agent(s) in human cell if the initial screen has been performed in a host cell other than human cell. Another screen may comprise testing the agent(s) in a host cell with native PARI and/or PAR4 function if the initial screen has been performed in a host cell without native PARI and/or PAR4 function but being introduced of such function. An infectivity assay using mice and other animal models (such as rat) are known in the art. In addition, a cytotoxicity assay would be performed as a further corroboration that an agent which tested positive in a primary screen would be suitable for use in living organisms. Any assay for cytotoxicity would be suitable for this purpose, including, for example the MTT assay (Promega).

Screening methods for PARI and/or PAR4 activating peptides (agonists) The invention provides methods for identifying an agent that activates PARI and/or PAR4. These methods may be in vitro or in vivo (i.e., cell-based, tissue-based, and/or animal based). In one embodiment, the invention provides a method for identifying an agent that activates PAR4, said method comprising (a) contacting a suitable host cell comprising PARI and/or PAR4 function with said agent to be tested and a PARI and/or PAR4 activating peptide; and (b) analyzing at least one characteristic wliich is associated with activation of PARI and/or PAR4 function in said host cell, wherein an agent is identified by its ability to modulate at least one such characteristic as compared to contacting the suitable host cell with said PARI and/or PAR4 activating peptide without the agent.

It is understood that such a host cell either has the native PARI and/or PAR4 function, such as a platelet cell, or is restored of PARI and/or PAR4 function by the expression of recombinant PARI and/or PAR4 or their functional equivalent(s), as described above.

In some embodiments, a host cell that has both PARI and PAR4 function is used to test both PARI and PAR4 activation simultaneously.

In one embodiment, PAR-deficient mouse lung fibroblasts (KOLFs) that has been stably transfected with cDNAs encoding human PARI (KOLF-PARl) and/or PAR4

(KOLF-PAR4) are used for functional assays of receptor activation and agonist specificity. To screen PARI and/or PAR4 activating peptides, KOLF-PAR4 is incubated with the agents to be screened. Total [³H]-inositol phosphate released is measured by the phosphoinositide hydrolysis assay as described in Example 1. The activating potency of the agents to be screened can be also measured by the intracellular calcium mobilization assay as described in Example 1. It is understood that the screening methods described above can be used to test the specificity ofthe agonists. In an instance that an agonist can inhibit PARI activity but not PAR4 activity, it is said that the agonist is a PARI -specific agonist. Conversely, in an instance that an agonist can inhibit PAR4 activity but not PARI activity, it is said that the agonist is a PAR4-specific agonist.

The agent can be any compound, complex or substance. It can be chemically synthesized or made by expression systems, using recombinant methods, or made by other procedures known in the art. In one embodiment, it can be made from a phage display library.

Screening methods for PARI and/or PAR4 inhibiting peptides (antagonists) The invention provides methods for identifying an agent that inhibits/blocks PARI and/or PAR4 activity/function. These methods may be in vitro or in vivo (i.e., cell-based, tissue-based, and/or animal based). In one embodiment, the invention provides a method for identifying an agent that inhibits PARI and/or PAR4, said method comprising (a) contacting a suitable host cell comprising PARI and/or PAR4 function with said agent to be tested and a PARI and/or PAR4 activating peptide disclosed herein, and analyzing at least one characteristic associated with the inhibition of PARI and/or PAR4 activation in said host cell, wherein an agent is identified by its ability to modify at least one such characteristic as compared to contacting the suitable host cell with said PARI and/or PAR4 activating peptide without the agent.

In another aspect, the invention provides methods for identifying an agent that inhibits/blocks PARI and PAR4 activities/functions simultaneously. These methods may be in vitro or in vivo (i.e., cell-based, tissue-based, and/or animal based). It is understood that the screening methods described above can be used to test the specificity ofthe antagonists. In an instance that an antagonist can inhibit PARI activity but not PAR4 activity, it is said that the antagonist is a PARI -specific antagonist. Conversely, in an instance that an antagonist can inhibit PAR4 activity but not PARI activity, it is said that the antagonist is a PAR4-specific antagonist. The agent can be any compound, complex or substance. It can be chemically synthesized or made by expression systems, using recombinant methods, or made by other procedures known in the art. In one embodiment, it can be made from a phage display library.

The general screening strategy is to introduce a pharmaceutical candidate into a host cell that has PARI and or PAR4 activity and then determine whether the effect (if any) is beneficial, and preferably specific. Application ofthe agent can be direct (such as determining whether a candidate binds to PARI and/or PAR4 polypeptide in the assay) in an in vitro system, but also be used in an in vivo system, such as cell culture. For example, an agent that modulates the activity of PARI and/or PAR4 described herein has the potential to block any associated pathology when administered. It is not necessary that the mechanism of modulation be known; only that the alteration affect desirable cells preferably without being significantly detrimental to other cells.

Modulation of PARI or PAR4 or platelet or any combination thereof function may occur at any level that affects the function of PARI or PAR4 or platelet or any combination thereof. An agent may modulate PARI and/or PAR4 function by binding to PARI and/or PAR4. An agent may modulate PARI and/or PAR4 function by preventing, reducing or increasing production of a PARI and/or PAR4 binding protein. An agent may modulate

PARI and/or PAR4 function by binding to a polypeptide that binds PARI and/or PAR4.

The agent can be any compound, complex or substance. Generally, the choice of agents to be screened is governed by several parameters, such as the particular polynucleotide or polypeptide target, its perceived function, its three-dimensional structure (if known or surmised), and other aspects of rational drug design. Techniques of combinatorial chemistry can also be used to generate numerous permutations of candidates. Those of skill in the art can devise and/or obtain suitable agents for testing.

Methods for Selectively modulating PAR4-mediated platelet activation

The invention provides a method for selectively modulating PAR4 and/or PAR4- mediated platelet activation. Unlike PARI, PAR4 couples to Gq but not to Gi, see Example 3. Accordingly, PAR4-mediated platelet activation can be selectively modulated by agents that modulate Gq activity but do not modulate Gi activity. Methods using PARI and PAR4 activating peptides: Cloning genes and gene products along the PARI and/or PAR4 signaling functional pathway

The invention also provides methods for cloning genes and gene products that are involved in, and/or associated with, a PARI and/or PAR4 function (i.e., a PARI and/or PAR4 functional/signaling pathway). Because PARI and PAR4 function has been shown to play an important role in platelet activation, genes that are involved in a PARI and/or PAR4 pathway may well be suitable and useful drug targets. Further, these gene(s) and gene product(s) may provide even more precise, specific targets for drug discovery and development, and hence therapy. Accordingly, the invention provides methods of isolating genes involved in a PARI and/or PAR4 pathway which entail the following step: (a) identification of polynucleotide sequences which are repressed upon PARI and/or PAR4 activation by PARI and/or PAR4 activating peptides. For these methods, the polynucleotides are identified using standard techniques in the art for determining differential expression, such as representational difference analysis (RD A).

Preferably, the methods include an additional step of identifying those sequences from step (a) above which are expressed when platelet is activated. Presumably, this sequence is then considered to be required for platelet activation. Platelet activation may be induced, for example, by thrombin. In this embodiment, the sequence(s) so identified may be said to be associated with platelet activation as well as associated with PARI and/or PAR4 function/signaling.

Still more preferably, the methods include an additional step of identifying those sequences from step (a) and/or step (b) above which, when deleted, mutated, substituted, or otherwise altered such that the function ofthe expression product is compromised, inhibits platelet activation In this embodiment, the sequence(s) so identified may be said to be required for platelet activation as well as associated with PARI and/or PAR4 function/signaling.

Similarly, the invention provides methods of isolating genes involved in a PARI and/or PAR4 pathway which are de-repressed upon PARI and/or PAR4 activation by PARI and/or PAR4 activating peptides. The following examples are provided to illustrate but not limit the present invention.

EXAMPLES

All chemicals unless otherwise stated were obtained from Sigma Biochemical Co. ³H-myoinositol and ³H-adenine were obtained from Amersham (Arlington Heights, IL). FURA-2 AM was obtained from Molecular Probes (Eugene, OR). Bordatella pertussis toxin islet-activating protein was obtained from List Biologicals (Campbell, CA). α- thrombin was obtained from Enzyme Research Laboratories (South Bend, IN). The thromboxane receptor agonist, U46619, was obtained from Calbiochem-Novabiochem, Corp. (San Diego, CA). Peptides were synthesized as carboxyl terminal amides, purified by high-pressure liquid chromatography, and characterized by mass spectroscopy.

Example 1

Structure-function requirements for PAR4-activating peptides

Cells and Cell Lines. Stable cell lines based on KOLFs, a cell line derived from lung fibroblasts from PARI knockout mice (see Connolly et al. (1996); Trejo et al. (1996)), were generated by transfecting these cells with bBJl -based mammalian expression vectors that directed hPARl or hPAR4 together with a hygromycin resistance vector. Kahn et al. (1999); Ishii et al. (1993). The PARI and PAR4 cDNAs used in these studies encoded receptors bearing an amino terminal FLAG epitope. KOLF clones resistant to hygromycin were screened for receptor expression by cell surface ELISA for the FLAG epitope using Ml monoclonal antibody. Ishii et al. (1993). KOLFs lines were maintained in Dulbecco's

Modified Eagle's medium containing 10% calf serum and 200 μg/ml of hygromycin. Analogous Rat 1 cell lines were derived by transfecting the same expression vectors and a neomycin resistance vector then screening G418-resistant clones for Ml antibody binding. Ishii et al. (1993). Phosphoinositide hydrolysis assay. Phosphoinositide hydrolysis was measured as following. Cells cultured in 24 well plates were labeled overnight with myo-[³H]-inositol (2 μCi/ml) in serum-free DME media containing 1 mg/ml BSA, 20 mM HEPES, and penicillin/streptomycin. Following labeling, the cells were incubated in the absence or presence of pertussis toxin (0.1 - 100 ng/ml) for 5 hours at 37°C. Agonists were then added with 20 mM LiCl in serum-free media and incubated at 37°C for 20-120 min. Cells were extracted and total [³H]-inositol phosphates quantitated as described in Hung et al. (1992b).

Intracellular calcium mobilization assay. Intracellular calcium mobilization was measured fluorometrically using FURA-2 as described in Ishihara et al. (1998); Sage (1996) in Platelets, A Practical Approach (Watson, S. P., and Authi, K. S., eds), pp. 67-90,

IRL Press, Oxford.

Results. To define the structure-function requirements for PAR4-activating peptides, we tested a series of hexapeptides based in the sequence GYPGKF. GYPGKF represents the first six amino acids ofthe new amino terminus unmasked when thrombin cleaves mouse PAR4 and was chosen as the starting sequence because it was more than the cognate human sequence GYPGQV on both the mouse and human receptors. PAR- deficient mouse lung fibroblasts (KOLFs) that had been stably transfected with cDNAs encoding human PAR4 (KOLF-PAR4) and PARI (KOLF-PARl) (see Kahn et al. (1999); Connolly et al. (1996)) were used for functional assays of receptor activation and agonist specificity. Untransfected KOLFs showed no responses to thrombin or to the peptides tested and thus responses in KOLF-PARl and KOLF-PAR4 cells were dependent upon PARI and PAR4, respectively.

Phosphoinositide hydrolysis. To examine phosphoinositide hydrolysis triggered by synthetic peptides in fibroblasts expressing PARI or PAR4, KOLF cells stably transfected with either human PARI or PAR4 cDNAs were incubated with the indicated peptide (500 μM) for 60 minutes and total [³H]-inositol phosphate released was measured. The results are shown in Table 2. (^α. Results were normalized to the maximum responses elicited by thrombin (30 nM); values shown are the means + S.E. of at least two experiments done in triplicate (Table 2).

Untransfected KOLF exhibited no increase in phosphoinositide hydrolysis in response to peptides or α-thrombin. The fold increase in [³H]-inositol phosphate release in response to α-thrombin ranged from 7-16 fold in PARI -expressing cells and from 10-36 fold in PAR4- expressing cells.

TABLE 2

Position of % of α-Thrombin % of α-Thrombin

Altered Synthetic Peptide Response for PAR4 Response for PARI

Amino Acid

None GYPGKF 55 ± 4.9 0

SFLLRN 0 ^' 120 ± 4.7

1 AYPGKF 92 ± 4.0 0

SYPGKF 92 ± 4.3 0

TYPGKF 21 ± 0.5 0

MprYPGKF 0 0

2 GFPGKF 53 ± 4.5 35 ± 2.5

G(F)PGKF 61 ± 18.0 111 ± 19.5

GLPGKF 0 0

3 GYAGKF 4 ± 0.3 4 ± 4.0 GYGGKF 0 0

GYLGKF 4 ± 0.5 4 ± 3.5

GYIGKF 4 ± 0.5 6 ± 4.5

GYKGKF 3 ± 0.0 7 ± 4.5

GYRGKF 4 ± 0.5 6 ± 5.5

GY(Cha)GKF 4 ± 0.5 7 ± 4.5

4 GYPAKF 21 ± 6.0 16 ± 4.5

GYP(Cha)KF 3 ± 0.0 111 ± 4.0

GYPLKF 4 ± 0.5 106 ± 9.0

GYPIKF 3 ± 0.0 41 ± 5.0

5 GYPGRF 63 ± 15.0 0

GYPG(homoR)F 73 ± 14.2 3 ± 3.3

GYPG(Orn)F 51 ± 1.0 0

6 GYPGKY 72 ± 14.5 5 ± 5.0

GYPGKW 47 ± 5.0 0

GYPGKK 24 ± 1.5 0

GYPGK(Orn) 0 0

Multiple Sites SYPAKF 45 ± 4.0 0

SYAGKF 20 ± 2.5 0

SYPGRF 76 ± 8.0 0

SYPG(homoR)F 87 ± 9.0 2 ± 1.5

S(F)(Cha)(Cha)(homoR)K 2 ± 1.5 121 ± 1.0

S(F)(Cha)(Cha)RK 2 ± 2.0 140 ± 10.0

(Cha), cyclohexylalanine; (F), parafluoro-phenylalanine; (homoR), homoarginine; (Orn), ornithine; Mpr, mercaptoproprionic acid; other letters represent the single letter amino acid code. Intracellular Calcium Mobilization

To examine intracellular calcium mobilization in response to synthetic agonist peptides, KOLF-PARl and KOLF-PAR4 cells were loaded with FURA-2/AM, and increases in cytoplasmic calcium in response to α-thrombin (30 nM), AYPGKF peptide (500 μM), SYPGKF (500 μM), or SFLLRN (100 μM) were measured fluorometrically. These data were representative of three replicate experiments done in duplicate.

Other examples of PARI and/or PAR4 and/or platelet modulators include A(F)PGKF, S(F)PGKF, AYP(Cha)KF, SYP(Cha)KF, AYPLKF, and SYPLKF, wherein (F) is parafluoro-phenylalanine and (Cha) is cyclohexylalanine.

We first compared the native PAR4 and PARI peptide GYPGKF and SFLLRN. GYPGKF elicited only 55% ofthe maximal response triggered by thrombin in PAR4-expressing cells, even when added at 500 μM. By contrast, in PARI -expressing cells, the maximal responses to SFLLRN and thrombin were of similar magnitude. Both peptides were specific for their respective receptors in this cell system (Table 2 and Figure 1).

We next performed an alanine scan and a series of other substitutions designed to identify sites important for activity and specificity at PAR4 vs. PARI . Surprisingly, alanine or serine substitution at position one of GYPGKF yielded a gain-of-function for PAR4 activation (Table 2). Threonine substitution caused a loss of function. These data suggest that small aliphatic side chains are preferred at position 1. Substitution of a mercaptoproprionic acid (Mpr) for serine in SFLLRN nearly abolished agonist activity for PARI. U.S. Patent 5,759,994; Seiler et al.(1995) Biochem. Pharmacol. 49:519-528. The cognate substitution of Mpr for glycine in GYPGKF also resulted in loss of agonist function. MprYPGKF and SYPGKF differ at only two positions. The Mpr peptide lacks an amino terminal protonated amino group and has a sulfur atom at the position corresponding to oxygen in the serine side in SYPGKF. The amino terminal protonated amino group of SFLLRN is critical for agonist function at PARI, thus it is likely that the protonated amino group in GYPGKF is critical for agonist activity at PAR4. This is consistent with the notion that the proteolytic switch in PARs involves both removal of sequence amino to the cleavage site and creation of a new protonated amino group that functions in the context ofthe tethered ligand. Scarborough et al. (1992); Coller et al. (1992)

Biochemistry 31:11713-11720; Coughlin (1999) Proc. Natl. Acad. Sci. USA 96:11023-11027.

In contrast to the gain-of-function seen with alanine substitution at position 1, substitution of alanine for the tyrosine at position 2 in GYPGKF resulted in a loss of agonist activity. Thus, as in the PARI agonist SFLLRN, the aromatic side chain at position 2 is critical for activity. Interestingly, substitution of phenylalanine or para- fluorophenylalanine for tyrosine did not decrease agonist activity for PAR4 but did cause a remarkable gain for function for PARI (Table 2). This result suggests that the presence of a tyrosine at position two is an important determinant of GYPGKF's activity and of its specificity for PAR4 over PARI . More importantly, this result shows that it is possible to develop agonists and perhaps antagonists, that act on both PARI and PAR4.

Substitution of alanine for proline at position three or for glycine at position four in GYPGKF resulted in substantial loss of agonist function for PAR4 (Table 2). This loss of function was also noted in the context ofthe SYPGKF better-than-native peptide. Proline and glycine are often found in beta-turns, thus proline three and glycine four may be important for a conformation necessary for agonist function.

Substitution of leucine, cyclohexylalanine, or isoleucine for glycine at position four all caused gain of function for PARI, consistent with what is taught in Scarborough et al. (1992); Seiler et al. (1996) Mol. Pharmacol. 49:190-197, but also caused loss of function at PAR4. Substitutions at positions five and six that might be expected to yield gain-of-function at PARI failed to do so (Table 2). Conversely, the better-than-native PARI agonists S(F)(Cha)(Cha)RK and S(F)(Cha)(Cha)(homoR)K had no activity at PAR4 (Table 2).

The results in Table 2 were confirmed in two ways. First, all ofthe phosphoinositide hydrolysis studies were verified at a semiquantitative level using peptide triggered increases in cytoplasmic calcium, measured fluorometrically, as an endpoint (see below). Second, important experiments were replicated using Rat 1 cell lines stably transfected with hPARl or hPAR4. These studies yielded results very similar to those obtained with the KOLF-based cell lines shown in Table 2. Specifically, in Rat 1 cells expressing PARI or PAR4, 30 nM thrombin caused 11- and 19-fold increases in phosphoinositide hydrolysis, respectively. Untransfected cells showed no significant response. In cells expressing PARI or PAR4, the response to 500μM SFLLRN was 130% or 3% ofthe maximal response to thrombin, respectively. The cognate responses to 500μM AYPGKF were 4% or 97% ofthe maximal response to thrombin, respectively. Thus, even at 500μM, SFLLRN and AYPGKF were relatively specific for their respective receptors. By contrast, the peptides GFPGKF and G(F)PGKF, which demonstrated an ability to activate both PARI and PAR4 expressed in KOLFs, showed similar activity in the Ratl system. The response to 500μM GFPGKF in Ratl cells expressing PARI or PAR4 was 72% or 81% ofthe maximal thrombin response, respectively. The cognate responses to G(F)PGKF were 131 or 76%, respectively.

Example 2

PAR4 activating peptides AYPGKF and SYPGKF are specific for PAR4 and intrinsically more active than GYPGKF at PAR4

The structure-activity scan above yielded two peptides SYPGKF and

AYPGKF that were more potent than the native GYPGKF sequence at stimulating PAR4-mediated phosphoinositide hydrolysis. In the phosphoinositide hydrolysis assay, cells were exposed to agonist for 60 minutes, raising the possibility that increased activity of a given peptide might be due to better stability in cell culture. To test this possibility and to further probe the specificity of these peptides, we examined their ability to trigger immediate increases in cytoplasmic calcium in KOLF-PAR4 and KOLF-PARl cell lines (Figure 1). AYPGKF and SYPGKF triggered increases in cytoplasmic calcium concentration in KOLF-PAR4; over several experiments, the peak calcium response elicited by peptide was similar to that elicited by thrombin in these cells. AYPGKF and SYPGKF triggered little response in KOLF-PARl . Conversely, SFLLRN induced calcium mobilization in KOLF- PARl but not in KOLF-PAR4 cells. We next compared the relative potencies of GYPGKF, SYPGKF, and

AYPGKF in the phosphoinositide hydrolysis and cytoplasmic calcium assays (Figures 2 and 3) in KOLF-PAR4 cells. GYPGKF appeared to be a partial agonist for PAR4-triggered phosphoinositide hydrolysis; even at 500 μM, GYPGKF elicited only approximately 50% ofthe maximal response to thrombin (Figure 2). By contrast, both AYPGKF and SYPGKF were full agonists for PAR4 activation and showed EC50S of 20 μM and 50 μM, respectively, in this assay. Similarly, AYPGKF stimulated calcium mobilization in KOLF-PAR4 cells with an EC50 of approximately 25 μM (Figure 3), while more than 200 μM GYPGKF was required to elicit similar responses in this assay. Thus in assays in which cellular effects are measured over either seconds or hours, AYPGKF was substantially more potent than GYPGKF.

These data militate against peptide stability as being responsible for the greater activity of AYPGKF and instead suggest that AYPGKF is intrinsically more active than GYPGKF at PAR4.

AYPGKF appeared to be potentially useful as a probe of PAR4 function. To further assess specificity, we examined the ability of this peptide to activate calcium mobilization in Xenopus oocytes expressing human PARI , PAR2, PAR3, or PAR4. AYPGKF (500 μM) elicited ~10-fold increases in ⁴⁵Ca release from PAR4- expressing oocytes, but only ~1.5 fold increase in PARI expressing oocytes and no responses in oocytes expressing hPAR2 or hPAR3. Oocytes expressing PARI, PAR2, and PAR3 did respond robustly to SFLLRN, SLIGRL, and thrombin, respectively. Thus even in our most sensitive assay system, AYPGKF was relatively specific for PAR4 vs. the other known PARs.

Example 3

PAR4 couples to G_q but not Gj

Adenylyl cyclase activity assay. The accumulation of ³H-cAMP in ³H- adenine-labeled cells was measured as described in Hung et al. (1992c) J Biol. Chem. 353:20831-20834. Briefly, cells were grown in 24 well plates and labeled with ³H-adenine (2 μCi/ml) overnight at 37°C in 1 ml HEPES-buffered serum-free DME media + 0.1% BSA. Cells were treated in the presence or absence of pertussis toxin (0.1 - 100 ng/ml) for 5 hours at 37°C, then incubated with forskolin and the phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine (IBMX) (1 mM) for 30 min at 37°C in the presence or absence of α-thrombin (30 nM) or agonist peptides. Cells were then extracted and ³H-cAMP and total ³H-adenine nucleotides were assayed as described in Hung et al. (1992c).

Transfection. COS-7 cells were transiently transfected for 5 hrs with 2 μg/ml DNA (human PARI or PAR4 cDNA in the mammalian expression vector, pBJl) using lipofectamine and OPTI-MEM media (Gibco BRL - Grand Island, NY) as per manufacturer's instructions. Following transfection, the cells were incubated in DME media containing 10% calf serum ovemight. The cells were then seeded onto 24-well plates for phosphoinositide hydrolysis assays. All cell culture was at 37°C with 5% CO₂. Results. PARI has been shown to couple to G-proteins ofthe Gj, G_q, and

G_12/i₃ subfamilies. Hung et al. (1992c); Offermanns et al. (1994) Proc. Natl. Acad. Sci. USA 91:504-508; Aragay et al. (1995) J. Biol. Chem. 270:20073-20077; Barr et al. (1997) J. Biol. Chem. 272:2223-2229. We first determined whether PAR4 might exhibit a different coupling pattern by measuring phosphoinositide hydrolysis and inhibition of adenylyl cyclase in the presence of absence of pertussis toxin, then compared signaling in response to thrombin vs. AYPGKF to determine whether such agonists recapitulated the signaling pattern elicited by thrombin.

Thrombin or SFLLRN inhibited forskolin-stimulated adenylyl cyclase activity in a pertussis toxin sensitive manner in PARI -KOLF cells (Figure 4), consistent with PARI 's known coupling to G^. Hung et al. (1992c). Moreover, phosphoinositide hydrolysis in response to thrombin or SFLLRN was slightly inhibited by pertussis toxin in KOLF-PARl cells and was more substantially inhibited by pertussis toxin in

COS-7 cells transiently transfected with PARI (Figure 5). This is consistent with a Gi-mediated contribution to phospholipase C activation. Blank et al. (1993) J Biol. Chem. 268:25184-25191. By contrast, neither thrombin, GYPGKF, nor AYPGKF inhibited adenylyl cyclase in PAR4-expressing KOLFs or COS-7 cells (Figure 4). Moreover, PAR4-mediated phosphoinositide hydrolysis was pertussis toxin- insensitive. Under the conditions used in these experiments, treatment with 100 ng/ml pertussis toxin ADP-ribosylates all detectable pertussis substrate in cell membranes as assayed by subsequent ADP-ribosylation of membrane preparations in vitro using activated pertussis toxin and ³²P-NAD. Hung et al. (1992b). Thus these data suggest that, unlike PARI, PAR4 does not couple to Gj. The observation that the responses to thrombin and the various agonist peptides were concordant, at least in terms of which G proteins were activated, suggests that these peptides are useful probes of PAR4 signaling in mammalian cells. The observation that the response of PAR4-expressing cells to GYPGKF, even at 500 μM, was less than 60% ofthe maximum response to thrombin again emphasizes the utility of better than native

PAR4 agonists. Example 4

PAR4 activating peptides are useful for probing PAR4 function

Platelet function assay. Platelet-rich plasma and washed human or mouse platelets were prepared and platelet ATP secretion and aggregation in response to various agonists was measured by lumiaggregometry (Chrono-log Corp.) as described in Kahn et al. (1999); Ishihara et al. (1998).

Results. Having identified a relatively potent and specific PAR4 agonist peptide, AYPGKF, we investigated its utility for PAR4 function in a differentiated cell that naturally expresses PAR4, the human platelet.

In platelet-rich plasma, AYPGKF caused platelet shape change at 20 μM (Figure 6, top left; shape change is indicated by the dip in the solid aggregation curve). At lOOμM and above, AYPGKF reliably stimulated platelet ATP secretion and aggregation (Figure 6). By comparison, under the conditions used in these experiments, GYPGKF elicited only shape change even at 500μM. The PARI agonist SFLLRN was considerably more potent than AYPGKF; threshold for SFLLRN for shape change and secretion was ~4 μM and substantial responses occurred 20μM. However, maximum responses to AYPGKF were at least as great at those to SFLLRN (Figure 6). AYPGKF also elicited robust increases in platelet cytoplasmic calcium concentration (Figure 7). Responses to AYPGKF were not inhibited by preincubating platelets with the PARI antagonist BMS200261 (lOOμM). Bematowicz et al. (1996). By contrast, SFLLRN responses were blocked by this reagent. Thus platelet responses to AYPGKF are not attributable agonist activity at PARI. These results are consistent with expression of both PARI and PAR4 on human platelets. Kahn et al. (1998); Xu et al. (1998); Kahn et al. (1999).

Desensitization with PAR-specific agonist peptides has been useful for probing the role of specific receptors in differentiated cells, and AYPGKF proved useful in this regard. Increases in cytoplasmic calcium were measured fluorometrically as an index of PAR signaling. In platelets in wliich PARI was blocked by the antagonist BMS200261, pretreatment with AYPGKF caused platelets to become refractory to both AYPGKF and to thrombin (Figure 7). Only limited heterologous desensitization to thromboxane receptor agonist U46619 was noted. These results indicate that AYPGKF effectively desensitized PAR4-mediated signaling in human platelets. Moreover, they confirm and extend previous studies that suggest PARI and PAR4 account for most if not all thrombin signaling in human platelets. Kahn et al. (1999).

Example 5

Mouse platelet secretion and aggregation in platelet-rich plasma - effectiveness of AYPGKF

Genetically manipulated mice are increasingly utilized to identify the molecules and mechanisms that regulate platelet function. In our hands, thrombin is the most effective agonist for activation of mouse platelets with ADP, collagen, epinephrine and U46619 being considerably less potent. However, because thrombin clots fibrinogen, thrombin cannot be used to study platelet function in platelet-rich plasma (PRP); washed platelet preparations must be used and preparation of washed platelets entails substantial additional ex vivo manipulation.. In the human system, the PARI agonist SFLLRN has proven useful as a strong agonist that can activate platelets in platelet-rich plasma. However, mouse platelets utilize PAR3 and PAR4 for thrombin signaling ~ PARI does not contribute. No peptide agonist capable of activating mouse PAR3 has been described, and recent evidence suggests that mPAR3 functions as a cofactor for thrombin cleavage of PAR4 rather than as a bonafide receptor. (Nakanishi-Matsui et al. (2000) Nature (In Press)).

AYPGKF proved to be a potent activator of mouse platelets in platelet-rich plasma, unlike the wild-type GYPGKF peptide (Figure 8). AYPGKF will therefore be useful for exploring platelet function in genetically manipulated mice. Indeed, AYPGKF caused shape change but not secretion or aggregation in platelets from Gα_q-deficient mice, suggesting that PAR4 mediates shape change through a G protein other than Gq, probably G₁₂ i3- Klages et al. (1999) J. Cell. Biol. 144:745- 754.

One specialized use of AYPGKF is probing mouse platelet function, as it elicits robust platelet activation in platelet-rich plasma. Such reliable activators of mouse platelets are needed given the increasing use of genetically modified mice to investigate platelet function.

Example 6

Screening for PAR4 antagonists and agonists

Agonists

Another aspect ofthe invention features screening for compounds that act as PAR4 agonists. Activation of PAR4 with thrombin or an agonist leads to a cascade of events (such as phosphoinositide hydrolysis, Ca²⁺ efflux, and platelet aggregation), providing a convenient means for measuring thrombin or other agonist activity.

The agonist screening assay ofthe invention utilizes recombinant cells expressing recombinant PAR4 (or a suitable receptor fragment or analog, as outlined herein) configured to permit detection of PAR4 function. Alternatively, a cell such as a leukocyte, a platelet, or a mesenchymal cell that naturally express PAR4 is used. Other elements ofthe screen include a detectable downstream substrate ofthe PAR4 activation, such as radiolabeled phosphoinositide, the hydrolysis of which to a detectable product indicates PAR4 activation by the candidate agonist. ⁴⁵Ca efflux from a cell expressing PAR4 is used as a means of measuring receptor activation by candidate agonists. Williams et al. (1988) PNAS 85:493904943; Vu et al. (1991a); USP 5, 256, 766; 09/032,397; 09/360, 482, which references are herein incorporated by reference in their entirety. ⁴⁵Ca release by oocyte expressing cRNA encoding PAR4 are assessed as follows. Briefly, intracellular calcium pools are labeled by incubating groups of 30 oocytes in 300 μl calcium-free MBSH containing 50 μCi ⁵CaCl₂ (10-40 mCi/mg Ca; Amersham) for 4 hours at room temperature. The labeled oocytes are washed, then incubated in MBSH II without antibiotics for 90 minutes. Group of 5 oocytes are selected and placed in individual wells in a 24-well tissue culture plate (Falcon 3047) containing

0.5 mg/ml MBSH II without antibiotics. This medium is removed and replaced with fresh medium every 10 minutes, the harvested medium is analyzed by scintillation counting to determine ⁴⁵Ca release by the oocytes during each 10-minute incubation. The 10-minute incubations are continued until a stable baseline of ⁴⁵Ca release per unit time is achieved. Two additional 10-minute collections are obtained, then test medium including agonist is added and agonist-induced ⁴⁵Ca release determined.

A voltage clamp assay provides an alternative method of monitoring agonist activity. Agonist-induced inward chloride currents are measured in voltage-clamped oocyte expressing thrombin receptor encoding cRNA essentially as previously described by Julius et al. (1988) Science 241 :558-563, herein incorporated by reference in its entirety, except that either the single electrode voltage-clamp technique or a two electrode technique is employed. Platelet aggregation is used as means for monitoring PAR4 receptor activation. For example, Connolly et al. (1996). Human platelets may use both PARI and PAR4. An agonist useful in the invention is one which imitates the normal thrombin- mediated signal transduction pathway leading, e.g., to an increase in phosphoinositide hydrolysis. Appropriate candidate agonists include thrombin analogs or PAR4 tethered ligand domains or other agents which mimic the action of thrombin or the PAR4 tethered ligand domain. An agonist is useful for aiding discovery of antagonists.

A PARI agonist can be similarly identified as described above. An ordinarily person skilled in the art can easily derive the procedure for screening PARI agonist after reading the disclosure above.

For screening an agonist that activates both PARI and PAR4 simultaneously, the procedures to screen PARI agonist and PAR4 agonist are carried out sequentially or concurrently, herein applying a single candidate agonist.

Antagonists

As discussed above, one aspect ofthe invention features screening for agents that inhibit the interaction between PAR4 activating peptide and the protease- activated receptor 4, thereby preventing or reducing the cascade of events that are mediated by that interaction. The elements ofthe screen are a PAR4 activating peptide (such as AYPGKF, or SYPGKF), a candidate antagonist, and recombinant

PAR4 (or a suitable receptor fragment or analog, as outlines above) configured to permit detection of PAR4 activator, antagonist, and PAR4 function. An additional element may be ⁴⁵Ca, Fura-2, ³H-inositol, or another indicator used to detect downstream signaling. Ishii et al. (1993); Nanevicz et al. (1996). Inhibition of PAR4 activating peptide-induced platelet aggregation is used as a means for monitoring an antagonist of PAR4 activation. PAR4 activating peptide is incubated with the candidate inhibitory compound (such as a peptide) for 5 minutes, then the mixture is added to washed platelets and platelet activation is followed as platelet ATP secretion by lumiaggregometry. See, for example, Connolly et al. (1996); USP 5, 256, 766; 09/032,397; 09/360, 482, which references are herein incorporated by reference in their entirety. Alternatively, platelets are incubated with a candidate PAR4 antagonist for 5 minutes. Thereafter the response to a PAR4 activating peptide is measured. Inclusion of potential antagonists in the screening assay along with a PAR4 activating peptide allows for the screening and identification of authentic receptor antagonists as those which decrease the PAR4 activating peptide-mediated events, such as platelet aggregation. A PARI antagonist is similarly identified as described above. An ordinarily person skilled in the art can easily derive the procedure for screening PARI antagonist after reading the disclosure above.

For screening an antagonist that blocks both PARI and PAR4 activation simultaneously, the procedures to screen PARI antagonist and PAR4 antagonist are carried out sequentially or concurrently, herein applying a single candidate antagonist.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding it, it will be apparent to those skill in the art that certain changes and modifications will be practiced.

Therefore, the description and examples should not be construed as limiting the scope ofthe invention, which is delineated by the appended claims.

Claims

CLAIMSWe claim:

1. An isolated peptide selected from the group consisting of GYPAKF, GYP(Cha)KF, GYPLKF, GYPIKF, S(F)(Cha)(Cha)(homoR)K, and S(F)(Cha)(Cha)RK, wherein (Cha) is cyclohexylalanine, (F) is parafluoro-phenylalanine, and (homoR) is homoarginine wherein the isolated peptide activates PARI .

2. An isolated peptide selected from the group consisting of GYPGRF, GYPG(homoR)F, GYPG(Orn)F, GYPGKY, GYPGKW, GYPGKK, SYPAKF, SYAGKF,

SYPGRF, SYPG(homoR)F, wherein (homoR) is homoarginine and (Orn) is ornithine wherein the isolated peptide activates PAR4.

3. An expression vector comprising a polynucleotide encoding an isolated peptide of claim 1 or claim 2.

4. A host cell comprising the expression vector of claim 3.

5. A pharmaceutical agent comprising an isolated peptide of claim 1 and a pharmaceutically acceptable excipient.

6. A pharmaceutical agent comprising an isolated peptide of claim 2, and a pharmaceutically acceptable excipient.

7. A pharmaceutical agent comprising an isolated peptide selected from the group consisting of peptides AYPGKF, SYPGKF, and TYPGKF, and a pharmaceutically acceptable excipient.

8. The pharmaceutical agent of claim 5 wherein said peptide is AYPGKF.

9. A method for modulating PAR4 activity in an individual, comprising administering an effective amount of said agent of claim 6 or 7 to said individual.

10. The method of claim 9 wherein said peptide is AYPGKF.

11. The method of claim 9, wherein the effective amount of said agent is between 0.001 and about 100 mg/kg body weight of said individual.

12. The method of claim 11 , wherein the effective amount of said agent is between 0.01 and about 10 mg/kg body weight of said individual.

13. A method for modulating platelet activation in an individual, comprising administering an effective amount of said agent of claim 6 or 7 to said individual.

14. The method of claim 13 wherein said peptide is AYPGKF.

15. The method of claim 13 , wherein the effective amount of said agent is between 0.001 and about 100 mg/kg body weight of said individual.

16. The method of claim 15, wherein the effective amount of said agent is between 0.01 and about 10 mg/kg body weight of said individual.

17. A pharmaceutical agent comprising an isolated peptide selected from the group consisting of peptides GFPGKF and G(F)PGKF, and a pharmaceutically acceptable excipient, wherein (F) is parafluoro-phenylalanine.

18. A method for modulating PARI activity in an individual, comprising administering an effective amount of said agent of claim 5 or 17 to said individual.

19. The method of claim 18 wherein said agent comprises the peptide S(F)(Cha)(Cha)(homo R)K.

20. A method for modulating both PARI and PAR4 activity in an individual, comprising administering an effective amount of said agent of claim 17 to said individual.

21. A method for modulating both PARI and PAR4 activity in an individual, comprising administering an effective amount of a pharmaceutical agent comprising GYPAKF and a pharmaceutically acceptable excipient.

22. The method of claim 18, 19, 20 or 21 , wherein the effective amount of said agent is between 0.001 and about 100 mg/kg body weight of said individual.

23. The method of claim 22, wherein the effective amount of said agent is between 0.01 and about 10 mg/kg body weight of said individual.

24. A method for modulating platelet activation in an individual, comprising administering an effective amount of said agent of claim 5 or 17 to said individual.

25. The method of claim 24, wherein the effective amount of said agent is between 0.001 and about 100 mg/kg body weight of said individual.

26. The method of claim 25, wherein the effective amount of said agent is between 0.01 and about 10 mg/kg body weight of said individual.

27. A method for identifying an agent that modulates PAR4 activity, said method comprising: (a) contacting a suitable host cell comprising PAR4 function with said agent to be tested and a PAR4 activating peptide selected from the group consisting of AYPGKF, SYPGKF, TYPGKF, GFPGKF and G(F)PGKF, GYPAKF, GYPGRF, GYPG(homoR)F, GYPG(Orn)F, GYPGKY, GYPGKW, GYPGKK, SYPAKF, SYAGKF, SYPGRF, S YPG(homoR)F, wherein (homoR) is homoarginine and (Orn) is ornithine; and

(b) analyzing at least one characteristic which is associated with PAR4 function in said host cell, wherein an agent is identified by its ability to modulate at least one such characteristic as compared to contacting the suitable host cell with said PAR4 activating peptide without the agent.

28. The method of claim 27, wherein said host cell has native PAR4 function.

29. The method of claim 27, wherein said host cell lacks native PAR4 function and comprises a recombinant polynucleotide encoding PAR4 or a functional fragment thereof, wherein PAR4 function is restored in said host cell.

30. The method of claim 27, wherein the host cell is a platelet cell.

31. The method of claim 27, wherein the characteristic which is associated with

PAR4 function is phosphoinositide hydrolysis.

32. The method of claim 27, wherein the characteristic which is associated with PAR4 function is intracellular calcium mobilization.

33. The method of claim 27, wherein the characteristic which is associated with PAR4 function is platelet shape change.

34. The method of claim 27 wherein the characteristic which is associated with PAR4 function is platelet ATP secretion.

35. The method of claim 27, wherein the characteristic which is associated with PAR4 function is platelet aggregation.

36. A method for identifying an agent that modulates PARI activity, said method comprising:

(a) contacting a suitable host cell comprising PARI function with said agent to be tested and a PARI activating peptide selected from the group consisting of

GYPAKF, GYP(Cha)KF, GYPLKF, GYPIKF, S(F)(Cha)(Cha)(homoR)K, and S(F)(Cha)(Cha)RK, GFPGKF, and G(F)PGKF, wherein (Cha) is cyclohexylalanine, (F) is parafluoro-phenylalanine, and (homoR) is homoarginine; and

(b) analyzing at least one characteristic which is associated with PARI function in said host cell, wherein an agent is identified by its ability to modulate at least one such characteristic as compared to contacting the suitable host cell with said PARI activating peptide without said agent.

37. The method of claim 36, wherein said host cell has native PARI function.

38. The method of claim 36, wherein said host cell lacks native PARI function and comprises a recombinant polynucleotide encoding PARI or a functional fragment thereof, wherein PARI function is restored in said host cell.

39. The method of claim 36, wherein the host cell is a platelet cell.

40. The method of claim 36, wherein the characteristic which is associated with PARI function is phosphoinositide hydrolysis.

41. The method of claim 36, wherein the characteristic which is associated with PARI function is intracellular calcium mobilization.

42. The method of claim 36, wherein the characteristic which is associated with PARI function is platelet shape change.

43. The method of claim 36 wherein the characteristic which is associated with PARI function is platelet ATP secretion.

44. The method of claim 36, wherein the characteristic which is associated with

PAR4 function is platelet aggregation.

45. A method for identifying an agent that modulates both PARI and PAR4 activity, said method comprising: (a) contacting a suitable host cell comprising PARI function with said agent to be tested and a PARI activating peptide selected from the group consisting of GYPAKF, GYP(Cha)KF, GYPLKF, GYPIKF, S(F)(Cha)(Cha)(homoR)K, S(F)(Cha)(Cha)RK, GFPGKF and G(F)PGKF wherein (Cha) is cyclohexylalanine, (F) is parafluoro- phenylalanine, and (homoR) is homoarginine, and analyzing at least one characteristic which is associated with PARI function in said host cell, wherein an agent is identified by its ability to modulate at least one such characteristic as compared to contacting the suitable host cell with said PARI activating peptide without the agent;

(b) contacting a suitable host cell comprising PAR4 function with said agent to be tested and a PAR4 activating peptide selected from the group consisting of GYPGRF, GYPG(homoR)F, GYPG(Orn)F, GYPGKY, GYPGKW, GYPGKK, SYPAKF, SYAGKF,

SYPGRF, SYPG(homoR)F, AYPGKF, SYPGKF, TYPGKF, GFPGKF, and G(F)PGKF, wherein (homoR) is homoarginine and (Orn) is ornithine, and analyzing at least one characteristic which is associated with PAR4 function in said host cell, wherein an agent is identified by its ability to modulate at least one such characteristic as compared to contacting the suitable host cell with said PAR4 activating peptide without the agent; and (c) selecting an agent that modulates at least one characteristic associated with PARI function and at least one characteristic associated with PAR4 function, wherein the steps (a) and (b) are performed in any order or simultaneously.

46. The method of claim 45, wherein said host cell of step (a) has native PARI function.

47. The method of claim 45, wherein said host cell of step (a) lacks native

PARI function and comprises a recombinant polynucleotide encoding PARI or a functional fragment thereof, wherein PARI function is restored in said host cell.

48. The method of claim 45, wherein said host cell of step (b) has native PAR4 function.

49. The method of claim 45, wherein said host cell of step (b) lacks native PAR4 function and comprises a recombinant polynucleotide encoding PAR4 or a functional fragment thereof, wherein PAR4 function is restored in said host cell.

50. The method of claim 45, wherein the host cell is a platelet cell.

51. The method of claim 45, wherein the characteristic which is associated with PARI and/or PAR4 function is selected from the group consisting of phosphoinositide hydrolysis, intracellular calcium mobilization, platelet shape change, platelet ATP secretion, and platelet aggregation.

52. A method for identifying an agent that antagonizes PAR4 activity, said method comprising: (a) contacting a suitable host cell comprising PAR4 function with said agent to be tested and a PAR4 activating peptide selected from the group consisting of GYPGRF, GYPG(homoR)F, GYPG(Orn)F, GYPGKY, GYPGKW, GYPGKK, SYPAKF, SYAGKF, SYPGRF, SYPG(homoR)F, AYPGKF, SYPGKF, TYPGKF, GFPGKF, and G(F)PGKF, wherein (homoR) is homoarginine and (Orn) is ornithine, and

(c) analyzing at least one characteristic associated with the inhibition of PAR4 activation in said host cell, wherein an agent is identified by its ability to modify at least one such characteristic as compared to contacting the suitable host cell with said PAR4 activating peptide without the agent.

53. The method of claim 52, wherein said host cell has native PAR4 function.

54. The method of claim 52, wherein said host cell lacks native PAR4 function and comprises a recombinant polynucleotide encoding PAR4 or a functional fragment thereof, wherein PAR4 function is restored in said host cell.

55. The method of claim 52, wherein the host cell is a platelet cell.

56. A method for identifying an agent that antagonizes PARI activity, said method comprising: (a) contacting a suitable host cell comprising PARI function with said agent to be tested and a PARI activating peptide selected from the group consisting of GYPAKF, GYP(Cha)KF, GYPLKF, GYPIKF, S(F)(Cha)(Cha)(homoR)K, S(F)(Cha)(Cha)RK, GFPGKF and G(F)PGKF wherein (Cha) is cyclohexylalanine, (F) is parafluoro- phenylalanine, and (homoR) is homoarginine; and (b) analyzing at least one characteristic associated with the inhibition of

PARI activation in said host cell, wherein an agent is identified by its ability to modify at least one such characteristic as compared to contacting the suitable host cell with said PARI activating peptide without the agent.

57. The method of claim 56, wherein said host cell has native PARI function.

58. The method of claim 56, wherein said host cell lacks native PARI function and comprises a recombinant polynucleotide encoding PARI or a functional fragment thereof, wherein PARI function is restored in said host cell.

59. The method of claim 56, wherein the host cell is a platelet cell.

60. A method for identifying an antagonist of both PARI and PAR4 activity, said method comprising: a) contacting a suitable host cell comprising PARI function with said agent to be tested and a PARI activating peptide selected from the group consisting of GYPAKF, GYP(Cha)KF, GYPLKF, GYPIKF, S(F)(Cha)(Cha)(homoR)K, S(F)(Cha)(Cha)RK, GFPGKF and G(F)PGKF wherein (Cha) is cyclohexylalanine, (F) is parafluoro- phenylalanine, and (homoR) is homoarginine and analyzing at least one characteristic associated with the inhibition of PARI activation in said host cell, wherein an agent is identified by its ability to modify at least one such characteristic as compared to contacting the suitable host cell with said PARI activating peptide without the agent;

(b) contacting a suitable host cell comprising PAR4 function with said agent to be tested and a PAR4 activating peptide selected from the group consisting of GYPGRF, GYPG(homoR)F, GYPG(Orn)F, GYPGKY, GYPGKW, GYPGKK, SYPAKF, SYAGKF,

SYPGRF, SYPG(homoR)F, AYPGKF, SYPGKF, TYPGKF, GFPGKF, and G(F)PGKF, wherein (homoR) is homoarginine and (Orn) is ornithine and analyzing at least one characteristic associated with the inhibition of PAR4 activation in said host cell, wherein an agent is identified by its ability to modify at least one such characteristic as compared to contacting the suitable host cell with said PAR4 activating peptide without the agent; and

(c) selecting an agent that modifies at least one characteristic associated with the inhibition of PARI activation and at least one characteristic associated with the inhibition of PAR4 activation, wherein the steps (a) and (b) are performed in any order or simultaneously.

61. The method of claim 60, wherein said host cell of step (a) has native PARI function.

62. The method of claim 60, wherein said host cell lacks native PARI function and comprises a recombinant polynucleotide encoding PARI or a functional fragment thereof, wherein PARI function is restored in said host cell.

63. The method of claim 60, wherein said host cell of step (b) has native PAR4 function.

64. The method of claim 60, wherein said host cell lacks native PAR4 function and comprises a recombinant polynucleotide encoding PAR4 or a functional fragment thereof, wherein PAR4 function is restored in said host cell.

65. The method of claim 60, wherein said host cell of step (a) or step (b) is a platelet cell.

66. A kit comprising an isolated peptide selected from the group consisting of GYPAKF, GYP(Cha)KF, GYPLKF, GYPIKF, S(F)(Cha)(Cha)(homoR)K, and

S(F)(Cha)(Cha)RK, wherein (Cha) is cyclohexylalanine, (F) is parafluoro-phenylalanine, and (homoR) is homoarginine wherein the isolated peptide activates PARI .

67. A kit comprising an isolated peptide selected from the group consisting of GYPGRF, GYPG(homoR)F, GYPG(Orn)F, GYPGKY, GYPGKW, GYPGKK, SYPAKF,

SYAGKF, SYPGRF, SYPG(homoR)F, wherein (homoR) is homoarginine and (Orn) is ornithine, wherein the isolated peptide activates PAR4.