WO2001060401A1

WO2001060401A1 - Methods of modulating protease-activated receptor-4 (par4) activation via cathepsin g

Info

Publication number: WO2001060401A1
Application number: PCT/US2001/004987
Authority: WO
Inventors: Gilberto Sambrano; Shaun R. Coughlin
Original assignee: University of California Berkeley; University of California San Diego UCSD
Current assignee: University of California Berkeley; University of California San Diego UCSD
Priority date: 2000-02-17
Filing date: 2001-02-14
Publication date: 2001-08-23
Anticipated expiration: 2002-08-17
Also published as: AU2001238366A1

Abstract

The present invention provides methods for modulating cathepsin G-mediated PAR4 activation and/or platelet activation. Further, the invention provides methods for screening agents which may modulate cathepsin G-mediate PAR4 activation and/or platelet activation.

Description

METHODS OF MODULATING PROTEASE-ACTIVATED RECEPTOR-4 (PAR4)

ACTIVATION VIA CATHEPSIN G

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH Work described herein was supported in part by a grant from the National Institutes of Health (HL 44907). The government may have certain rights in the invention.

TECHNICAL FIELD This invention relates to the field of platelet activation via protease activated receptors (PAR). More specifically, this invention relates to cathepsin G-mediated protease activated receptor 4 (PAR4) activation, and methods for modulating cathepsin G-mediated PAR4 activation and/or platelet activation and methods for screening agents that modulate cathepsin G-mediated PAR4 activation and/or platelet activation.

BACKGROUND ART

Platelet activation is critical for normal hemostasis and plays key roles in dealing with 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, editor. 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 proliferative responses to injury, as occur in normal wound healing and a variety of diseases (atherosclerosis, resternosis, pulmonary inflammation (ARDS), glomerulosclerosis, etc.). A thorough understanding of how thrombin activates cells is an important goal. Characterization of the 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 of the 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 of the 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 PARl is the prototype. Nu et al. (1991a);

USP 5,256,766; Rasmussen et al. (1991) FEBS Lett. 288:123-128. Thrombin activates PARl by binding to and cleaving PARl amino-terminal exodomain at the R41/S42 peptide bond. The cleavage serves to unmask a new receptor amino terminus beginning with the sequence SFLLRΝ. This new amino terminus then serves as a tethered peptide ligand, binding intramolecularly to the body of the receptor to effect transmembrane signaling. Nu et al. (1991a); Nu 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.

Our understanding of the role of PARs in platelet activation is evolving rapidly. Four distinct PARs are now known. PARl, PAR3, and PAR4 can be activated by thrombin. Nu 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; 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. PARl mRNA and protein were detected in human platelets. Nu 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. PARl-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 PARl in activation of human platelets by thrombin but held open the possibility that other receptors might contribute. On contrary, PARl appeared to play no roles in mouse platelet activation. PARl expression was difficult to detect in rodent platelets, and PAR1- activating peptides did not activate rodent platelets. Moreover, platelets from PAR1- deficient mice responded like wild-type platelets to thrombin. PAR3 expressed in mouse platelets but could not be detected in human platelets. Ishihara et al. (1997); 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 PAR3 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); Νystedt et al. (1994). Thus in both mouse and human, platelets utilize two thrombin receptors. A "high-affinity" thrombin receptor (PARl 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 these receptors account for thrombin activation of platelets. Activation of either PARl and PAR4 triggered platelet activation. Inhibition of PARl 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 PARl 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 of the new

PARl amino terminus unmasked by receptor cleavage, functions as an agonist for PARl 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. 267:13146-13149. Such peptides have been used as pharmacological probes of PARl function in various cell types, as well as a starting point for PARl antagonist development. Bematowicz et al. (1996) J. Med. Chem. 39:4879-4887; Andrade-Gordon et al. (1999) Proc. Natl Acad. Sci. USA 96:12257-12262. The cognate Pl'-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 PARl . Kahn et al. (1998); Xu et al. (1998); Kahn et al. (1999). 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). The synthetic peptide GYPGQV functions as an agonist for PAR4 at a concentration of 200-500μM.

Available data suggest that PAR4 activation is not necessary for robust responses in human platelets when PARl function is intact. Aside from providing a backup signaling device, PAR4 might allow platelets to respond to proteases other than thrombin, mediate thrombin signaling to distinct effectors or with a tempo different from that of PARl, or function in platelet responses beyond simple secretion and aggregation. The existence of two genes and gene products also raises the possibility of differential regulation at many levels in platelets or other cell types. Most interestingly, it is possible that PARs interact with each other.

A link between inflammation and coagulation is well established and occurs at multiple levels. Endothelial activation promotes both neutrophil and platelet adhesion and rolling via stimulation of vWF release and P-selectin expression on the endothelial surface. Subramaniam et al. (1996) Blood 87:1238-1242; Frenette et al. (1996) Cell 84:563-574; Hattori et al. (1989) J Biol. Chem. 264:7768-7771 ; Norman et al. (1995) Blood 86:4417- 4421. Thus, endothelial activation provides a mechanism to concentrate platelets and neutrophils at the same site, perhaps promoting neutrophil-platelet interaction in the setting of graded local inflammatory responses. Interactions between marginated neutrophils and rolling platelets might become especially prominent in the setting of profound endothelial activation as occurs in the Shwartzman reaction and related systemic phenomena associated with sepsis and disseminated intravascular coagulation. Brozna et al. (1990) Semin. Thromb. Hemostas. 16:326-332. In the local Shwartzman reaction, for example, a cytokine stimulus at an endotoxin-primed skin site triggers robust margination of neutrophils and microvascular thrombosis within 15 minutes. Whether interrupting platelet-neutrophil interactions would be beneficial in such settings is unknown.

The foregoing discussion casts neutrophil-platelet interactions as proinflammatory and/or prothrombotic. That is, activated neutrophils might induce platelet activation as a means of further promoting inflammatory cell recruitment by causing release of platelet cytokines, (see Weksler (1988) in Inflammation: Basic Principles and Clinical Correlates (Gallin et al. eds.) p. 543-557, New York), stimulating thrombin generation via platelet procoagulant activity (thereby further activating the endothelium locally), or perhaps inciting local thrombosis to isolate a site of infection. Similarly, activated neutrophils might bind and activate platelets as a means of promoting their own incorporation into nascent thrombi at sites of injury. Conversely, it is conceivable that activated neutrophils might bind and activate platelets as part of a shutoff mechanism that limits the systemic effects of local inflammation by preventing activated neutrophils from circulating. Experimental models of inflammation in mice may provide a means of testing these hypotheses.

Platelet PARs may be exposed to a variety of proteases at sites of inflammation or coagulation, and leukocytes are one potential source. For example, neutrophils and platelets are both concentrated at sites of inflammation and thrombus formation. Moreover, aggregates of cells containing both neutrophils and platelets have been found in experimental models of inflammation or thrombosis suggesting that interactions between these cell types may be physiologically relevant. Cerletti et al. (1995) Thromb. Haemostas. 74, 218-223. Secreted platelet products act on neutrophils to stimulate adhesion, migration and/or degranulation. Weksler (1988) in Inflammation: Basic Principles and Clinical Correlates (Gallin et al. eds.) p. 543-557, New York. A role for neutrophil-derived factors acting on platelets is less well-established, but interestingly, the neutrophil proteases cathepsin G and elastase have been shown to act on platelets. Cerletti et al. (1995); LaRosa et al. (1994) J Vase. Surg. 19:306-319; Si-Tahar et al. (1997) J. Biol. Chem. 272:11636- 11647.

Cathepsin G is a neutral serine protease found in neutrophil azurophilic granules and is released upon stimulation of cells by chemotactic agents such as fMet-Leu-Phe (N- formyl-methionyl-leucyl-phenylalanine; fMLP) or platelet activating factor. Owen and

Campbell (1999) J. Leukoc. Biol. 65:137-150. The protease is known to hydrolyze matrix components such as laminin and fibronectin after methione, leucine, phenylalanine, lysine or arginine residues.

Cathepsin G has been found to cause platelet secretion and aggregation ex vivo. LaRosa et al. (1994); Molino et al. (1992) Biochem. J. 288:741-745; Selak and Smith

(1990) Biochem. J. 266:55-62. This activity was shown to be dependent upon the active site of cathepsin G. Studies of PARl as a candidate cathepsin G receptor suggested that this receptor did not account for cathepsin G signaling in platelets. Selak (1994) Biochem. J. 297:269-275; Molino et al. (1995) J Biol. Chem. 270:11168-11175. Molino et al. showed that cathepsin G could cleave PARl either productively at the R41/S42 peptide bond or non-productively at the F55/W56 peptide bond in the PARl amino terminal exodomain. The latter cleavage served to remove the PARl tethered ligand rendering it unresponsive to thrombin while still responsive to SFLLRN.

In view of the alarming prevalence of life-threatening disorders related to platelet activation such as heart attack, stroke, atherosclerosis, restemosis, pulmonary inflammation

(ARDS), and glomerulosclerosis, hemostasis, thrombosis, and normal wound healing, there is a pressing need for developing therapeutic agents and methods to modulate platelet activation.

All publications and patent applications cited herein are hereby incorporated by reference in their entirety. DISCLOSURE OF THE INVENTION The invention provides methods for modulating cathepsin G-mediated PAR4 activation and/or platelet activation.

Accordingly, in one aspect, the invention provides methods for inhibiting cathepsin G-mediated PAR4 activation comprising contacting any of PAR4, a PAR4 expressing cell, and/or cathepsin G with an agent which interferes with cathespin G/PAR4 interaction, such that PAR4 activation is inhibited.

In another aspect, the invention provides methods for inhibiting cathepsin G- mediated PAR4 activation in an individual comprising administering to the individual a composition comprising an agent which interferes with cathepsin G/PAR4 interaction, such that PAR4 activation is inhibited.

In another aspect, the invention provides methods of inhibiting PAR4-mediated platelet activation comprising contacting a PAR4 expressing cell (such as platelet) and/or cathepsin G with an agent which interferes with cathepsin G/PAR4 interaction, such that PAR4-mediated platelet activation is inhibited.

In another aspect, the invention provides methods of inhibiting PAR4-mediated platelet activation in an individual comprising administering to the individual a composition comprising an agent which interferes with cathepsin G/PAR4 interaction in an amount sufficient to inhibit PAR4-mediated platelet activation. In another aspect, the invention provides methods of inhibiting neutrophil-mediated platelet activation in an individual comprising administering to the individual a composition which inhibits cathepsin G/PAR4 interaction in an amount sufficient to inhibit neutrophil-mediated platelet activation.

In another aspect, the invention provides methods of stimulating cathepsin G- mediated PAR4 activation comprising contacting any of PAR4, a PAR4 expressing cell, and/or cathepsin G with an agent which enhances cathepsin G/PAR4 interaction, such that PAR4 activation is stimulated.

In another aspect, the invention provides methods of stimulating cathepsin G- mediated PAR4 activation in an individual comprising administering to the individual a composition comprising an agent which enhances cathepsin G/PAR4 interaction, such that

PAR4 activation is stimulated. In another aspect, the invention provides methods of stimulating PAR4-mediated platelet activation comprising contacting a PAR4-expressing cell and/or cathepsin G with an agent which enhances cathepsin G/PAR4 interaction such that PAR4-mediated platelet activation is stimulated. In another aspect, the invention provides methods of stimulating neutrophil- mediated platelet activation in an individual comprising administering to the individual a composition which enhances cathepsin G/PAR4 interaction in an amount sufficient to stimulate neutrophil-mediated platelet activation.

In another aspect, the invention provides screening methods for identifying agents that modulate cathepsin G-mediated PAR4 activation. One in vivo screening method entails the steps of combining at least one agent to be tested with a PAR4-expressing cell and cathepsin G and analyzing at least one characteristic which is associated with modulation of cathepsin G-mediated PAR4 function in the cell, wherein an agent is identified by its ability to elicit at least one such characteristic as compared with control conditions. In vitro screening methods, which, inter alia, detect interference with cathepsin

G/PAR4 binding, are also included in the invention.

BRIEF DESCRIPTION OF THE DRA WINGS Figure 1 is a bar graph depicting activation of PARs by thrombin (solid bars) and cathepsin G (gray bars) as measured by calcium release.

Figures 2A and 2B are bar graphs depicting effect of serine protease inhibitors on thrombin-induced calcium release (Figure 2A) from Xenopus oocytes that express human PARl (solid bars) or PAR4 (gray bars) and on cathepsin G-induced calcium release (Figure 2B) from Xenopus oocytes that express human PARl (solid bars) or PAR4 (gray bars). The control did not receive any protease inhibitors.

Figure 3 is a bar graph depicting effect of thrombin (white bars), cathepsin G (gray bars) and PAR-activating peptides (AP) (solid bars; PARl -activating peptide for PARl mutant and PAR4-activating peptide for PAR4 mutant) on calcium release from Xenopus oocytes that express a mutant PARl (PARl F/A) or a mutant PAR4 (PAR4 G/P). Figures 4 (A) - (C) are graphs depicting calcium mobilization induced by: 100 nM cathepsin G in mammalian cells expressing hPARl (Figure 4A); 500 nM cathepsin G in mammalian cells expressing hPAR4 (Figure 4B); 500 nM cathepsin G in untransfected mammalian cells (Figure 4C).

Figures 5 (A) - (H) are graphs depicting agonist-induced calcium mobilization in human platelets loaded with Fura-2 under various conditions. Calcium mobilization induced by 500 nM cathepsin G was measured in naϊve platelets (Figure 5A), and in platelets treated with 100 μM PARl -blocking peptide BMS200261 (Figure 5B), 1 mg/ml PAR4-blocking antibody (Figure 5C), or 1 mg/ml pre-immune IgG (Figure 5D). Responses in platelets desensitized for 30 minutes with 100 μM SFLLRN (Figure 5E) or 500 μM AYPGKF (Figures 5F, 5G and 5H) were also measured. Agonists were added as indicated by arrows and responses plotted as fluorescence ratio versus time (sec).

Figures 6 (A) - (C) are graphs depicting platelet aggregation in: 1 μM cathepsin G (Figure 6A); and 100 μM BMS200261 (Figure 6B); and 1 mg/ml PAR4-blocking antibody (Figure 6C).

Figures 7(A) - (C) are graphs depicting neutrophil-dependent calcium mobilization in platelets that were not treated with any antibodies (Figure 7A); in the presence of 1 mg/ml PAR4-blocking antibody (Figure 7B); in the presence of 1 mg/ml pre-immune IgG (Figure 7C). The arrows indicate the stimulation of platelets with 100 nM fMLP.

MODES FOR CARRYING OUT THE INVENTION We have discovered that cathepsin G mediates protease-activated receptor 4

(PAR4) activation which in turn triggers platelet activation, raising the possibility that cathepsin G mediates neutrophil-platelet interaction at sites of vascular injury or inflammation. This PAR4-specific activation, as mediated by cathepsin G, provides useful methods for selective induction or inhibition of platelet activation-associated pathways as well as a basis for discovering useful agents which modulate cathepsin G/PAR4 interaction.

For example, inhibition of PAR4 may suppress, disrupt, or prevent neutrophil-platelet interaction, thereby interrupting a positive feedback loop important for the link between coagulation and inflammation. Because PARl signaling would still be intact, PAR4 inhibition would likely not disrupt normal hemostasis. Cathepsin G triggered calcium mobilization in PAR4-transfected fibroblasts, PAR4- expressing Xenopus oocytes and washed human platelets. An antibody raised against the PAR4 thrombin cleavage site blocked platelet activation by cathepsin G but not other agonists. Desensitization with a PAR4 activating peptide had a similar effect. By contrast, inhibition of PARl function had no effect on platelet responses to cathepsin G. When neutrophils were present, the neutrophil agonist fMet-Leu-Phe triggered calcium signaling in Fura-2-loaded platelets. Strikingly, this neutrophil-dependent platelet activation was blocked by the PAR4 antibody. These observations strongly suggest that PAR4 is the "cathepsin G receptor" on human platelets and that activation of PAR4 by cathepsin G is necessary for neutrophil-dependent platelet activation.

There are several significant implications of inhibiting cathepsin G-mediated PAR4 activation in the context of neutrophils. For example, there are many states in which neutrophil-platelet interaction likely occurs with undesirable results. In sepsis and endotoxic shock there is massive neutrophil margination, activation of the coagulation cascade with thrombin generation, and platelet consumption, which are intimately related. Activated neutrophils can promote platelet activation; activated platelets promote thrombin formation; thrombin activates both platelets and endothelial cells; activated endothelial cells bind neutrophils and platelets. It is likely that both infarction due to microvascular thrombosis and tissue damage from activated neutrophils contribute to organ failure. Thus, interventions that disrupt the positive feedback loops between neutrophils, platelets, and coagulation factor activation could be desirable. PAR4-specific inhibition could be especially desirable because it could be relatively safe, in the sense that PARl is sufficient for platelet activation by thrombin, thus leaving hemostasis unaffected. Other states in which blocking neutrophil-platelet interaction might be efficacious includes reperfusion injury which can occur in stroke, myocardial infarction, unstable angina, vascular procedures, transplantation, and states such as lung injury (ARDS) and other states involving inflammation and tissue injury.

Accordingly, the invention provides methods based on these discoveries: (a) modulation of PAR4 activation (including interfering, inhibiting, stimulating or enhancing) by modulating the interaction of PAR4 with cathepsin G; (b) activation of PAR4 by interaction with cathepsin G; (c) modulating neutrophil-mediated platelet activation via cathepsin G/PAR4 interaction; (d) screening methods to identify agents which modulate cathepsin G-mediated PAR4 activation. Such agents, whether agonists or antagonists, could be useful, as they would presumably mediate platelet activation specifically through PAR4 (as opposed to through PARl). This inhibition of platelet activation could be especially useful in the context of, for example, inflammation, thrombosis, or reperfusion injury.

General Techniques

The practice of the 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 of the art. Such techniques are explained fully in the literature, such as: Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); 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, 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). PAR4 is characterized as having any one or more of 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 ('-hybrid" mPAR4); PCT/US99/19158. PAR4 may refer to a naturally occurring form of the receptor and/or a recombinantly produced form of the receptor (or, when suitable, a fragment thereof). In addition, the term may include variants of PAR4 that retain the at least one activity and/or property of naturally-occurring PAR4. A "functionally preserved" variant or "functionally equivalent" variant of PAR4 (or polynucleotide encoding PAR4) is a PAR4 sequence which retains at least one aspect of PAR4 function. Functionally preserved variants may arise, for example, by conservative and/or non-conservative amino acid substitutions, amino acid analogs, and deletions. The function that it preserved depends upon the relevant function being considered. For example, a PAR4 polypeptide is considered for its ability to bind to a particular entity (such as an cathepsin G or other serine protease), then the ability of a variant sequence to encode a polypeptide with equivalent binding characteristics that is relevant.

"PAR4 function" refers to any activity or characteristic associated with expression of PAR4 (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 exerts control over a number of other genes, it is understood that the term "PAR4 function" encompasses results and characteristics that stem from PAR4 activation which include affecting gene expression of any gene(s) that is regulated by PAR4 gene product or an active fragment thereof. For example, if gene A is repressed by expression and/or activation of PAR4, then lack of expression of gene A is a function of PAR4. Conversely, expression of gene A indicates a compromise of PAR4 function. As used herein, a characteristic which is associated with a "modulation of PAR4 function" is a characteristic which is associated with an alteration or change in PAR4 function. The modulation can be an increase or decrease. Decrease may range from partial to total loss, or knockout, of PAR4 function. Modulation of PAR4 function can occur as a result of an effect at any point along any pathway in which PAR4 exerts control, from transcription of the PAR4 gene, to PAR4 expression (i.e., transcription and/or translation), to affecting regulation of any gene(s) under PAR4 control, to activity associated with regulation of these gene(s). Other PAR4 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. "Cathepsin G" refers to an intact cathepsin G molecule or a fragment or region of cathepsin G which is able to bind to PAR4 and initiate one or more effector functions of PAR4. "Cathepsin G" includes any variants which exhibit the requisite function. A fragment or derivative of cathepsin G which is said to be an antagonist or inhibitor of cathepsin G/PAR4 interaction is a fragment or derivative which inhibits cathepsin G- mediated PAR4 activation.

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

"PAR4/cathepsin G interaction", "cathepsin G/PAR4 interaction" or "interaction of PAR4 and cathepsin G" refer to any aspect of this interaction, such as binding (which may be due to direct binding of cathepsin G to PAR4 and/or due to involvement of other intermediate moieties), cleavage, and one or more effector functions caused by this interaction. Cathepsin G/PAR4 interaction also includes upstream aspects and events which affect cathepsin G binding to PAR4 and its subsequent activation. For example, lowering the effective amount (biologically available) cathepsin G by, for instance, suppressing cathepsin G release from a cell affects cathepsin G/PAR4 interaction.

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, disulfide 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") is a polypeptide comprising an amino acid sequence of PAR4 that has at least about any of the following lengths of contiguous amino acids of a sequence of PAR4:

5, 10, 15 25 30, 40, 50, 80. A PAR4 fragment may be characterized as having any of the 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; (d) ability to elicit a characteristic associated with PAR4 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.

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, cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidine bases, or other natural, chemically, biochemically modified, non-natural or derivatized nucleotide bases. The backbone of the 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 of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be a 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 of the 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 of the 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 of the 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 of the 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 of the 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 of the DNA or RNA. Also included are vectors that provide more than one of the 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 supernatants, 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 of the 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" is an agent that reduces the extent of PAR4 activation mediated by cathepsin G (i.e., the extent of activation of PAR4 in the presence of agent and cathepsin G is reduced when compared to the extent of activation in the presence of cathepsin G without presence of agent). The inhibition or suppression of PAR4 activation may be partial or total. Methods of indicating PAR4 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 PARl . 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 cathepsin G; agents which bind PAR4 and block tethered ligand binding and/or transmembrane signaling; agents which are cathepsin G inhibitors.

An agent that "inhibits or suppresses platelet activation" is an agent that reduces the extent of platelet activation mediated by cathepsin G (i.e., the extent of platelet activation in the presence of agent and cathepsin G is reduced when compared to the extent of activation in the presence of 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 PARl. Examples include, but are not limited to, agents which inhibit activation of cathepsin G. For instance, dipeptidyl peptidase I is necessary for generating active cathepsin. 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.

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

An agent that "suppresses or inhibits" or "interferes with" cathepsin G/PAR4 interaction is an agent which reduces the extent of cathepsin G-mediated PAR4 activation compared to otherwise same conditions except for absence of the agent(s). The inhibition or reduction can be partial or total. It is understood that an agent that interferes with cathepsin G/PAR4 interaction can act on any one or more biological levels which reduces the amount of bioavailable cathepsin G with respect to PAR4 activation. "Bioavailable" cathepsin G means the local amount of cathepsin G (i.e., the amount of cathepsin G in proximity to a PAR4 receptor) that, when contacted with PAR4, binds to and activates PAR4. For example, in addition to interfering with the binding per se of cathepsin G to PAR4 (by, for example, binding to cathepsin G such that cathepsin G cannot bind and activate PAR4), an agent which interferes with cathepsin G/PAR4 interaction can cause any of the following: (a) reduction in cathepsin G-encoding RNA (for example, an antisense transcript, which can be a partial anti-sense oligomer or full-length transcript); (b) reduction in the amount of active cathepsin G (for example, an agent which interferes with an enzyme which activates cathepsin G); (c) reduction in amount of cathepsin G which is translated; (d) reduction in local amounts of cathepsin G (with respect to PAR4), effected by, for example, suppression of cathepsin release from a cell such as a neutrophil; (e) decreases cathepsin G half-life.

Conversely, an agent which "enhances" cathepsin G/PAR4 interaction is one which increases such interaction. Such an agent either mimics the action of cathepsin G (i.e., acts as a substitute for cathepsin G) or increases the effect of cathepsin G, with respect to binding to and activating PAR4. As such, an agent which enhances cathepsin G/PAR4 interaction may cause any of the following: (a) increase in cathepsin G-encoding RNA; (b) increase (local) amount of active cathepsin G (by, for example, increasing amount and/or activity of an enzyme that activates cathepsin G); (c) increase in amount of cathepsin G which is translated; (d) increase in local amounts of cathepsin G, such as effected by, for example, an agent which promotes or stimulates cathepsin G release from a cell such as a neutrophil; (e) increases cathepsin G half-life.

Agent which "increases cathepsin G activity" is an agent which effects any one of following: (a) increase in cathepsin G-encoding RNA; (b) increase in amount of cathepsin G which is translated; (c) increase in amount of active cathepsin G (such as by enzymatic cleavage or other modification); (d) increase in amount of cathepsin G by stimulating release of cathepsin G from cells; (e) increase of half-life of cathepsin G. An agent which

"decreases cathepsin G activity" has any one or more of the opposite effect(s).

A "PAR4-expressing cell" is a cell which produces PAR4, preferably in a form such that PAR4 is able to bind to cathepsin G (in the absence of any agents which inhibit 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.

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 of the following as compared to control conditions: 1.5 fold, 2-fold, 5-fold, 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 of the following percentages as compared to control conditions: 80%, 50% , 25%, 10%.

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 fimction 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 of the 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.

An "agonist" refers a molecule which mimics a particular activity, such as a ligand, or interaction of cathepsin G or PAR4 which activates PAR4 thereby triggering the biological events which normally result from the interaction (e.g., phosphoinositide hydrolysis, Ca 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.

An "antagonist" refers a molecule which blocks or suppresses activation of PAR4 activation as mediated by 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 cathepsin G. In another aspect, an antagonist may bind to cathepsin G and thereby block the activation of PAR4. "Modulating" a characteristic, activity or interaction includes increase or decrease of the characteristic, activity or interaction as compared to control conditions.

"Stimulating" PAR4 activation means an increase in the level of PAR4 activation. Such an increase may be as compared to no activation or as compared to a previously lower level of activation. "Stimulating" PAR4-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 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 an individual, preferably 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 of the 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 modulator(s) is an amount sufficient to modulate cathepsin-mediate PAR4 activation and/or platelet activation. In terms of treatment, an "effective amount" of a modulator (s) is an amount sufficient to palliate, ameliorate, stabilize, reverse, slow or delay progression of a PAR4-associated disease state (i.e., a state in which PAR4 indicate potential or actual pathology). Detection and measurement of indicators of efficacy are generally based on measurement of PAR4 and/or clinical symptoms associated with the disease state, such as heart attack, stroke, atherosclerosis, restemosis, 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 references unless the contexts 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 of the same general type, and reference to "the method" includes one or more methods or steps of the type described herein. "Comprising" means including.

Methods of the invention With respect to all methods described herein, reference to substances such as PAR4, cathepsin G, agonists or antagonists of cathepsin G (or cathepsin G/PAR4 interaction), and agents also include compositions comprising one or more of these substances. These compositions may further comprise suitable excipients, such as pharmaceutically acceptable excipients including buffers, which are well known in the art. Methods of inhibiting cathepsin G-mediated PAR4 activation

The invention provides methods of inhibiting, or suppressing, PAR4 activation, particularly, cathepsin G-mediated PAR4 activation.

In one embodiment, these methods entail contacting PAR4 and/or cathepsin G with an agent that interferes with cathepsin G/PAR4 interaction, such that cathepsin G-mediated activation of PAR4 is inhibited. In some embodiments, PAR4-expressing cells are used, which may be recombinant or naturally-occurring. In some embodiments, an agent that interferes with cathepsin G/PAR4 interaction is administered to an individual in an amount sufficient to inhibit cathepsin G-mediated PAR4 activation.

Any agent which inhibits cathepsin G/PAR4 interaction such that cathepsin G- mediated PAR4 activation is inhibited is suitable. In some embodiments, an agent other than a PAR4 antagonist is used. Determining that an agent inhibits cathepsin G/PAR4 interaction may be determined using standard techniques in the art. For example, PAR4 activation is measured in the presence of cathepsin G (controlling for other sources of possible PAR4 activation, such as thrombin), with and without agent. A reduction in level of activation in the presence of agent under these reaction conditions indicates that the agent specifically inhibits cathepsin G-mediated PAR4 activation. Agents which inhibit cathepsin G are known, as are agents which inhibit PAR4 (such as an anti-PAR4 antibody which inhibits binding of cathepsin G). Examples of agents which inhibit cathepsin G include, but are not limited to, ecotin, AMCI-1, AMCI-2, AMCI-3, oxidized mucus proteinase inhibitor (MPI), squamous cell carcinoma antigen 2 (SCCA2), suramin, alpha 1- antichymotrypsin, alpha 1 -proteinase inhibitor, eglin c, squash inhibitors, sulfated polymer MDL 101,028. Bania et al. (1999) Eur. J. Biochem. 262:680-687; Boudier et al. (1999) Biochemistry 38:8451-8457; Silverman et al. (1998) Tumour. Biol. 19:480-487; Ermolieff et al. (1998) Biochem. J. 330:1369-1374; Cadene et al. (1997) J. Biol. Chem. 272:9950- 9955; Schick et al. (1997) J. Biol Chem. 272:1849-1855; Kalsheker (1996) Int. J. Biochem. Cell. Biol 28:961-964; Otlewski and Krowarsch (1996) Ada. Biochim. Pol. 43:431-444; aXsXon (1995) Inflammation 19:75-81 ; Ermolieff et al. (1994) J. Biol. Chem. 269:29502- 29508; Janusz and Hare (1994) Int. J. Immunopharmacol. 16:623-632. Another example of an inhibitor of cathepsin G is fragment or derivative of cathepsin G which inhibits cathepsin G-mediated PAR4 activation, i.e., competes with intact cathepsin G for binding to PAR4 but does not activate PAR4, or causes reduction in extent of activation of PAR4.

Another example of an agent which inhibits cathepsin G is an antibody (For example, the antibody binds to cathepsin G such that cathepsin G binding to and/or cleavage of PAR4 is impaired.). Examples of agents which inhibit PAR4 include, but are not limited to, anti- PAR4 antibodies which block binding and cleavage by cathepsin G. Methods of producing antibodies and testing for such specificity are well known in the art. Preferably, the antibody is homogeneous with respect to sequence (for example, is monoclonal). It is understood, however, that for the practice of these methods, the inhibitor specifically inhibits cathepsin G/PAR4 interaction, which can be shown using standard methods in the art. For example, using a culture system an assay is conducted with and without cathepsin G, using cells which do or do not express PAR4 (by, for example, transfection with a

PAR4-expressing polynucleotide).

In another embodiment, suppression of cathepsin G-mediated PAR4 activation is effected by an agent which reduces cathepsin G activity by reducing the amount of cathepsin G available for interacting with PAR4. For example, such an agent could reduce the amount of bioavailable cathepsin G by inhibiting the amount of cathepsin G (either by inhibiting transcription and or translation of cathepsin G mRNA or by decreasing half-life). As another example, an agent could inhibit activation of cathepsin G such as by dipeptidyl peptidase I. As another example, an agent could inhibit release of cathepsin G from a cell, such as from a neutrophil cell. The methods may be in vitro, ex vivo, or in vivo. For in vitro methods, an agent is contacted with cathepsin G and/or PAR4 (or PAR4-expressing cell) under suitable reaction conditions such that cathepsin G-mediated PAR4 activity is reduced (or would be reduced if cathepsin G were present). As an example, PAR4-expressing cells may be used, which are combined with the agent(s). Examples of naturally-occurring cells which express PAR4 (or at least produce PAR4 RNA transcripts) include platelets, small intestine, lung, placenta, liver, pancreas, thyroid, prostate, and testis. Xu et al. (1998). In the presence of cathepsin G and the agent, cathepsin G-mediated PAR4 activation is inhibited. In other in vitro embodiments, PAR4 expressing cells and cathepsin G expressing cells (such as neutrophils) are used. An agent is introduced into the cell culture in an amount sufficient to inhibit cathepsin G-mediated PAR4 activation. For ex vivo methods, the cells are removed from an individual, are contacted with the agent(s), and then are returned to the individual.

Preferably, suitable cells or cell classes are isolated (such as platelets). For in vivo methods, the agent is administered (in a suitable formulation and dosage) to an individual. A suitable individual for the ex vivo or in vivo methods is one who has a condition and/or disorder which would likely benefit from inhibition of PAR4 activation, such as a thrombosis or inflammation. Preferably, but not necessarily, the individual is human.

Administration options are discussed below. Preferred modes of administration are local although non-local methods may be used.

Assays and indicia of PAR4 activation (and thus assays and indicia for inhibition) are known in the art and described in the Examples. For in vivo use, inhibition of PAR4 activation may be measured and/or indicated by several criteria, such as evaluating an appropriate biological sample which contains PAR4 expressing cells, testing hemostatis, and/or other clinical indicia of reduction in extent of PAR4 activation, depending on the individual's condition.

Methods of inhibiting PAR4-mediated platelet activation

The invention also provides methods of inhibiting PAR4-mediated platelet activation. In one embodiment, these methods entail contacting PAR4 and/or cathepsin G with an agent that interferes with cathepsin G/PAR4 interaction, such that PAR4-mediated platelet activation is inhibited. In some embodiments, PAR4-expressing cells are used, which may be recombinant or naturally-occurring. In some embodiments, an agent that interferes with cathepsin G/PAR4 interaction is administered to an individual in an amount sufficient to inhibit PAR4-mediated platelet activation.

Any agent which inhibits cathepsin G/PAR4 interaction such that platelet activation is inhibited is suitable. In some embodiments, an agent is other than a PAR4 antagonist. Determining that an agent inhibits cathepsin G/PAR4 interaction may be determined using standard techniques in the art. For example, PAR4 activation is measured in the presence of cathepsin G (controlling for other sources of possible PAR4 activation, such as thrombin), with and without agent. A reduction in level of activation in the presence of agent under these reaction conditions indicates that the agent specifically inhibits cathepsin G-mediated PAR4 activation. Agents which inhibit cathepsin G are known, as are agents which inhibit PAR4 (such as anti-PAR4 antibody). Examples of agents which inhibit cathepsin G include, but are not limited to, ecotin, AMCI-1, AMCI-2, AMCI-3, oxidized mucus proteinase inhibitor (MPI), squamous cell carcinoma antigen 2 (SCCA2), suramin, alpha 1 -antichymotrypsin, alpha 1 -proteinase inhibitor, eglin c, squash inhibitors, sulfated polymer MDL 101 ,028. Another example of an inhibitor of cathepsin G is fragment or derivative of cathepsin G which inhibits cathepsin G-mediated PAR4 activation or an appropriate anti-cathepsin G antibody, as described above. Preferably, the antibody is homogeneous with respect to sequence (for example, is monoclonal). Examples of agents which inhibit PAR4 include, but are not limited to, anti-PAR4 antibodies (particularly, but not necessarily, those which block the PAR4 thrombin/cathepsin G cleavage site).

Methods of producing antibodies and testing for such specificity are well known in the art. Such an antibody can be further tested to determine whether it suppresses cathepsin G- mediated platelet activation (via PAR4 activation). It is understood, however, that for the practice of these methods, the inhibitor specifically inhibits cathepsin G/PAR4 interaction, which can be shown (as discussed above) using standard methods in the art. In some embodiments, an agent which suppresses cathepsin G-mediated platelet activation is used.

In another embodiment, suppression of PAR4-mediated platelet activation is effected by an agent which reduces cathepsin G activity by reducing the amount of cathepsin G available for interacting with PAR4. For example, such an agent could reduce the amount of bioavailable cathepsin G by inhibiting the amount of cathepsin G (either by inhibiting transcription and/or translation of cathepsin G mRNA or by decreasing half-life). As another example, an agent could inhibit dipeptidyl peptidase I-mediated activation of cathepsin G.

The methods may be in vitro, ex vivo, or in vivo. For in vitro methods, an agent is contacted with cathepsin G and/or a PAR4 expressing cell (whether recombinant or naturally-occurring). The three components are combined under suitable reaction conditions such that PAR4-mediated platelet activation is reduced. As an example, PAR4- expressing cells may be used, which are combined with the agent(s). In the presence of cathepsin G and the agent(s), platelet activation mediated by cathepsin G is inhibited. For ex vivo methods, the cells are removed from an individual, are contacted with the agent(s), and then are returned to the individual. Preferably, suitable cells or cell classes are isolated

(such as platelets). For in vivo methods, the agent is administered (in a suitable formulation and dosage) to an individual. A suitable individual for the ex vivo or in vivo methods is one who has a condition and/or disorder which would likely benefit from inhibition of PAR4 activation, such as a thrombosis or inflammation. Preferably, but not necessarily, the individual is human. Administration options are discussed below.

Preferred modes of administration are local although non-local methods may be used.

Assays and indicia of platelet activation are known in the art and described in the Examples. For in vivo use, inhibition of platelet activation may be measured and/or indicated by several criteria, such as evaluating an appropriate biological sample which contains PAR4 expressing cells, testing hemostatis and/or other clinical indicia of reduction in extent of platelet activation, depending on the individual's condition.

Methods of inhibiting neutrophil-mediated platelet activation The invention also provides methods of inhibiting neutrophil-mediated platelet activation in an individual, comprising administering to the individual a composition comprising an agent which inhibits cathepsin G-PAR4-mediated platelet activation in an amount sufficient to inhibit cathepsin G-PAR 4-mediated platelet activation. Examples of suitable agents, which include agents which inhibit cathepsin G/PAR4 interaction, been described above. Because these methods are designed for settings in which neutrophils are found in significant concentrations, generally these methods entail administering the composition such that the agent(s) will reach these site(s), and the individuals will have a condition(s) in which neutrophil-mediated platelet activation is perceived to be, or believed to be, an undesirable occurrence. For example, the individual may have a condition in which significant neutrophil margination occurs or may occur, such as sepsis, endotoxic shock, infarction due to microvascular thrombosis and tissue damage, and reperfusion injury.

Accordingly, the methods can be used to treat, delay and/or prevent such a condition(s).

As discussed above and below, a variety of formulations and administration protocols can be used, from topical to systemic (such as IN.).

Methods of stimulating cathepsin G-mediated PAR4

The invention provides methods of stimulating PAR4 activation using cathepsin G, comprising contacting PAR4, (e.g., a PAR4-expressing cell) with cathepsin G (or a composition comprising cathepsin G) in an amount sufficient to effect stimulation of PAR4 activation. In other embodiments, PAR4 is contacted with an agent that mimics cathepsin G (or a composition comprising such a mimic (or mimetic)). In other embodiments, cathepsin G is induced such that the amount of biologically available (or biologically active) cathespin G is increased (for example, the activity of cathepsin G is increased and/or amount of cathepsin G release from cells is increased). In some embodiments, PAR4-expressing cells are used, which may be recombinant or naturally-occurring. In some embodiments, a composition comprising cathepsin G or a mimetic of cathepsin G is administered to an individual in an amount effective to stimulate PAR4 activation.

Examples of mimics of cathepsin G are peptides which activate PAR4, such as AYPGKF and GYPGQV (for GYPGQV, see Kahn et al. (1998); Xu et al. (1998)). In some embodiments, the invention provides methods of stimulating P AR4 activation using AYPGKF. For these methods, AYPGKF is contacted with a PAR4 expressing cell, whereby PAR4 is activated. In other embodiments, AYPGKF is administered to an individual in an amount sufficient to stimulate PAR4 activation.

Examples of agents which increase (biologically available or active) cathepsin G is dipeptidyl peptidase I (Pham et al. (1999)), fMLP and other neutrophil activators which cause cathepsin G release. In some embodiments, the mimic of cathepsin G is other than a

PAR4 activating peptide (such as AYPGKF and GYPGQV). These methods can be in vitro, ex vivo, or in vivo. For in vitro methods, cathepsin G (or an mimic of cathepsin G) may be contacted with, for example, a cell expressing PAR4 (whether naturally occurring or recombinant, such as those described herein and in the examples). For ex vivo methods, the cells are removed from an individual, are contacted with cathepsin G (or an mimic of cathepsin G), and then are returned to the individual. For in vivo methods, cathepsin G (or an mimic of cathepsin G) is administered (in a suitable formulation and dosage) and/or cathepsin G is induced by adding an agent which elicits cathepsin G production or otherwise enhances cathepsin G activity, such as fMLP (via promoting cathepsin G release) and dipeptidyl peptidase I. A suitable individual for the ex vivo or in vivo methods is one who has a condition and/or disorder which would likely benefit from PAR4 activation, such as a wound, improper or inappropriate hemostasis response (e.g., a bleeding condition), or a condition in which an enhanced and/or localized inflammatory response is desirable. Administration options are discussed below. Preferred modes of administration are local (such as directly to the wound), although non-local methods may be used. The amount(s) to be administered depends, inter alia, on the condition to be treated as well as the agent to be used. An agent (or composition comprising one or more agents) is administered (which could occur in one or more administrations) in a sufficient amount to achieve the desired result, which may be any manifestation of PAR4 activation. Assays and indicia of PAR4 activation (such as calcium efflux and platelet activation) are known in the art and described in the Examples.

Methods of stimulating PAR4-mediated platelet activation The invention also provides methods of stimulating PAR4-mediated platelet activation comprising contacting PAR4 (e.g., a PAR4-expressing cell) with cathepsin G (or mimic of cathepsin G) in an amount sufficient to effect activation of platelets. In some embodiments, PAR4-expressing cells are used, which may be recombinant or naturally- occurring. In some embodiments, a composition comprising cathepsin G or a mimetic of cathepsin G is administered to an individual in an amount effect to stimulate PAR4- mediated platelet activation. In other embodiments, a composition comprising an agent which stimulates cathepsin G production, activation and/or release in cells is administered.

Examples of cathepsin G mimics have been discussed above. In some embodiments, the cathepsin G mimic is other than a PAR4 activating peptide. With respect to PAR4-activating peptide AYPGKF, in some embodiments, the invention provides methods of stimulating PAR4-mediated platelet activation using AYPGKF. For these methods, AYPGKF is contacted with a PAR4 expressing cell (such as a platelet), thereby stimulating stimulation of PAR4-mediated platelet activation. In other embodiments, AYPGKF is administered to an individual in an amount sufficient to stimulate PAR4- mediated platelet activation.

These methods can be in vitro, ex vivo, or in vivo. For in vitro methods, cathepsin G (or mimic) may be contacted with, for example, a cell expressing PAR4 (such as those described in the examples). For ex vivo methods, the cells are removed from an individual, are contacted with cathepsin G (or a mimic), and then are returned to the individual. Preferably, suitable cells or cell classes are isolated (such as platelets). For in vivo methods, cathepsin G (or a mimic) is administered (in a suitable formulation and dosage) and/or induced by adding an agent which elicits its production or otherwise enhances cathepsin G activity (for example, by stimulating release) such as fMLP. A suitable individual for the ex vivo or in vivo methods is one who has a condition and/or disorder which would likely benefit from PAR4 activation, such as a wound, improper or inappropriate hemostasis response (e.g., a bleeding condition), or a condition in which an enhanced and/or localized inflammatory response is desirable. Administration options are discussed below. Preferred modes of administration are local (such as directly to the wound), although non-local methods may be used. In some embodiments, platelet activation is stimulated by stimulating neutrophil production (in an individual or ex vivo or in vitro) and/or introducing neutrophils to an individual. Other embodiments include the additional step of adding (or administering) an agent which stimulates cathepsin G release, such as fMLP.

Assays and indicia of platelet activation (such as aggregation) are known in the art and described in the Examples. The invention provides methods of stimulating neutrophil-mediated platelet activation in an individual comprising administering to the individual a composition which enhances cathepsin G/PAR4 interaction in an amount sufficient to stimulate neutrophil- mediated platelet activation. Such agents, which include agents which enhance cathepsin G/PAR4 interaction, have been discussed above.

Formulations and administration

With respect to all the above methods, any one or more appropriate modulating agents may be used. Accordingly, compositions may also contain a variety of, for example, other conventional antiplatelet or anti-thrombin or anti-cathepsin G compounds. 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. Alternative anti- thrombin compounds are hirudin derivatives. These compositions may be in single or multiple dosage forms.

A composition may be administered to an individual using any convenient means capable of resulting in the desired modulation. Thus, the a composition can be incorporated into a variety of formulations for 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, com starch or potato starch; binders, such as com 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 by using, for example, 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 for rectal administration. 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 of the 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.

Agents for use in the methods of the 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 of the functional chemical groups. The candidate compounds are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivative, stmctural 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 of the 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 of the following ranges: 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 of the following ranges: 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 of the 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 of the 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 of the clotting, thus eliminating some of the 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 of the clinical indication), by IN.

Screening assays for agents that modulate cathepsin G-mediated PAR4 activation The present invention encompasses methods of identifying agents that modulate cathepsin G-mediated PAR4 activation based on their ability to elicit a characteristic associated with PAR4 function, or activity. These methods, which are useful for identifying agents which may modulate PAR4-specific platelet activation, 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 a cathepsin G- mediated PAR4 activity. In the in vivo embodiments, living cells having PAR4 function are used for testing agents in conjunction with cathepsin G. For purposes of this invention, an agent may be identified on the basis of only partial loss of cathepsin G-mediated PAR4 function, although characteristics associated with total loss of cathepsin G-mediated PAR4 function may be preferable for antagonist. An agent may also be identified by its ability to enhance cathepsin G-mediated PAR4 function. Accordingly, the screening methods of the invention encompass methods of identifying agonists which increase or elicit activation as well as methods of identifying antagonists which inhibit activity. The general screening strategy is to introduce a pharmaceutical candidate and then determine whether the effect (if any) is beneficial, and preferably specific. Application of the agent can be direct (such as determining whether a candidate binds to cathepsin G 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 cathepsin G/PAR4 interaction has the potential to inhibit a pathology associated with cathepsin G/PAR4 interaction when administered. It is not necessary that the mechanism of modulation be known; only that the alteration affect infected cells preferably without being significantly detrimental to other, uninfected cells.

Modulation of cathepsin G-mediated PAR4 activation and/or platelet activation may occur at any level that affects the function of cathepsin G or PAR4 or platelet or any combination thereof. For instance, an agent may modulate cathepsin G-mediated PAR4 activation and/or platelet activation by binding to cathepsin G and/or PAR4. An agent may modulate cathepsin G-mediated PAR4 activation and/or platelet activation by preventing, reducing or increasing production of a cathepsin G and/or PAR4 binding protein. An agent may modulate cathepsin G-mediated PAR4 activation and/or platelet activation by binding to a polypeptide that binds cathepsin G and/or PAR4. The assays described herein include those that examine effects on unoccupied receptors as well as assays that utilize displacement of a ligand from an occupied receptor.

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 dmg 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.

As indicated in the definition of "agent" above, agents which may be used in the screening methods described herein encompass numerous chemical classes. Candidate agents 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 which the cDNA molecules may be cloned are conventional techniques. With respect to the screening methods described herein, it is understood that (intact) PAR4, functional fragments of PAR4, and/or functional equivalents (including functional equivalent fragments) of PAR4 may be used, as appropriate. For example, if cathepsin G binds to a region(s) of PAR4 which may be isolated from intact PAR4, 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 constmcted, for example, by employing conservative amino acid substitutions. Accordingly, as the definition of "PAR4" makes clear, reference to "PAR4" in this section and throughout this application applies to any of the above PAR4 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 modulate interaction between cathepsin

G and PAR4 which could indicate an ability to modulate cathepsin G-mediated PAR4 activation. Generally, these in vitro screening assays entail determining whether an agent interferes with binding of cathepsin G to PAR4 using standard techniques in the art. A preliminary indication of whether an agent interferes with cathepsin G/PAR4 interaction is assaying for agents which bind to cathepsin G and/or PAR4.

For an assay for an agent that binds to cathepsin G and/or PAR4, cathepsin G 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 cathepsin G and/or PAR4 or fragment of cathepsin G and/or PAR4 is conjugated with a well-characterized epitope or protein. Recombinant cathepsin G and or PAR4 is then purified by, for instance, immunoprecipitation using anti-cathepsin G and/or anti-PAR4 antibodies or anti-epitope antibodies or by binding to immobilized ligand of the conjugate. Alternatively, cathepsin G and/or PAR4 are available by other means, such as through commercial sources. An affinity column made of cathepsin G and/or PAR4 or cathepsin G 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. See, for example, Lechner and Carbon (1991) Cell 64:717-725. 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 cathepsin G and/or PAR4 or cathepsin G and/or PAR4-fusion proteins, may be performed to determine the cathepsin G and/or PAR4 binding activity of different agents.

Similar methods can be used for screening for an agent(s) that competes for binding to cathepsin G and/or PAR4. Competitive assays are known in the art, and generally involve measuring degree of binding in the presence of increasing amounts of the 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 compete for binding to cathepsin G and/or PAR4 polypeptides.

It is also understood that the in vitro screening methods of this invention include stmctural, or rational, dmg design, in which the amino acid sequence, three-dimensional atomic structure or other property (or properties) of cathepsin G and/or PAR4 provides a basis for designing an agent which is expected to bind to cathepsin G and/or PAR4. Generally, the design and/or choice of agents in this context is governed by several parameters, such as the perceived function of the cathepsin G and/or PAR4 target, its three- dimensional stmcture (if known or surmised), and other aspects of rational dmg 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 dmg designed may also be tested in the in vivo assays described below. In vivo screening methods In in vivo screening assays, a living cell containing a functioning PAR4 gene, or a living cell containing a polynucleotide constmct comprising a PAR4 encoding sequence are exposed to an agent in the presence of cathepsin G under conditions which permit cathepsin G/PAR4 interaction. In contrast (as described above), conventional dmg 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 dmg 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 modulation of cathepsin G-mediated P AR4 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 cathepsin G-mediated 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, which in this instance is any characteristic associated with PAR4 function, 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 cathepsin G-mediated modulation of PAR4 is measured, i.e., the modulation must be specific interference, mimicry, and/or enhancement of cathepsin G-mediated PAR4 activation. This specificity may be determined using experimental methods and designs standard in the art.

For 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 PAR4 expressing cell in the presence of agent without cathepsin G. As 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. 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 elicit a characteristic associated with modulation of cathepsin G-mediated PAR4 function in a suitable host cell. In some embodiments, the modulation is an increase or activation of cathepsin G-mediated

PAR4 activation (i.e., the screening methods identify agonists). In other embodiments, the modulation is a decrease (which can be partial to total loss) of cathepsin G-mediated PAR4 activation (i.e., the screening methods identify antagonists).

In some embodiments, the screening methods identify agents which may modulate cathepsin G-mediated PAR4 activation. In some embodiments, the screening methods identify agents which may modulate PAR4-mediated platelet activation (by modulating cathepsin G/PAR4 interaction).

Accordingly, in one embodiment, the invention provides methods for identifying an agent that may modulate cathepsin G-mediated PAR4 activation comprising the following steps: (a) combining, or contacting, at least one agent to be tested with a PAR4-expressing cell and cathepsin G (i.e., contacting said host cell with cathepsin G in the presence of agent), and (b) analyzing at least one characteristic which is associated with modulation of cathepsin G-mediated PAR4 function in said cell, wherein an agent is identified by its ability to elicit at least one such characteristic, as compared to control conditions (i.e., without agent). For these methods, the (host) cell may be any cell in which PAR4 function has been demonstrated. PAR4 function may arise due to naturally-occurring PAR4 encoding sequences in the host cell or due to recombinant expression (i.e., expression of recombinant 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 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, CN1 cells, L cells, and HeLa cells.

The steps of the screening methods described herein (including any portions of steps or substeps) may be practiced in any order, as long as an appropriate result is obtained. For example, a PAR4-expressing cell may be contacted first with cathepsin G, and then an agent, or the reverse. As another example, a PAR4-expressing cell may be simultaneously contacted with agent and cathepsin G.

In one embodiment, these methods comprise the following steps: (a) introducing a polynucleotide encoding PAR4 into a suitable host cell that otherwise lacks PAR4 function, wherein PAR4 function is restored (or established) in said host cell; (b) combining said host cell with cathepsin G and at least one agent to be tested; and (c) analyzing at least one characteristic which is associated with modulation of PAR4 function, wherein an agent is identified by its ability to elicit at least one said characteristic.

In another embodiment, PAR 1 -deficient mouse lung fibroblasts (KOLFs) that have been stably transfected with cDΝA encoding human PAR4 (KOLF-PAR4) are used for the screening assays. To screen for agents that modulate cathepsin G-mediated PAR4 activation, KOLF-PAR4 is incubated with the agent to be screened. The modulating potency of the agents to be screened can be measured by, for example, the intracellular calcium mobilization assay as described in Example 3. The host cell used for these methods initially lacks PAR4 function (i.e., lacks PAR4 function before introduction of polynucleotide encoding PAR4). Lacking PAR4 function may be partial to total. A suitable host cell in this context is any host cell in which recombinant PAR4 complements a defect of host cell PAR4 function. For example, PAR1- 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 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 of the 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 of the art.

Determining whether recombinant PAR4 product can substitute for the host cell's PAR4 (or homologue) gene product is within the skill of the art. For example, the host cell's PAR4 (or homologue) function may be deleted by, for instance, recombinant methods. A polynucleotide encoding PAR4 or a functional fragment thereof, is then introduced into the cell, depending on the particular host cell used, by using any of the many methods known in the art, including but not limited to electroporation, CaCl₂ precipitation, and lipofectamine treatment of the host cells. Polynucleotides introduced into a suitable host cell(s) are polynucleotide constmcts comprising a polynucleotide encoding PAR4 or a functional fragment thereof. These constmcts contain elements (i.e., functional sequences) which, upon introduction of the constmct, allow expression (i.e., transcription, translation, and post-translational modifications, if any) of PAR4 amino acid sequence in the host cell. Exemplary methods and procedures for generating such host cells either transiently or stably expressing recombinant PAR4 are described in Kahn et al.

(1999); Connolly et al. (1996), and are generally known to the practitioners in the art.

Restoring 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 PAR4 function (i.e., wild type). Such parameters include, but are not limited to, response to thrombin or activating peptides.

The screening methods described above represent primary screens, designed to detect any agent that may exhibit modulation activity on 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 cells if the initial screen has been performed in a host cell other than a human cell. Another screen may comprise testing the agent(s) in a host cell with native PAR4 function if the initial screen has been performed in a host cell without native PAR4 function but being introduced of such function. 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).

Kits of the invention The invention also provides kits comprising cathepsin G and/or an mimic of cathepsin G (or compositions comprising cathepsin G and/or an mimic of cathepsin G) in suitable packaging, which further comprise instmctions for administration to an individual in order to effect, or stimulate, PAR4 activation in an individual. The instmctions can be for any of the following: effecting PAR4 activation; stimulation of PAR4-mediated platelet activation; and/or treatment of a condition for which PAR4 activation or stimulation of

PAR4-mediated platelet activation is indicated. In some embodiments, the mimic of cathepsin G is other than a PAR4-stimulating peptide. In some embodiments, the mimic of cathepsin G is AYPGKF. In some embodiments, the agent enhances cathepsin G/PAR4 interaction. In some embodiments, the kits comprise an agent which interferes with cathepsin

G/PAR4 interaction (or a composition comprising one or more such agents) in suitable packaging. The kits further comprises instmctions for administering the composition to an individual to effect inhibition of PAR4 activation, inhibition of PAR4-mediated platelet activation, and/or treatment of a condition for which inhibition of PAR4 activation or inhibition of PAR4-mediated platelet activation is indicated. In some embodiments, the agent is other than a PAR4 antagonist.

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 labeled calcium was obtained from Molecular Probes (Eugene, OR). 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. Cathepsin G was obtained from Athens Research (Athens, GA). Thrombin was obtained from Enzyme Research Labs (South Bend, IN). Hi din was purchased from Sigma Chemical Co. and PPACK from Calbiochem (San Diego, CA).

Example 1

Activation of PAR receptors by thrombin and cathepsin G and effect of thrombin and serine protease inhibitors on the induced activation

Oocyte experiments. cRNA encoding human PARl , PAR3 and PAR4 and mouse PAR2 were microinjected into Xenopus oocytes, respectively. 24 hours later, oocytes were radiolabeled with ⁴⁵Ca and responses to 10 nM thrombin or 100 nM cathepsin G stimulation were assessed as calcium mobilization, i.e., ⁴⁵Ca release from radiolabeled oocytes, as described in Vu et al. (1991a). Unless indicated otherwise, in all oocyte microinjection experiments, relative expression levels of FLAG epitope-tagged wild type and mutant PAR receptors expressed on the oocytes surface were determined using binding of Ml monoclonal antibodies that were raised against FLAG epitope.

Determination of Ecotin K^* Values. K^* values were determined by titration of cathepsin G (6 nM) or thrombin (8.5 nM) with various ecotin concentrations. Yang and

Craik (1998) J. Mol. Biol. 279:1001-1011. Enzyme activity was monitored by cleavage of the chromogenic cathepsin G substrate N-succinyl-alanyl-alanyl-prolyl-phenylalanyl- thiobenzyl ester (SynPep) and the chromogenic thrombin substrate Spectrozyme-TH (American Diagnostica). The apparent K* for cathepsin G was determined by non-linear regression analysis fit of the data to an equation derived for kinetics of reversible tight binding inhibitors. Morrison (1969) Biochim. Biophys. Acta 185:269-286; Williams and Morrison (1979) Methods Enzymol. 63:437-467. The apparent Kj for thrombin was determined by linear regression analysis fit of the data to an equation derived for Michaelis-Menten kinetics of competitive inhibitors. Results. Neither thrombin nor cathepsin G triggered calcium signaling in Xenopus oocytes that had not been injected with any PAR receptor cRNAs. Cathepsin G purified from human neutrophils, as thrombin did, triggered consistent calcium signaling in Xenopus oocytes expressing human PARl or PAR4, but not those expressing PAR2 or PAR3 (Figure 1).

The activation of PARl or PAR4 induced by cathepsin G was not the result of thrombin contamination in the cathepsin G preparation. Oocytes expressing human PARl or PAR4 were stimulated with 10 nM thrombin or 100 nM cathepsin G in the absence (Control) or presence of the thrombin inhibitors, PPACK (1 μm), hirudin (10 units/ml), or ecotin (500 nM). Thrombin inhibitors such as himdin or PPACK did not prevent PAR activation by cathepsin G (Figure 2B) but did block activation by thrombin (Figure 2A). By contrast, the macromolecular serine protease inhibitor, ecotin, blocked cathepsin G- induced calcium responses (Figure 2B) but had no effect on thrombin (Figure 2A).

The apparent Kj for inhibition of cathepsin G by wild type ecotin was determined to be 15 + 6 pM while the K_j for the inhibition of thrombin was 1.1 + 0.2 μM. This gives ecotin a specific index of 73,000 in favor of blocking cathepsin G and not thrombin.

Example 2 Dependence of cathepsin G activation on cleavage site and tethered ligand in PARl and PAR4

A mutant PARl with a phenylalanine to alanine substitution at position 2 of the tethered ligand (PARl F/A) or a mutant PAR4 with a glycine to proline substitution at position 1 of the tethered ligand (PAR4 G/P) was expressed in Xenopus oocytes as described in Example 1. PARl F/A ablates tethered ligand function in PARl, while PAR4 G/P renders PAR4 uncleavable at the R⁴⁷/G⁴⁸ peptide bond. ⁴⁵Ca release was measured in response to stimulation with 10 nM thrombin, 100 nM cathepsin G, or the PAR-activating peptides (AP) SFLLRN at 100 μM for PARl F/A and AYPGKF at 500 μM for PAR4 G/P.

Cells expressing a PARl mutant, PARl F/A, in which alanine was substituted for phenylalanine at position 2 of the activation peptide, failed to respond to either thrombin or cathepsin G but did respond to PARl -activating peptide (AP) SFLLRN (Figure 3). Similarly, a PAR4 mutant, PAR4 G/P, in which a glycine to proline mutation at position 1 of the activation peptide, rendered the receptor uncleavable by thrombin or cathepsin G but still responsive to the PAR4-activating peptide (AP). Thus activation of PARl and PAR4 by cathepsin G and thrombin almost certainly occur by the same mechanism: unmasking of the respective tethered ligands by cleavage at the R ¹/S⁴² peptide bond in PARl and the R⁴⁷/G⁴⁸ peptide bond in PAR4.

Example 3

Cathepsin G activated PARl andPAR4 stably expressed in mammalian cells

Calcium measurement. Lung fibroblasts derived from PARl null mice (KOLF) were cultured in DME containing 10%> bovine calf semm at 37°C in a humidified CO incubator, and were transfected to stably express FLAG epitope-tagged hPARl or hPAR4. Trejo et al. (1996). Calcium mobilization in Fura-2-loaded cells was measured fluorometrically after loading with 4 μg/ml Fura-2/AM (Molecular Probes) for 30 minutes at 37°C using a Hitachi F2000 fluorometer, and plotted as fluorescence ratio versus time

(sec). For mixed cell experiments neutrophils (4x10 /ml) and platelets (5x10 /ml) were suspended together in a cuvette in the presence of 1 mM EGTA and 2.5 μg/ml cytochalasin B just prior to measurement.

Results. Optimal responses in cells expressing hPARl (Figure 4A) were obtained at 100 nM cathepsin G. Cells expressing hPAR4 (Figure 4B) or untransfected cells (Figure

4C) were stimulated with 500 nM cathepsin G.

Cathepsin G (100 nM) triggered increases in cytoplasmic calcium in PAR1- transfected cells (Figure 4A). Cells transfected with PAR4 required 500 nM cathepsin G to produce a similar response (Figure 4B). No response was detected in untransfected cells (Figure 4C). Similar results were observed in transfected Ratl fibroblasts expressing

PARl and PAR4. Thus, cathepsin G can activate PARl and PAR4 in mammalian cell expression systems. These results raised the possibility that PARl and/or PAR4 might mediate cathepsin G activation of human platelets. Example 4

Activation of and signaling via PAR4, but not PARl, are required and necessary for activation of platelets by cathepsin G

Blood cells. Human platelets were isolated from donor blood collected in 1/5 volume citrate buffer. The first one milliliter drawn was discarded. Blood was centrifuged at 200g for 15 minutes to separate PRP, buffy coat and erythrocytes. Platelets were then washed in buffer (134 mM NaCI, 12 mM NaHCO₃, 2.9 mM KC1, 0.34 mM Na₂HPO₄, 1 mM MgCl2, 10 mM Hepes, 5 mM glucose and 0.3%> BSA) containing postaglandin Ej (1 μM) and EDTA (10 mM), then resuspended in buffer alone prior to use. Neutrophils were isolated from buffy coat of the same donor blood as platelets. The buffy coat was layered over a 63%) and 72% Percoll solution and centrifuged for 30 minutes at 500g. The polymorphonuclear leukocyte layer was removed, washed with PBS and resuspended in platelet buffer. Aggregometry. Agonist-induced platelet aggregation was measured using a

Chrono-Log lumiaggregometer. Platelets suspended in buffer described under stirring conditions in the presence of 1 mM CaCl2 were introduced to agonist and change in light transmission measured.

Results. Agonist-induced calcium mobilization was studied in washed human platelets loaded with Fura-2. Platelets were studied in the presence of 0.1 mM EGTA to prevent aggregation. Calcium mobilization induced by 500 nM cathepsin G was measure in naϊve platelets (Figure 5 A), and platelets treated with 100 μM BMS200261, a peptide- based antagonist that blocks PARl activation, but not PAR4 activation (Figure 5B), 1 mg/ml PAR-4 blocking antibody (Figure 5C), or 1 mg/ml pre-immune IgG (Figure 5D). Responses in platelets desensitized for 30 minutes with 100 μM SFLLRN (Figure 5E) or

500 μM AYPGKF (Figure 5F, Figure 5G, Figure 5H) were also measured. Agonists were added as indicated by arrows and responses plotted as fluorescence ratio versus time (sec). Aggregation induced by 1 μM cathepsin G was tested in naϊve platelets (Figure 6A) and platelets treated with 100 μM BMS200261 (Figure 6B), a peptide-based antagonist that blocks PARl activation, but not PAR4 activation, or 1 mg/ml PAR4-blocking antibody (Figure 6C). Platelets treated with BMS200261 were unresponsive to 100 mM SFLLRN.

Cathepsin G (500 nM) triggered both aggregation and robust increases in cytoplasmic calcium in washed human platelets (Figure 5 and Figure 6). The roles of PARl and PAR4 in this response were first examined by desensitization studies. Platelets preincubated with the PARl -activating peptides SFLLRN (100 μM) for 30 minutes failed to respond to subsequent challenge with SFLLRN but did respond to cathepsin G (Figure 5E). By contrast, platelets preincubated with AYPGKF (100 μM) were refractory to stimulation with cathepsin G but did respond robustly to SFLLRN (Figure 5F). These desensitization studies suggest that signaling via PAR4 but not PARl is required for cathepsin G responses in human platelets.

A PAR4-blocking antibody and a PARl -specific antagonist were also used to probe the roles of PARl and PAR4 in platelet responses to cathepsin G. Activation of PARl by thrombin or SFLLRN can be blocked with BMS200261, a peptide-based antagonist that does not block PAR4 activation. Kahn et al. (1999). Similarly, a polyclonal antibody raised to a receptor peptide that spans the thrombin cleavage site of PAR4 blocks cleavage and activation of the receptor by thrombin. Because the antiserum works by blocking cleavage, it does not block PAR4 activation by AYPGKF. Treatment of platelets with the PARl antagonist BMS200261 blocked calcium mobilization and aggregation in response to SFLLRN but not cathepsin G (Figure 5B, Figure 6). By contrast, presence of the antibody to PAR4 effectively blocked the cathepsin G-triggered platelet responses but not responses to AYPGKF or SFLLRN (Figure 5C, Figure 6). Pre-immune rabbit IgG was without effect (Figure 5D), and the specificity of the PAR4 antibody for blockade of PAR4 but not PARl was reported by Kahn et al. (1999). These data strongly suggest that activation of PAR4 but not PARl is necessary for activation of platelets by cathepsin G.

These data are concordant with previous studies concluding that cathepsin G does not activate platelets via PAR 1. Selak (1994); Molino et al. (1995). Molino et al. teach that cathepsin G could cleave PARl either productively at the R41/S42 peptide bond or non-productively at the F55/W56 peptide bond in the PARl amino terminal exodomain. The latter cleavage served to remove the PARl tethered ligand rendering it unresponsive to thrombin while still responsive to SFLLRN. In the context of the model that PARl and PAR4 mediate platelet activation by thrombin, this observation predicts that if PAR4 function were inhibited, cathepsin G might actually block platelet activation triggered by thrombin. This prediction is based on the observation that cathepsin G cleaves PARl at two sites: site 1 (R41/S42), which is the usual activation site (same site used by thrombin), and site 2 (F55/W56), carboxyl to site 1, which is the preferred cleavage site by cathepsin G. Preferential cleavage at site 2 by cathepsin G removes the amino terminal exodomain, essentially disabling the receptor in terms of ability to respond to protease. If PAR4 function were also lost, the platelets should become thrombin refractory. Platelets were first desensitized with AYPGKF then challenged with thrombin or cathepsin G. As expected, thrombin induced a response, presumably via PARl , while cathepsin G elicited no response (Figure 5G and Figure 5H). After both desensitization with AYPGKF and exposure to cathepsin G, platelets became refractory to thrombin but still responded to SFLLRN (Figure 5H).

Taken together, these data are consistent with the model that cathepsin G cleaves PARl at two (or more) sites, with a small fraction of cleavage events occurring at the activating site but the majority occurring at the inhibitory site(s). Molino et al. (1995). Signaling responses to cathepsin G in cultured cells that express high levels of PARl (Figure 4) is perhaps due to cleavage of a small fraction but still adequate absolute number of PARl receptors at the activating site. The absent PARl -mediated thrombin signaling but retained responsiveness to SFLLRN in cathepsin-treated platelets is likely due to cleavage of the majority of receptors at the inhibitory site.

Example 5

Action of cathepsin G on platelet PAR4 mediates neutrophil-dependent platelet activation

Fura-2-loaded platelets (5xl0⁷/ml) and unloaded neutrophils (4xl0⁶/ml) were mixed in the presence of 0.1 mM EGTA and 2.5 μg/ml cytochalasin B. Calcium mobilization in platelets following stimulation with 100 nM fMLP was measured in the absence (Figure 7A) or presence (Figure 7B) of 1 mg/ml PAR4-blocking antibody or 1 mg/ml pre-immune IgG (Figure 7C). Responses shown were plotted as fluorescence ratio versus time (sec).

In cell suspensions containing both neutrophils and platelets, the neutrophil agonist fMLP induced activation of platelets, presumably by release of cathepsin G from neutrophil azurophilic granules. Del Maschio et al. (1990) Am. J. Physiol. 258:H870-H879. This model predicts that, in such a mixed cell system, fMLP-triggered platelet activation should be PAR4-dependent. We tested this prediction. Washed human platelets were loaded with Fura-2 and mixed with neutrophils isolated from the same donor prior to measurement of calcium mobilization. Stimulation with fMLP (100 nM) resulted in reproducible increases in cytoplasmic calcium in Fura-2 loaded platelets (Figure 7A). In the absence of neutrophils, addition of fMLP to platelets alone did not induce an observable signal in Fura-2-loaded platelets. Pretreatment of platelets with the PAR4-blocking antibody (Figure 7B) but not pre-immune IgG (Figure 7C) prevented platelet activation in the mixed cell experiment. These data strongly suggest that, in this system, neutrophil-dependent platelet activation is mediated by the action of cathepsin G on platelet PAR4.

These observations strongly suggest that PAR4 is the "cathepsin G receptor" on human platelets and that activation of PAR4 by cathepsin G is necessary for neutrophil- dependent platelet activation. The ratio of neutrophils to platelets required to achieve robust neutrophil-dependent platelet calcium mobilization was approximately 1/12, one half to one log higher than that likely to be achieved in whole blood. However, endothelial cells that have been activated by thrombin or cytokines bind neutrophils and platelets, thereby concentrating both at sites of inflammation and thrombosis. In addition, shear alone can induce neutrophil-platelet aggregates. Such mechanisms presumably create microenvironments that favor neutrophil-platelet interactions. In this regard, minimal free cathepsin G is detectable in solution after neutrophil stimulation and like many other proteases including thrombin, cathepsin G is inhibited by circulating protease inhibitors. LaRosa et al. (1994); Owen et al. (1995a) J. Immunol. 155:5803-5810. However, a significant proportion of cathepsin G released by neutrophils remains bound to the extracellular membrane where it is catalytically active and relatively resistant to inactivation by protease inhibitors. Owen et al. (1995a); Owen et al. (1995b) J Cell Biol.

131 :775-789; Evangelista et al. (1991) Blood 77:2379-2388. Thus platelet activation might be induced by neutrophil-bound rather than fluid-phase cathepsin G via direct cell-cell contact. Indeed, when P-selectin antibodies were used to block the formation of neutrophil-platelet aggregates, neutrophil-dependent platelet activation became more sensitive to inhibition by protease inhibitors. Evangelista et al. (1993) Blood 81 :2947- 2957. Thus microenvironments created at sites of thrombosis or inflammation may provide a setting where close proximity or direct contact of activated neutrophils and platelets allows cathepsin G-mediated activation of platelet PAR4 to occur.

Cathepsin G-deficient mice showed only a mildly augmented inflammatory response during wound healing but were otherwise without apparent phenotype suggesting that cathepsin G activation of platelet PAR4 is not necessary for survival in an unstressed setting. Brozna (1990) Semin. Thromb. Hemostas. 16:326-332. However, in a stressed, clinical setting, cathepsin G-PAR4 mediated platelet activation could be significant. Clearly, multiple ligand-receptor systems regulate inflammatory and thrombotic responses, and whether deficiencies in other pathways that serve redundant functions might unmask an important role for neutrophil cathepsin G is unknown. Moreover, as noted above, activation of platelets by neutrophil cathepsin G may become important only at sites where activated neutrophils and platelets are substantially concentrated, and the effect of cathepsin G deficiency in models that might engender interactions between activated neutrophils and platelets such as the Shwartzman reaction has not been reported. One general point is that PARs that were originally described as thrombin receptors may also confer responsiveness to proteases other than thrombin. The differential importance of PARl vs. PAR4 for thrombin vs. cathepsin G signaling in human platelets supports the idea that these receptors may serve, at least in part, distinct roles. Thus PAR4 might serve not only to mediate thrombin responsiveness; it might expand the platelet' s repertoire by mediating responses to other proteases.

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 constmed as limiting the scope of the invention, which is delineated by the appended claims.

Claims

CLAIMSWe claim:

1. A method of inhibiting cathepsin G-mediated PAR4 activation comprising contacting cathepsin G or a PAR4-expressing cell with an agent which interferes with cathepsin G/PAR4 interaction, such that PAR4 activation is inhibited.

2. The method of claim 1, wherein the agent is a cathepsin G inhibitor.

3. The method of claim 1, wherein the agent inhibits activation of cathepsin G.

4. The method of claim 1, wherein the agent is an antibody.

5. A method of inhibiting cathepsin G-mediated PAR4 activation in an individual comprising administering to the individual a composition comprising an agent which interferes with cathepsin G/PAR4 interaction in an amount sufficient to inhibit PAR4 activation.

6. The method of claim 5, wherein the agent is a cathepsin G inhibitor.

7. The method of claim 5, wherein the individual is human.

8. The method of claim 7, wherein the individual has a condition selected from the group consisting of thrombosis, organ failure, stroke, myocardial infarction, unstable angina, organ transplantation, inflammation, and pulmonary embolism.

9. A method of inhibiting PAR4-mediated platelet activation comprising contacting cathepsin G or a PAR4-expressing cell with an agent which interferes with cathepsin G/PAR4 interaction, such that PAR4-mediated platelet activation is inhibited.

10. The method of claim 9, wherein the agent is a cathepsin G inhibitor.

11. The method of claim 9, wherein the agent is an antibody.

12. A method of inhibiting PAR4-mediated platelet activation in an individual, comprising administering to the individual a composition comprising an agent which interferes with cathepsin G/PAR4 interaction in an amount sufficient to inhibit PAR4- mediated platelet activation.

13. The method of claim 12, wherein the agent is a cathepsin G inhibitor.

14. The method of claim 13, wherein the individual is human.

15. The method of claim 14, wherein the individual has a condition selected from the group consisting of thrombosis, organ failure, stroke, myocardial infarction, unstable angina, organ transplantation, inflammation, and pulmonary embolism.

16. A method of inhibiting neutrophil-mediated platelet activation in an individual, comprising administering to the individual a composition comprising an agent which inhibits cathepsin G-PAR4-mediated platelet activation.

17. The method of claim 16, wherein the individual is human.

18. The method of claim 17, wherein the individual has a condition selected from the group consisting of thrombosis, organ failure, stroke, myocardial infarction, unstable angina, organ transplantation, inflammation, and pulmonary embolism.

19. The method of claim 16, wherein the agent is a cathepsin G inhibitor.

20. A method of stimulating PAR4 activation comprising contacting a PAR4- expressing cell with cathepsin G such that PAR4 is activated.

21. A method of stimulating PAR4 activation in an individual comprising administering to the individual a composition comprising cathepsin G in an amount sufficient to stimulate activation of PAR4.

D

22. The method of claim 21, wherein the individual is human.

23. A method of stimulating PAR4 activation in an individual comprising administering to the individual a composition comprising an agent which increases 0 cathepsin G activity in an amount sufficient to stimulate PAR4 activation.

24. The method of claim 24, wherein the agent stimulates release of cathepsin G from neutrophils.

5 25. The method of claim 24, wherein the agent is fMLP.

26. A method of stimulating PAR4-mediated platelet activation in an individual comprising administering to the individual a composition comprising an agent which increases cathepsin G activity in an amount sufficient to stimulate PAR4-mediated platelet 0 activation.

27. The method of claim 26, wherein the agent stimulates release of cathepsin G from neutrophils.

5 28. The method of claim 27, wherein the agent is fMLP.

29. A method for identifying an agent that may modulate cathepsin G-mediated PAR4 activation, said method comprising:

(a) combining at least one agent to be tested with a PAR4-expressing cell 0 and cathepsin G; and (b) analyzing at least one characteristic which is associated with modulation of cathepsin G-mediated PAR4 function in said cell, wherein an agent is identified by its ability to elicit at least one such characteristic as compared with control conditions.

30. The method of claim 29, wherein said PAR4-expressing cell is a platelet cell.

31. The method of claim 29, wherein said characteristic is phosphoinositide hydrolysis.

32. The method of claim 29, wherein said characteristic intracellular calcium mobilization.

33. The method of claim 30, wherein said characteristic is change in platelet shape.

34. The method of claim 30, wherein said characteristic is platelet ATP secretion.

35. The method of claim 30, wherein said characteristic is platelet aggregation.

36. The method of claim 29, wherein the modulation is inhibition of cathepsin G- mediated PAR4 activation.

37. A kit comprising a composition comprising cathepsin G, said kit further comprising instmctions for administering the composition to an individual to effect PAR4 activation, stimulation of PAR4-mediated platelet activation, and/or treatment of a condition for which PAR4 activation or stimulation of PAR4-mediated platelet activation is indicated.

38. A kit comprising a composition comprising an agent which interferes with cathepsin G/PAR4 interaction, said kit further comprising instmctions for administering the composition to the individual to effect inhibition of PAR4 activation, inhibition of PAR4- mediated platelet activation, and/or treatment of a condition for which inhibition of PAR4 activation or inhibition of PAR4-mediated platelet activation is indicated.

39. The method of claim 29, wherein the agent to be tested is an antibody.

40. The method of claim 30, wherein the agent to be tested is an antibody.