AU2672899A - Protease-activated receptor 4 and uses thereof - Google Patents

Description

WO 99/43809 PCT/US99/02983 PROTEASE-ACTIVATED RECEPTOR 4 AND USES THEREOF FIELD OF THE INVENTION This invention relates to nucleic acids, their encoded protease-activated receptor 4 5 proteins, and screening assays for agonists and antagonists of the protease activated receptor 4 proteins. BACKGROUND OF THE INVENTION Thrombin, a coagulation protease generated at sites of vascular injury, activates 10 platelets, leukocytes, and mesenchymal cells (Vu, T.-K.H. et al. (1991) 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 suggests that thrombosis in arteries can be blocked by inhibitors of platelet function and by thrombin inhibitors. Thus it 15 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, restenosis, pulmonary inflammation (ARDS), glomerulosclerosis, etc.). A 20 thorough understanding of how thrombin activates cells is an important goal. A receptor that mediates thrombin signaling has been previously identified (Vu, T.

K.H. et al. (1991) Cell 64:1057-1068; USP 5,256,766). This receptor revealed a novel proteolytic mechanism of activation and is referred to as PARI (protease-activated receptor 1). PARI is activated by the binding of thrombin to and cleavage of PARI's amino terminal 25 exodomain at a specific site. Receptor cleavage unmasks a new amino terminus, which then functions as a tethered peptide ligand by binding intramolecularly to the body of the receptor to effect transmembrane signaling (Vu, T.-K.H. et al. (1991) Cell 64:1057-1068). Synthetic peptides that mimic this tethered ligand domain function as PAR1 agonists and activate it independent of thrombin and receptor cleavage (Vu, T.-K.H. et al. (1991) Cell 64:1057 30 1068). To identify which of thrombin's known cellular actions are mediated by PARI, a PARI knockout mouse was recently generated (Connolly, A. et al. (1996) Nature 3_81:516 -1- WO 99/43809 PCT/US99/02983 519). Analysis of mice in which both alleles of the PARI gene were disrupted provided definitive evidence for a second platelet thrombin receptor and for tissue specific roles of distinct thrombin receptors. Specifically, in mice, PARI is not important for platelet responses but is critical for fibroblast responses. 5 Since the identification of PAR1, two other protease-activated receptors have been cloned. A second protease-activated receptor (PAR2) was cloned during a search for relatives of the Substance K receptor (Nystedt, S., et al. (1994) PNAS USA, 91:9208-9212). The physiological activator of PAR2 remains unknown; it is not activated by thrombin. A third protease-activated receptor (PAR3) has also been cloned, but available data suggests 10 that this receptor is involved in thrombin-mediated platelet responses in mice but not in humans. There is a need for a better understanding of thrombin-mediated platelet activation. There is also a need for the identification and characterization of factors involved in platelet mediated pathologies, such as platelet-dependent arterial thrombosis. The understanding of 15 the mechanisms of such offer new mechanisms for treating associated pathologies. SUMMARY OF THE INVENTION Protease-activated receptor 4 (PAR4) is disclosed. PAR4 is useful in assaying libraries of compounds for their activity as thrombin agonists and antagonists. DNA encoding 20 PAR4 is also disclosed as is its insertion into a functional expression vector, DNA expressed in a cell line, and the use of the DNA expression product in an assay to identify compounds as agonists or antagonists of thrombin's effect on PAR4. The invention comprises substantially pure DNA (cDNA or genomic DNA) encoding a protease-activated receptor 4 (PAR4) from vertebrate tissues (SEQ ID NO:1, SEQ ID 25 NO:3, AND SEQ ID NO:4) and degenerate sequences thereof, substantially pure protease activated receptor 4 polypeptides encoded thereby; as well as amino acid sequences substantially identical to the amino acid sequences SEQ ID NO:2 and SEQ ID NO:5 from mouse and human, respectively. The invention also features DNA sequences that hybridize under stringent conditions to DNA encoding PAR4, or to DNA complementary to DNA 30 encoding PAR4. Such DNA sequences are preferably at least 25 nucleotides in length, more preferably 50 nucleotides in length. The invention further comprises fragments of the PAR4 receptor which are activated by thrombin. Such fragments may have the same amino acid -2- WO 99/43809 PCT/US99/02983 sequence as SEQ ID NO:2 and SEQ ID NO:5 or be at least 80% identical to the amino acid sequences SEQ ID NO:2 and SEQ ID NO:5. Such fragments are preferably at least 10 amino acids in length, more preferably at least 30 amino acids in length. In various embodiments, the DNA, receptor or receptor fragment is derived from a 5 vertebrate animal, preferably, human or mouse. However, the gene can be chemically synthesized. An object of the invention is to provide a nucleotide sequence encoding a novel receptor, preferably PAR4 and its functional equivalents. Another object is to provide a cell line genetically engineered to express the 10 nucleotide sequence encoding PAR4. Another object is to provide an antibody which selectively binds the PAR 4 receptor. Another object is to provide a method whereby a compound or library of compounds can be assayed for their ability to activate or block the receptor expressed by the nucleotide sequence. 15 An advantage of the present invention is that a novel thrombin receptor PAR4 is disclosed making it possible to identify novel thrombin agonists and antagonists which may not be identifiable via PARI, PAR2, or PAR3 receptors. A feature of the invention is that it makes it possible to obtain additional information regarding thrombin activation and the sequence of biochemical events initiated by such. 20 These and other objects, advantages and features of the present invention will become apparent to those skilled in the art upon reading the disclosure. BRIEF DESCRIPTION OF THE DRAWINGS Figs. 1A and lB are the complete nucleotide sequences (SEQ ID NO: 1) of the mouse 25 protease-activated receptor 4 gene coding region cDNA. Fig. 2 is the deduced amino acid sequence (SEQ ID NO:2) of the receptor encoded by the nucleotide sequence of Fig. 1. The amino acid sequence encoding mouse PAR4 contains 397 amino acids. The deduced amino acid sequence begins at nucleotides 1-3 (ATG = Met) and ends at nucleotides 1192-1194 (TGA = stop). 30 Fig. 3 is the nucleotide sequence of genomic mouse PAR4 (SEQ ID NO:3) demonstrating a small intron (250 bp) between the signal peptide and cleavage site of the receptor coding sequence. The intronic sequence is underlined. -3- WO 99/43809 PCTIUS99/02983 Figs. 4A and 4B are the complete nucleotide sequences (SEQ ID NO:4) of the human protease-activated receptor 4 gene coding region cDNA. Fig. 5 is the deduced amino acid sequence (SEQ ID NO:5) of the receptor encoded by the nucleotide sequence of Fig. 4. The amino acid sequence encoding human PAR4 contains 5 385 amino acids. The deduced amino acid sequence begins at nucleotides 3-5 (ATG = Met) and ends at nucleotides 1157-1159 (TGA = stop). Figs. 6A and 6B show the alignment of the deduced amino acid sequences (SEQ ID NO:2, 6-8) of the mouse PAR4, mouse PAR3, mouse PAR2, and mouse PARI genes. To indicate homology, gaps (represented by blank spaces) have been introduced into the five 10 sequences. Exact amino acid matches between at least three of the PARs have been enclosed in a shaded box. Fig. 7 shows the structure of the mouse PAR genes, revealing a small (250bp) intron separating exon 1, which encodes the signal peptide, from exon 2, which encodes the mature receptor protein (SEQ ID NO:3, 9-11). 15 Fig. 8 pictorially describes the thrombin cleavage site and activating peptide of the mouse PAR4 receptor. Fig. 9 is a bar graph showing the calcium response of oocytes expressing mouse PAR4 upon exposure to different agonists. Agonists include thrombin, the predicted activating peptide for PAR4 (GYPGKF), and the predicted activating peptide for PARI 20 (SFLLRN). Fig. 10 is a bar graph showing the calcium response of oocytes expressing human PAR4 upon exposure to different agonists. Agonists include thrombin, the predicted activating peptide for human PAR4 (GYPGQV), the predicted activating peptide for mPAR4 (GYPGKF), and the predicted activating peptide for PARI (SFLLRN). 25 Fig. 11 is a bar graph showing the calcium response of oocytes expressing mouse PAR4 upon exposure to various serine proteases. Fig. 12 is a bar graph representing the activation of PAR4 in Xenopus laevis oocytes cell types upon exposure to the tethered ligand peptides of the PARs. Fig. 13 is a graph showing the aggregation of PAR3 knockout mouse platelets in 30 response to mPAR4 activating peptide GYPGKF. Fig. 14 is a graph showing ATP secretion (top) and aggregation (bottom) of wild-type mouse platelets in response to mPAR4 activating peptide GYPGKF. -4- WO 99/43809 PCT/US99/02983 Fig. 15 is a graph depicting the activation of human platelets desensitized to the PARI activating peptide by the PAR4 predicted activating peptide. DESCRIPTION OF THE PREFERRED EMBODIMENTS 5 Before the present protease-activated receptor assays and methods of using such are described, it is to be understood that this invention is not limited to the particular DNA sequences, materials, methods, or processes described as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting since the scope of the present invention 10 will be limited only by the appended claims. It must be noted that as used in this specification and the appended claims, the singular forms "a", "and," and "the" include plural referents 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 15 type, and reference to "the method" includes one or more methods or steps of the type described herein. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. 20 Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials, similar or equivalent to those described herein, can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications cited herein are incorporated herein by 25 reference for the purpose of disclosing and describing specific aspects of the invention for which the publication is cited in connection with. DEFINITIONS By "protease-activated receptor 4", "PAR4", "PAR4 receptor" and the like, is meant 30 all or part of a vertebrate cell surface protein which is specifically activated by thrombin or a thrombin agonist thereby activating PAR4-mediated signaling events (e.g., phosphoinositide hydrolysis, Ca efflux, platelet aggregation). The polypeptide is characterized as having the -5- WO 99/43809 PCT/US99/02983 ligand activating properties (including the agonist activating and antagonist inhibiting properties) and tissue distribution described herein. Specifically, PAR4 receptors are expressed by the DNA sequences of SEQ ID NOs: 2, 4, and 5. By a "polypeptide" is meant any chain of amino acids, regardless of length or post 5 translational modification (e.g., glycosylation). By "substantially pure" is meant that the protease-activated receptor 4 polypeptide provided by the invention is at least 60%, by weight, free from the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, 10 PAR4 polypeptide. A substantially pure PAR4 polypeptide may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding a PAR4 polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. The protein is substantially pure if it can be isolated to a band in a gel. 15 By a "substantially identical" amino acid sequence is meant an amino acid sequence which differs only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for leucine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the biological activity of the 20 receptor. Such equivalent receptors can be isolated by extraction from the tissues or cells of any animal which naturally produces such a receptor or which can be induced to do so, using the methods described below, or their equivalent; or can be isolated by chemical synthesis; or can be isolated by standard techniques of recombinant DNA technology, e.g., by isolation of cDNA or genomic DNA encoding such a receptor. Substantially identical receptors have the 25 same biological function, e.g. are activated by the same compound. By "derived from" is meant encoded by the genome of that organism and present on the surface of a subset of that organism's cells. By "isolated DNA" is meant DNA that is not in its native environment in terms of not being immediately contiguous with (i.e., covalently linked to) the complete coding sequences 30 with which it is immediately contiguous (i.e., one at the 5' end and one at the 3' end) in the naturally-occurring genome of the organism from which the DNA of the invention is derived. The term therefore includes, for example, recombinant DNA which is incorporated into a -6- WO 99/43809 PCT/US99/02983 vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes any recombinant DNA which is part of a hybrid gene 5 encoding additional polypeptide sequence. "Isolated DNA" can mean the DNA is in vectors which are preferably capable of directing expression of the protein encoded by the DNA in a vector-containing cell and further includes cells containing such vectors (preferably eukaryotic cells, e.g., CHO cells (ATCC; Cat. No. CCL 61 or COS-7 cells (ATCC; Cat. No. CRL 1651; and the Xenopus 10 oocytes of the type described in the above cited reference Vu, T.-K.H. et al. (1991) Cell 64:1057-1068). Preferably, such cells are stably transfected with such isolated DNA. By "transformed cell" and "transfected cell", "genetically engineered cell", and the like, is meant a cell into which (or into an ancestor of which) has been introduced, by means of genetic engineering, a DNA molecule encoding a PAR4 (or DNA encoding a biologically 15 active fragment or analog, thereof). Such a DNA molecule is "positioned for expression" meaning that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of the PAR4 protein, or fragment or analog, thereof). By "antibody" is meant an immunoglobulin protein which is capable of binding an 20 antigen. Antibody as used herein is meant to include the entire antibody as well as any antibody fragments (e.g. F(ab') 2 , Fab', Fab, Fv) capable of binding the epitope, antigen or antigenic fragment of interest. Antibodies of the invention are immunoreactive or immunospecific for and therefore specifically and selectively bind to a PAR4 protein. Antibodies for PAR4 are preferably 25 immunospecific -- i.e., not substantially cross-reactive with related materials. Although the term "antibody" encompasses all types of antibodies (e.g., monoclonal) the antibodies of the invention are preferably produced using the phage display methodology described herein. The preferred antibody of the invention is a purified antibody. By purified antibody is meant one which is sufficiently free of other proteins, carbohydrates, and lipids with which it is naturally 30 associated. Such an antibody "preferentially binds" to a PAR4 protein (or an antigenic fragment thereof), i.e., does not substantially recognize and bind to other antigenically unrelated molecules. -7- WO 99/43809 PCT/US99/02983 By "specifically activates", as used herein, is meant an agent, such as thrombin, a thrombin analog, a PAR4 agonist or other chemical agent including polypeptides such as an antibody, which activates protease-activated receptor 4, receptor polypeptide or a fragment or analog thereof to initiate PAR4-mediated biological events as described herein, but which 5 does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally includes a protease-activated receptor 4 polypeptide. By "specifically inhibits", as used herein, is meant an agent, such as a thrombin analog, a PAR4 antagonist or other chemical agent including polypeptides such as an antibody, which inhibits activation of protease-activated receptor 4, receptor polypeptide or a fragment or 10 analog thereof, such as by inhibiting thrombin or by blocking activation of PAR4 by thrombin or other PAR4 activator. Preferably, the agent activates or inhibits the biological activity in vivo or in vitro of the protein to which it binds. By "biological activity" is meant the ability of the protease-activated receptor 4 to bind thrombin or a PAR4 agonist and signal the appropriate cascade of biological events (e.g., 15 phosphoinositide hydrolysis, Ca2+ efflux, and platelet aggregation, and the like, as described herein. By "substantial increase" is meant an increase in activity or other measurable phenotypic characteristic that is at least approximately a 2-fold increase over control level (where control assays are performed in the absence of activator), preferably at least 20 approximately a 5-fold increase, more preferably at least approximately a 10-fold increase in activity over a control assay. By "substantial decrease" or "substantial reduction" is meant a decrease or reduction in activity or other measurable phenotypic characteristic that is approximately 80% or the control level, preferably reduced to approximately 50% of the control level, or more 25 preferably reduced to approximately 10% or less of the control level. The terms "screening method" and "assay method" are used to describe a method of screening a candidate compound for its ability to act as an agonist or antagonist of a PAR4 ligand. The method involves: a) contacting a candidate agonist compound with a recombinant protease-activated receptor 4 (or PAR4 agonist-binding fragment or analog); 30 b) measuring activation of the receptor, the receptor polypeptide or the receptor fragment or analog; and c) identifying agonist compounds as those which interact with the recombinant receptor and trigger or block PAR4 activation. Interaction may be cleavage of the receptor -8- WO 99/43809 PCT/US99/02983 to unmask an intramolecular receptor activating peptide or by mimicking the intramolecular receptor-activating peptide. A tethered ligand may be more difficult to block than a free agonist. Thus, blocking thrombin is the acid test for an antagonist which will block responses by other thrombin substrates. These terms include assays that examine effects on unoccupied 5 receptors as well as assays that utilize displacement of a ligand from an occupied receptor. By an "agonist" is meant a molecule which mimics a particular activity, in this case, interacting with a PAR4 ligand in a manner which activates thereby triggering the biological events which normally result from the interaction (e.g., phosphoinositide hydrolysis, Ca2+ efflux, and platelet aggregation). Preferably, an agonist initiates a substantial increase in 10 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 PAR4 ligand. The terms "antagonist assay", "antagonist screening" and the like, refer to a method of screening a candidate compound for its ability to antagonize interaction between a naturally 15 occurring activating ligand or an agonist and the PAR4. The method involves: a) contacting a candidate antagonist compound with a first compound which includes a recombinant PAR4 (or agonist-binding fragment or analog) on the one hand and with a second compound which includes thrombin or a PAR4 agonist on the other hand; b) determining whether the first and second compounds interact or are prevented from interaction by the candidate compound; 20 and c) identifying antagonistic compounds as those which interfere with the interaction of the first compound (PAR4 receptor) to the second compound (PAR4 agonist) and which thereby substantially reduce thrombin or PAR4 agonist-activated biological events (e.g., phosphoinositide hydrolysis, Ca efflux, and platelet aggregation). By an "antagonist" is meant a molecule which blocks activation of a PAR4 receptor. 25 This can be done by inhibiting a particular activity such as the ability of thrombin, for example, to interact with a protease-activated receptor 4 thereby triggering the biological events resulting from such an interaction (e.g., phosphoinositide hydrolysis, Ca efflux, and platelet secretion, or platelet aggregation). An antagonist may bind to and thereby block activation of a PAR4 receptor. 30 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 -9- WO 99/43809 PCT/US99/02983 and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, particular a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be 5 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. PREFERRED EMBODIMENTS 10 In preferred embodiments of both screening methods, the recombinant PAR4 is stably expressed by a vertebrate cell which normally presents substantially no PAR4 on its surface (i.e., a cell which does not exhibit any significant thrombin-mediated phosphoinositide hydrolysis or Ca efflux in the presence of a PAR activator); the vertebrate cell is a mammalian cell, is a Rat 1 cell, or a COS 7 cell; and the candidate antagonist or candidate 15 agonist is a thrombin analog, PAR4 peptide fragment or analog or other chemical agent including a polypeptide such as an antibody. The receptor proteins of the invention are likely involved in the activation of vertebrate platelet, leukocyte, and mesenchymal cells in response to wounding, as well as mediating signaling in embryonic development. Such proteins and in particular PAR4 20 antagonists are useful therapeutics for the treatment of such conditions as thrombosis, atherosclerosis, restenosis, and inflammation associated with normal wound healing and a variety of diseases including atherosclerosis, restenosis, pulmonary inflammation (ARDS) and glomerulosclerosis. Preferred therapeutics include 1) agonists, e.g., thrombin analogs, PAR4 peptide fragments or analogs thereof, or other compounds which mimic the action of 25 thrombin upon interaction with the protease-activated receptor 4 or mimic the action of an intramolecular receptor activating peptide; and 2) antagonists, e.g., thrombin analogs, antibodies, or other compounds, which block thrombin or protease-activated receptor 4 function by interfering with the thrombin:receptor interaction or by interfering with the receptor intramolecular activating peptide. The dosage would be expected to be comparable 30 with current antiflammatory drugs and should be adjusted based on the age, sex, weight and condition of the patient beginning with small doses and increasing gradually based on responsiveness and toxicity. -10- WO 99/43809 PCT/US99/02983 Because the receptor component may now be produced by recombinant techniques and because candidate agonists and antagonists may be screened using transformed, cultured cells, the instant invention provides a simple and rapid approach to the identification of useful therapeutics. Isolation of the PAR4 gene (as cDNA or genomic DNA) allows its expression 5 in a cell type which does not normally bear PAR4 on its surface, providing a system for assaying a thrombin:receptor interaction and receptor activation. EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art 10 with a complete disclosure and description of how to make receptor proteins and sequences encoding such proteins and carry out the methodology for finding such DNA sequences and proteins, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to insure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated 15 otherwise, parts or parts by weight, molecular weight is weight average molecular weight; temperature is in degrees centigrade; and pressure is at or near atmospheric. There now follows a description of the cloning and characterization of the cDNA, genomic DNA and the receptor protein of the protease-activated receptor 4 from mouse and human. Expression vectors containing and capable of expressing the PAR4 DNA, as well as 20 transformed cells containing and expressing the DNA of the invention are also described. Also described are possible PAR4 agonists and antagonists as well as screening assays for receptor agonists and receptor antagonists. EXAMPLE 1: ISOLATION OF THE MOUSE AND HUMAN PROTEASE 25 ACTIVATED RECEPTOR 4 The public expressed sequence tag (EST) database was searched for potential protease-activated receptor sequences by identifying sequences with homology to PAR1, PAR2 and PAR3. One particular EST, clone 400689, was identified in a database search using PAR2 sequences, showing similarity over an eleven amino acid stretch. The EST was 30 further characterized. The EST sequence was used to obtain mouse and human cDNA and genomic clones by a combination of PCR and hybridization techniques (see, for example, Sambrook, J. et al. -11- WO 99/43809 PCT/US99/02983 (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). The nucleotide sequences for the mouse and human PAR4, and deduced amino acid sequences corresponding to these nucleotide sequences, are shown in Figs. 1-4. The mouse PAR4 cDNA contained an open reading frame encoding a 397 amino acid putative G protein 5 coupled receptor (Fig. 2). The 5' sequence of the PAR4 cDNAs encoded a predicted thrombin cleavage site, suggesting that this new receptor was a novel thrombin receptor. The gene was characterized further as a potential protease-activated receptor. The predicted protein structure of the mouse PAR4 product showed significant homology in conserved regions when compared to other mouse protease-activated receptors. 10 Homology between the deduced mouse PAR4 amino acid sequence and the amino acid sequences of mouse PARI, PAR2, and PAR3 are shown by alignment in Figs. 6A and 6B. Amino acids shared by at least three of the receptor molecules are boxed and shaded. The genomic region containing the mouse PAR4 gene was sequenced, revealing an exon organization characteristic of protease-activated receptors: a first exon encoding the 15 signal peptide region separated from a second exon encoding the majority of the structure of the mature receptor molecule by a characteristic intron of approximately 250 bp (Fig. 7). The mPAR4 thrombin cleavage site is found at the amino terminus of the protein, and the amino acid sequence corresponding to the mPAR4 tethered ligand peptide, or "activating peptide", is directly adjacent to the cleavage site (Fig. 8). 20 Cloning of mouse PAR4 The mouse PAR4 cDNA used for the functional studies presented below was cloned from a mouse embryo day 14-15 library. The sequence of EST clone 400689 was used to generate two reverse primers, CCAGTCACAGAAGTGTAGAGGAGCAAATGG (R2) and 25 CAAGCCAGACCCCCTTCCCAC (R3), for 5'RACE using cDNA from mouse embryo day 14-15 (Marathon cDNA, Clonetech). The forward primers used were Clonetech API and AP2. Two "nested" PCR reactions were performed, the first using primers API and R2, the second with AP2 and R3. The first PCR reaction conditions were as follows: 95*C for 5 seconds, 72*C for 4 minutes for the first five cycles, and 95*C for 5 seconds, 70*C for 30 4 minutes for the next five cycles, and 95*C for 2 seconds, 68*C for 4 minutes for the following 25 cycles. The second PCR reaction conditions were 95'C for 1 minute, followed by 95*C for 30 seconds, 68'C for 3 minutes for 25 cycles. A dominant band of 950 bp was -12- WO 99/43809 PCT/US99/02983 seen following the nested (AP2-R3) reaction. This product was sequenced, providing mPAR4 sequence 5' to the first transmembrane domain. Sequence to the start codon was obtained with a second series of 5'RACE reactions again using the Clonetech Marathon cDNA and API and AP2 primers. The primary reaction 5 used API and reverse primer CCACAGCCACCACAAGCCCATAGAG (RACE 1) and the second reaction used AP2 and CCCCAGCAAGCAGTGCTTGAGAGCTG (RACE 2). The reaction conditions for API-RACEl were: 95'C for 30 seconds, 68'C for 3 minutes for 25 cycles. The conditions for the nested AP2-RACE 2 reaction were the same except the PCR went for 30 cycles. A dominant 300 bp band was obtained and sequenced. 10 The EST clone 400689 was sequenced from both ends to confirm the sequence in the EST database. This sequence, however, differed from sequence which was later obtained both from a cDNA obtained by hybridization screening of a bEND (mouse brain endothelial cell line) library and from a BAC genomic clone (Genome Systems). We believe the latter sequence to be correct. 15 The final functional clone was obtained by generating a PCR product from the start codon to the stop codon indicated by the EST clone using bEND cDNA as a template. The 3' end of this clone was subsequently replaced by subcloning with a genomic fragment at the Nco I site. Both the original product and the clone containing the 3' genomic fragment were tested in oocytes and found to be equally active. 20 Human PAR4 The human PAR4 sequence was determined using a degenerate PCR scheme. Human PAR4 sequence was obtained using degenerate PCR primers to amplify a 900 bp dominant product from total human genomic DNA. The primers used for the PCR reactions were: 25 TA(A/G)TA(A/G)TA(A/G/T)AT(A/G)AAIGG(G/A)TCIAC(G/A)CA (designated DR1), GTIGGIA(C/T)TICCIGCIAA(C/T)GG(A/C/G/T)(C/T)T (designated DF 1), where "I" designates Inosine. Reaction conditions were: 95*C for 4 minutes, followed by 95*C for 1 minute, 50*C for 2.5minutes, 72*C for 1.5 minutes for 50 cycles. Sequencing of the 900 bp product revealed a novel amino acid sequence that was 88% identical to mouse PAR4. 30 Human megakaryocytic cell lines were screened for PAR4 expression by Northern using the above 900 bp product as a probe and the K562 erythroleukemia cell line found to be positive. 5'RACE was then performed using the GIBCO 5'RACE kit and K562 mRNA per -13- WO 99/43809 PCT/US99/02983 the manufacturer's instructions. Nested PCR reactions were performed using two reverse primers: CGAGGTTCATCAGCAGCATGG (GSP2A) and TGCGTGTCCAGCAGGGACAG (GSP2B). Conditions for the first reaction using the GIBCO forward anchor primer and GSP2A were: 95*C for 4 minutes, followed by 95*C for 5 45 seconds, 56*C for 1 minute, and 72 for 1 minute for 35 cycles. The hemi-nested second reaction was performed with the GIBCO anchor primer and GSP2B as follows: 95*C for 4 minutes, followed by 95*C for 45 seconds, 50'C for 1 minute, and 72*C for 1 minute for 30 cycles. A dominant 350 bp band was observed, subcloned and sequenced, providing sequence to the hPAR4 start codon. 10 The same kit and template was used for 3'RACE. The forward primers used were: CCTTCTTCGTGCCCAGCAAC (3'GSPA) and GCTGCTGCTGCATTACTCGG (3'GSPB). The GIBCO UAP primer was used as a reverse primer and hemi-nested PCR performed. The first reaction consisted of 95'C for 4 minutes followed by 95'C for 45 seconds, 50'C for 1 minute, and 72*C for 4 minutes for 35 cycles. The second reaction consisted of 95*C for 45 15 seconds, 50'C for 1 minute, and 72'C for 1 minute for 30 cycles. A dominant 1.6 kb band was observed, subcloned and sequenced, providing sequence to a stop codon. A functional hPAR4 clone was created by PCR using Vent polymerase with primers from the start codon to 50 bp beyond the stop codon using 25 cycles of PCR. The template was K562 cDNA. The PCR product was sequenced and subcloned into an oocyte expression 20 vector for generating cRNA (pFROGGY). Human PAR4 cRNA was microinjected into Xenopus oocytes to demonstrate function. EXAMPLE 2: POLYPEPTIDE EXPRESSION Polypeptides according to the invention may be produced by transformation of a 25 suitable host cell with all or part of a PAR3 encoding cDNA fragment (e.g., the cDNAs described above) in a suitable expression vehicle, and expression of the receptor. Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems may be used to provide the recombinant receptor protein. The precise host cell used is not critical to the invention. The receptor may be produced in a 30 prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiae or mammalian cells, e.g., COS-6M, COS-7, NIH/3T3, or Chinese Hamster Ovary cells). Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, -14- WO 99/43809 PCTIUS99/02983 Rockville, MD). The method of transfection and the choice of expression vehicle will depend on the host system selected. Transformation and mammalian cell transfection methods are described, e.g., in Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, (1989)); expression vehicles may be chosen from those provided, e.g., in 5 Cloning Vectors: A Laboratory Manual (Pouwels, P.H. et al., (1985), Supp. 1987). Particularly preferred expression systems are the Xenopus oocyte cells of Vu et al. (Vu et al., Cell (1991) supra) and insect cells (SF9-baculovirus) transfected with an expression vector containing and expressing a receptor protein or biologically active fragment thereof DNA encoding the human or mouse PAR4 or an appropriate receptor fragment or 10 analog (as described above) is inserted into the expression vector in an orientation designed to allow expression. Alternatively, the PAR4 (or biologically active receptor fragment or analog) is expressed by a stably-transfected mammalian cell line. Other preferable host cells which may be used in conjunction with the expression vehicle include NIH/3T3 cells (ATCC Accession No. 1658). The expression may be used in a screening method of the invention 15 (described below) or, if desired, the recombinant receptor protein may be isolated as described below. A number of vectors suitable for stable transfection of mammalian cells are available to the public, e.g., see Pouwels et al. (supra); methods for constructing such cell lines are also publicly available, e.g., in Ausubel et al. (supra). In one example, cDNA encoding the 20 receptor (or receptor fragment or analog) is cloned into an expression vector which includes the dihydrofolate reductase (DHFR) gene. Integration of the plasmid and, therefore, the PAR 4-encoding gene into the host cell chromosome is selected for by inclusion of 0.01-3 00 yM methotrexate in the cell culture medium (as described in Ausubel et al., supra). This dominant selection can be accomplished in most cell types. Recombinant protein expression 25 can be increased by DHFR-mediated amplification of the transfected gene. Methods for selecting cell lines bearing gene amplifications are described in Ausubel et al. (supra); such methods generally involve extended culture in medium containing gradually increasing levels of methotrexate. DHFR-containing expression vectors commonly used for this purpose include pCVSEII-DHFR and pAdD26SV(A) (described in Ausubel et al., sp). Any of the 30 host cells described above or, preferably, a DHFR-deficient CHO cell line (e.g., CHO DHFR cells, ATCC Accession No. CRL 9096) are among the host cells preferred for DHFR selection of a stably-transfected cell line or DHFR-mediated gene amplification. -15- WO 99/43809 PCT/US99/02983 One particularly preferred stable expression system is a Rat 1 cell (ATCC) stably transfected with a pcDNAI/NEO (InVitrogen, San Diego, CA) expression vector. Expression of the recombinant receptor (e.g., produced by any of the expression systems described herein) may be assayed by immunological procedures, such as Western blot 5 or immunoprecipitation analysis of recombinant cell extracts, or by immunofluorescence of intact recombinant cells (using, e.g, the methods described in Ausubel et al., supra). Recombinant receptor protein is detected using an antibody directed to the receptor. Described below are methods for producing anti-protease-activated receptor 4 antibodies using, as an immunogen, the intact receptor or a peptide which includes a suitable protease 10 activate receptor 4 epitope. To detect expression of a PAR3 fragment or analog, the antibody is preferably produced using, as an immunogen, an epitope included in the fragment or analog. Once the recombinant PAR4 protein (or fragment or analog, thereof) is expressed, it is isolated, e.g., using immunoaffinity chromatography. In one example, an anti-PAR4 15 antibody may be attached to a column and used to isolate intact receptor or receptor fragments or analogs. Lysis and fractionation of receptor-harboring cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al., supr). Once isolated, the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry 20 And Molecular Biology, eds., Work and Burdon, Elsevier, (1980)). Receptors of the invention, particularly short receptor fragments, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, (1984) 2nd ed., The Pierce Chemical Co., Rockford, IL). 25 EXAMPLE 3: ACTIVATION STUDIES OF THE RECOMBINANT PROTEASE ACTIVATED RECEPTOR 4 PAR4 was demonstrated to be activated by thrombin when expressed in the Xenopus oocyte cells of Vu et al. (Vu et al., Cell (1991) supra). The ability of PAR4 to mediate signaling by a-thrombin was tested. Xenopus oocytes were microinjected with cRNA 30 encoding epitope-tagged mouse PAR4 (mPAR4). Thrombin-triggered 45Ca release was measured as described in Vu et al. (Vu, T.-K. H. et al. (1991) supra). The oocytes were microinjected with either 2.5 or 25 ng of the mPAR4 cRNA. A control set of oocytes were -16- WO 99/43809 PCTIUS99/02983 injected with 25 ng of the mouse PARI cDNA, WT5. Surface expression of receptors may be confirmed by M1 antibody binding by the method of Ishii, K. et al. (Ishii, K. et al. (1995) J. Biol. Chem. 270:16435-16440; and Ishii, K. et al. (1993) J. Biol. Chem. 268:9780-9786, which references are herein incorporated by reference in their entirety). 5 Microinjection ofXenopus oocytes with mouse PAR4 cRNA conferred thrombin dependent 45Ca mobilization (Fig. 9) which reflects agonist-triggered phosphoinositide hydrolysis in this system. a-thrombin was able to induce calcium mobilization in both the PARI and PAR4 expressing oocytes, even at injection levels of lnM. mPAR4 expressing cells treated showed approximately a 25-fold increase of Ca release at both concentrations, 10 and this did not vary significantly between the cells expressing higher or lower concentration of mPAR4. In addition, exposure of mPAR4-injected oocytes to mPAR4's activation peptide, GYPGKF (SEQ ID NO: 12 ), induced PAR4-mediated 45Ca mobilization to approximately a 33-fold increase, which is a 2-fold increase over PAR1-mediated mobilization. Exposure to the PARI activating peptide, SFLLRN (SEQ ID NO: 13), failed to activate PAR4 even at a 15 concentration of 1 OOM. Human PAR4 cRNA was also transcribed in vitro from hPAR4 cDNA using standard techniques as described above. Xenopus oocytes were microinjected with 2ng of cRNA/oocyte, and calcium signaling in response to agonist peptides was measured (Fig. 10). 1 OnM thrombin caused a 30-fold increase in calcium mobilization in oocytes expressing 20 PAR4, consistent with hPAR4 as a functional thrombin receptor. The human PAR4 activating peptide GYPGQV (SEQ ID NO: 14) also showed calcium mobilization at 100,yM. The mouse activating peptide, GYPGKF, was even more potent than the human activating peptide, whereas the human PARI activating peptide showed no significant activity. The negative controls, uninjected cells and cells expressing irrelevant receptors, also showed no 25 activity. EXAMPLE 4: SPECIFICITY OF THE RECOMBINANT PROTEASE ACTIVATED RECEPTOR 4 The specificity of activation of PAR4 was examined by the introduction of a number 30 of the arginine/lysine specific serine proteases and PAR4 activating peptides to Xenopus oocytes expressing PAR4 (Fig. 11). Various concentrations of the arginine/lysine specific serine proteases plasmin, trypsin, tissue plasminogen activator (APC), Factor VIla, Factor -17- WO 99/43809 PCTIUS99/02983 Xa, and thrombin were tested. A variant of the PAR4 activating peptide was also tested. The mPAR4 receptor was activated upon treatment with thrombin, its activating peptide GYPGKF, and the relatively less specific serine protease trypsin. No significant mPAR4 activation was seen in response to the other proteases tested, nor with the treatment of the 5 variant activating peptide GAPGKF, which is expected to lack activity. The specificity of mPAR4, mPAR1, and mPAR2 signaling was also examined. Protease-triggered "Ca release was measured in Xenopus oocytes expressing mouse PARI, PAR2 or PAR4 stimulated with the activating peptides of each receptor and the proteases thrombin and trypsin (Fig. 12). Each PAR was activated specifically by its respective 10 activating peptide, and not by the activating peptides of the other mPARs. mPAR2 and mPAR3 showed a small level of activation upon treatment with trypsin, but PAR4 displayed a much greater response with a 24-fold increase in "Ca release. Finally, PARI and PAR4 showed a significant response to thrombin, with PAR4 expressing oocytes exhibiting a greater response to thrombin than thrombin receptor mPAR1. 15 EXAMPLE 5: PAR4 TISSUE EXPRESSION IN MOUSE AND HUMAN Northern analysis of mouse tissues revealed that PAR4 mRNA was strongest in the mouse spleen cells. The levels of splenic expression of PAR4 are similar to the expression of PAR3 in megakaryocytes, the predicted site of action for PAR3. The role of PAR4 in mouse 20 tissues awaits elucidation, but the finding of PAR4 in spleen is consistent with a role for PAR4 in mediating activation of platelets and other hematopoietic cells by thrombin. In situ hybridization of mouse tissue reveals the presence of PAR4 mRNA in splenic megakaryocytes, the platelet precursor cells. Control samples in which hybridization is performed with a sense strand probe control are a negative control for all cell types. 25 Northern analysis of mouse tissues for PAR4 mRNA show signal in spleen, with low levels seen in brain, heart, and other tissues. The spleen is a hematopoietic organ in mouse, and both Northern and in situ hybridization data suggest that PAR4 is most abundantly expressed in megakaryocytes in the mouse. The in situ hybridization studies are performed as follows. Anesthetized adult 30 C57BL/6 mice are perfusion-fixed with 4% paraformaldehyde. Organs to be tested are dissected, trimmed, and immersion-fixed for 4 hours in 4% paraformaldehyde. Processed tissues are embedded in paraffin, and 5 mm sections were cut. Sense or antisense 355_ -18- WO 99/43809 PCT/US99/02983 riboprobe are transcribed in vitro from mouse PAR2 cDNA subcloned into the EcoR1 site of pBluescript II SK- (Stratagene, San Diego, CA). Hybridization, wash, and development conditions are as reported for mouse PARI (Soifer, S.J. et al. (1993) Am. J. Pathol. 144:60-69). To carry out Northern analysis, a 32P-labeled probe for the mouse message is 5 generated by random priming (Prime-It II kit; Stratagene) of PCR-amplified DNA fragments corresponding to mouse cDNA codons representing transmembrane domains 2 to 3. High stringency hybridizations and washes were performed as per the Clonetech protocol for Northern analysis. 10 EXAMPLE 6: ASSAYS FOR PAR4 FUNCTION Useful receptor fragments or analogs of the invention are those which interact with thrombin and are activated to initiate the cascade of events associated with thrombin receptor interaction. Such an interaction may be detected by an in vitro functional assay method (e.g., the phosphoinositide hydrolysis assay, 45Ca efflux assay, or platelet aggregation assay 15 described herein). This method includes, as components, thrombin and a recombinant protease-activated receptor 4 (or a suitable fragment or analog) configured to permit thrombin binding (e.g., those polypeptides described herein). Thrombin may be obtained from Sigma Chemical Co. (St. Louis, MO) or similar supplier. Preferably, the protease-activated receptor 4 component is produced by a cell that 20 naturally presents substantially no receptor on its surface, e.g., by engineering such a cell to contain nucleic acid encoding the receptor component in an appropriate expression system. Suitable cells are, e.g., those discussed above with respect to the production of recombinant receptor, such as Rat I cells or COS-7 cells. 25 EXAMPLE 7: SCREENING FOR PROTEASE-ACTIVATED RECEPTOR 4 ACTIVATOR ANTAGONISTS AND AGONISTS Antagonists As discussed above, one aspect of the invention features screening for compounds that inhibit the interaction between thrombin (or other PAR4 activating compound) and the 30 protease-activated receptor 4, thereby preventing or reducing the cascade of events that are mediated by that interaction. The elements of the screen are a PAR4 activator (such as thrombin), a candidate antagonist, and recombinant PAR4 (or a suitable receptor fragment or -19- WO 99/43809 PCT/US99/02983 analog, as outlined above) configured to permit detection of PAR4 activator, antagonist, and PAR4 function. An additional element may be 45 Ca, Fura-2, 3 H-inositol, or another indicator used to detect downstream signaling (Ishii, K. et al. (1993) supra; and Nanevicz, T. et al. (1996) supra). 5 Inhibition of thrombin-induced platelet aggregation may also be used as a means of monitoring an antagonist of PAR4 receptor activation. Thrombin is incubated with the candidate inhibitory compound (such as a peptide) for 5 minutes, then the mixture is added to washed platelets and platelet activation is followed as platelet ATP secretion by lumiaggregometry (see, for example, Connolly, A.J. et al. Nature 381:516-519 (1996); and 10 USPN 5,256,766). Alternately, platelets are incubated with a candidate PAR 4 antagonist for 5 minutes. Thereafter the response to thrombin is measured. Inclusion of potential antagonists in the screening assay along with thrombin allows for the screening and identification of authentic receptor antagonists as those which decrease thrombin-mediated events, such as platelet aggregation. 15 Appropriate candidate thrombin antagonists include PAR4 fragments, particularly, fragments of the protein predicted to be extracellular and therefore likely to bind thrombin or the tethered ligand; such fragments would preferably include five or more amino acids. Candidate PAR 4 antagonists include thrombin analogs as well as other peptide and non peptide compounds and anti-PAR4 antibodies. 20 Agonists Another aspect of the invention features screening for compounds that act as PAR4 agonists. Activation of the PAR4 with thrombin or an agonist leads to a cascade of events (such as phosphoinositide hydrolysis, Ca efflux, and platelet aggregation), providing a 25 convenient means for measuring thrombin or other agonist activity. The agonist screening assay of the invention utilizes recombinant cells expressing recombinant PAR4 (or a suitable receptor fragment or analog, as outlined herein) configured to permit detection of PAR4 function. Alternatively, a cell such as a leukocyte, a platelet, or a mesenchymal cell that naturally expresses PAR4 may be used. Other elements of the screen 30 include a detectable downstream substrate of the PAR4 activation, such as radiolabelled phosphoinositide, the hydrolysis of which to a detectable product indicates PAR4 activation by the candidate agonist. -20- WO 99/43809 PCT/US99/02983 45 Ca efflux from a cell expressing PAR4 may be used as a means of measuring receptor activation by candidate agonists (Williams, J.A. et al., (1988) PNAS USA 85:4939 4943; Vu, T.-K. H., et al. (1991) Cell M:1057-1068; and USPN 5,256,766, which references are herein incorporated by reference in their entirety). 45Ca release by oocytes 5 expressing cRNA encoding PAR4 are assessed as follows. Briefly, intracellular calcium pools are labeled by incubating groups of 30 oocytes in 300 pl calcium-free MBSH containing 50 pCi 45CaCl 2 (10-40 mCi/mg Ca; Amersham) for 4 hours at room temperature. The labeled oocytes are washed, then incubated in MBSH II without antibiotics for 90 minutes. Groups of 5 oocytes are selected and placed in individual wells in a 24-well tissue culture plate 10 (Falcon 3047) containing 0.5 ml/well MBSH II without antibiotics. This medium is removed and replaced with fresh medium every 10 minutes, the harvested medium is analyzed by scintillation counting to determine 45 Ca released by the oocytes during each 10-minute incubation. The 10-minute incubations are continued until a stable baseline of 45Ca release per unit time is achieved. Two additional 10-minute collections are obtained, then test 15 medium including agonist is added and agonist-induced 45 Ca release determined. A voltage clamp assay provides an alternative method of monitoring agonist activity. Agonist-induced inward chloride currents are measured in voltage-clamped oocytes expressing thrombin receptor encoding cRNA essentially as previously described (Julius, D. et al. Science (1988) 241:558-563, herein incorporated by reference in its entirety) except 20 that either the single electrode voltage-clamp technique or a two electrode technique may be employed. Platelet aggregation may also be used as a means of monitoring PAR4 receptor activation (see, for example, Connolly, A.J. et al. Nature 381:516-519 (1996). Human platelets may use both PAR 1 and PAR 4. An agonist useful in the invention is one which imitates the normal thrombin-mediated 25 signal transduction pathway leading, e.g., to an increase in phosphoinositide hydrolysis. Appropriate candidate agonists include thrombin analogs or PAR4 tethered ligand domains or other agents which mimic the action of thrombin or the PAR 4 tethered ligand domain. Agonists would be useful for aiding discovery of antagonists. 30 EXAMPLE 8: ANTI-PROTEASE-ACTIVATED RECEPTOR 4 ANTIBODIES Protease-activated receptor 4 (or immunogenic receptor fragments or analogs) may be used to raise antibodies useful in the invention. Receptor fragments preferred for the -21- WO 99/43809 PCT/US99/02983 production of antibodies are those fragments deduced or shown experimentally to be extracellular. Antibodies directed to PAR4 peptides are produced as follows. Peptides corresponding to all or part of the PAR4 protein are produced using a peptide synthesizer by 5 standard techniques. The peptides are coupled to KLH with m-maleimide benzoic acid N-hydroxysuccinimide ester. The KLH-peptide is mixed with Freund's adjuvant and injected into animals, e.g. guinea pigs or goats, to produce polyclonal antibodies. Monoclonal antibodies may be prepared using the PAR4 polypeptides described above and standard hybridoma technology (see, e.g., Kohler et al., Nature (1975) 256:495, 1975; Kohler et al., 10 Eur. J. Immunol. (1976) 6:292; Kohler et al., Eur. J. Immunol. (1976).6:511; Hammerling et al., in Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, (1981); and Ausubel et al., supra). Antibodies are purified by peptide antigen affinity chromatography. Once produced, antibodies are tested for their ability to bind PAR4 by specific binding to the surface of PAR4-transfected cells or by Western blot or immunoprecipitation analysis 15 (such as by the methods described in Ausubel et al., supra). Antibodies which specifically recognize PAR4 are considered to be likely candidates for useful antagonists; such candidates are further tested for their ability to specifically interfere with the interaction between thrombin and PAR4 (using the functional antagonist assays described herein). Antibodies which antagonize thrombin:PAR4 binding or PAR4 20 function are considered to be useful antagonists in the invention. EXAMPLE 9: ACTIVATION OF MOUSE PLATELETS USING THE PAR4 ACTIVATING PEPTIDE Blood was collected from mice anesthetized with pentobarbitol by cannulating the 25 inferior vena cava at the level of the renal veins. The blood was mixed with 3.15% citrate (15% of total final volume) and spun at 200g for 7 minutes to obtain platelet rich plasma (PRP). EDTA was added to a final concentration of 10mM, and PGE1 added to a final concentration of luM. The PRP was spun at 500g for 10 minutes. The platelet pellet was resuspended in platelet buffer (20mM Tris-HCI pH 7.4, 140 mM NaCl, 2.5 mM KCL, 1.0 30 mM MgCl 2 , 1 mg.ml glucose, 0.2% BSA) and 1 mM EDTA and luM PGE1 in a volume equal to the original PRP volume, and again spun at 500 g for 10 minutes. The second platelet pellet was resuspended in platelet buffer without EDTA or PGE1. The volume was -22- WO 99/43809 PCT/US99/02983 adjusted such that the OD 500 was 1.0. After a minimum of 30 minutes on ice, 300 ul of platelet suspensions were used to measure aggregation and secretion in a Chronolog aggregometer according to manufacturer's instructions. The PAR4 peptide GYPGKF was added to a final concentration of 500 uM. Secretion 5 and aggregation in response to this peptide were measured over 4-10 minutes (Fig. 13). Since mouse platelets also express PAR3, responses to PAR4 peptide were tested in platelets from PAR3 gene knockout mice to exclude the possibility that PAR3 might be mediating responses to the PAR4 peptide. Note persistent responses to PAR4 peptide were noted in these platelets (Fig. 14). 10 EXAMPLE 10: ACTIVATION OF HUMAN PLATELETS DESENSITIZED TO THE PARI ACTIVATING PEPTIDE To test whether PAR4 might be functionally expressed in human platelets, platelets were prepared by standard techniques and analyzed by lumiaggregometry as above. Because 15 the mouse peptide GYPGKF was a stronger agonist at human PAR4 than the cognate human peptide GYPGQV, GYPGKF was used. Human platelets were activated by GYPGKF. Human platelets also express PARI (like PAR3 in mouse platelets). To exclude the possibility that the PAR4 peptide might be cross-reacting with PARI, we desensitized human platelets with the PARI agonist SFLLRN. Note that these platelets failed to respond to a 20 second challenge with SFLLRN, but did respond to GYPGKF (Fig. 15); thus, the PAR4 peptide is not acting through PARI, strengthening the conclusion that PAR4 is functionally expressed in human platelets. EXAMPLE 11: THERAPEUTIC USES OF PAR4 25 Particularly suitable therapeutics for the treatment of wound healing, thrombosis, atherosclerosis, restenosis, inflammation, and other thrombin-mediated signaling disorders are the agonists and antagonists described above formulated in an appropriate buffer such as physiological saline. Where it is particularly desirable to mimic a receptor fragment conformation at the membrane interface, the fragment may include a sufficient number of 30 adjacent transmembrane residues. In this case, the fragment may be associated with an appropriate lipid fraction (e.g., in lipid vesicles or attached to fragments obtained by disrupting a cell membrane). Alternatively, anti-PAR4 antibodies produced as described -23- WO 99/43809 PCT/US99/02983 above may be used as a therapeutic. Again, the antibodies would be administered in a pharmaceutically-acceptable buffer (e.g., physiological saline). If appropriate, the antibody preparation may be combined with a suitable adjuvant. Antibodies to PAR 4 are useful antagonists which can be formulated as indicated 5 above. Other therapeutically useful antagonists are peptides derived from PAR4 that bind to and block thrombin and include formulation comprising a pharmaceutically acceptable carrier and one or more of the following: a) the isolated sequence PAPRGYPGQVCANDSDTLELPD (SEQ ID NO:15); 10 b) uncleavable thrombin inhibitor PAPRPYPGQVCANDSDTLELPD (SEQ ID NO: 16), wherein the PAR 4 cleavage site is mutated to block cleavage; c) uncleavable thrombin inhibitor PAP(hR)GYPGQVCANDSDTLELPD (SEQ ID NO: 17), wherein the 15 PAR 4 cleavage site P1 is mutated to block cleavage, and hR is beta homoarginine (the extra methylene group is in the main chain); d) uncleavable thrombin inhibitor (dF)PRPYPGQVCANDSDTLELPD (SEQ ID NO: 18), wherein the good active site binding sequence dFPR is substituted for PNPR and 20 dF is D-Phenylalanine; e) any of a)-d) above where all or part of the sequence corresponding to GYPGQVCAN is replaced with spacer sequences such as GGG; f) variations and combinations of a)-e) which 25 act as antagonists. The therapeutic preparation is administered in accordance with the condition to be treated. Ordinarily, it will be administered intravenously, at a dosage, of a duration, and with the appropriate timing to elicit the desired response. Appropriate timing refers to, for 30 example, time relative to wounding, time intervals between therapeutic administrations, and the like, at which administration of therapeutic preparation elicits the desired response. Alternatively, it may be convenient to administer the therapeutic orally, nasally, or topically, -24- WO 99/43809 PCT/US99/02983 e.g., as a liquid or a spray. The dosages are determined to be an amount of the therapeutic agent delivered to an animal that substantially reduces or alleviates disease symptoms. Treatment may be repeated as necessary for substantial reduction or alleviation of disease symptoms. 5 PAR4 activator agonists can be used for the treatment of bleeding. Antagonists may be useful in controlling the formation of clots that cause heart attack and stroke, mediating inflammation and the proliferative responses to injury in normal wound healing and a variety of diseases including atherosclerosis, restenosis, pulmonary inflammations (ARDS), glomerulosclerosis, etc. 10 The methods of the invention may be used to screen therapeutic receptor activator agonists and antagonists for their effectiveness in altering thrombin-mediated biological events, such as phosphoinositide hydrolysis or other cell signaling events by the assays described above. Where a non-human mammal is treated or where a therapeutic for a non human animal is screened, the PAR4 or receptor fragment or analog or the antibody 15 employed is preferably specific for that species. OTHER EMBODIMENTS Polypeptides according to the invention include any protease-activated 4 receptors (as described herein). Such receptors may be derived from any source, but are preferably derived 20 from a vertebrate animal, e.g., a human or mouse. These polypeptides are used, e.g., to screen for antagonists which disrupt, or agonists which mimic, a thrombin:receptor interaction. Polypeptides of the invention also include any analog or fragment of a PAR4 capable of interacting with thrombin. Such analogs and fragments may also be used to screen for 25 PAR4 ligand antagonists or agonists. In addition, that subset of receptor fragments or analogs which bind thrombin and are, preferably, soluble (or insoluble and formulated in a lipid vesicle) may be used as antagonists to reduce the in vivo concentration of endogenous thrombin, either circulating concentration or local concentration. The efficacy of a receptor analog or fragment is dependent upon its ability to interact with thrombin; such an interaction 30 may be readily assayed using PAR4 functional assays (e.g., those described herein). Specific receptor analogs of interest include full-length or partial receptor proteins including an amino acid sequence which differs only by conservative amino acid substitutions, -25- WO 99/43809 PCT/US99/02983 for example, substitution of one amino acid for another of the same class (e.g., valine for leucine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the receptors' ability to signal thrombin-mediated events (e.g., as assayed above). 5 Specific receptor fragments of interest include any portion of the PAR4 which is capable of interacting with thrombin, for example, all or part of the extracellular domains predicted from the deduced amino acid sequence. Such fragments may be useful as antagonists (as described above), and are also useful as immunogens for producing antibodies which neutralize the activity of PAR4 in vivo (e.g., by interfering with the interaction between 10 the receptor and thrombin). The area as illustrated in Fig. 8 is most likely to bind thrombin. For the human PAR4 protein, modification of the (R47/G48) cleavage site, e.g. substitution of proline for G48, will render peptides mimicking this site uncleavable. Such peptides will bind thrombin with high affinity. Extracellular regions of novel protease-activated receptors may be identified by 15 comparison with related proteins of similar structure (e.g., other members of the G-protein-coupled receptor family); useful regions are those exhibiting homology to the extracellular domains of well-characterized members of the family. Alternatively, from the primary amino acid sequence, the secondary protein structure and, therefore, the extracellular domain regions may be deduced semi-empirically using a 20 hydrophobicity/hydrophilicity calculation such as the Chou-Fasman method (see, e.g., Chou and Fasman, Ann. Rev. Biochem. (1978) -4:251). Hydrophilic domains, particularly ones surrounded by hydrophobic stretches (e.g., transmembrane domains) present themselves as strong candidates for extracellular domains. Finally, extracellular domains may be identified experimentally using standard enzymatic digest analysis, e.g., tryptic digest analysis. 25 Candidate fragments (e.g., any extracellular fragment) are tested for interaction with thrombin by the assays described herein (e.g., the assay described above). Such fragments are also tested for their ability to antagonize the interaction between thrombin and its endogenous receptor, such as PAR4, using the assays described herein. Analogs of useful receptor fragments (as described above) may also be produced and tested for efficacy as screening 30 components or antagonists (using the assays described herein); such analogs are also considered to be useful in the invention. -26- WO 99/43809 PCTIUS99/02983 Identification of the receptor(s) that mediate thrombin signaling provides potential targets for the development of drugs that block thrombin's undesirable actions or mimic its desirable activities. Thrombin receptor antagonists may be used for inhibition of platelet dependent thrombosis in the setting of unstable angina and myocardial infarction or for 5 blocking thrombin's proinflammatory actions on endothelial cells in the setting of vascular injury. Thrombin receptor agonists may be used to promote hemostasis and fibroblast proliferation at wound sites. Unmasked tethered ligand domain peptides may provide lead structures for the development of PAR4 agonists or antagonists. 10 The instant invention is shown and described herein in what is considered to be the most practical, and preferred embodiments. It is recognized, however, that departures may be made therefrom, which are within the scope of the invention, and that obvious modifications will occur to one skilled in the art upon reading this disclosure. 15 -27-

Claims (16)

1. Substantially pure DNA encoding a protease-activated receptor 4 (PAR4). 5

2. The substantially pure DNA of claim 1, wherein the DNA encodes the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5.

3. The substantially pure DNA of claim 2, wherein the DNA is SEQ ID NO: 1, 10 SEQ ID NO:4, or degenerate variants thereof

4. Substantially pure DNA having 50% or greater sequence identity to the DNA sequence of claim 1, wherein the DNA selectively hybridizes to sequences complementary to the DNA of claim 1 under stringent conditions. 15

5. Substantially pure DNA complementary to the DNA of claim 1, wherein the DNA has 50% or greater sequence identity to the DNA of claim 1 and the DNA selectively hybridizes to the DNA of claim 1 under stringent conditions. 20

6. An isolated PAR4 polypeptide.

7. The isolated polypeptide of claim 9 having an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5. 25

8. A fragment or analog of a polynucleotide according to Claim 6 or 7.

9. A substantially pure polypeptide having an amino acid sequence wherein said polypeptide is activated by thrombin; and said polypeptide mediates phosphoinositide hydrolysis in a cell expressing said polypeptide on its surface. 30

10. A substantially pure PAR4 activating peptide. -28- WO 99/43809 PCTIUS99/02983

11. An antibody which selectively binds to the polypeptide of claim 9.

12. A vector comprising the DNA of claims 1-5. 5

13. A cell comprising the vector of claim 12.

14. An assay device, comprising: a support surface; and a cell of claim 13 or membranes derived therefrom. 10

15. A therapeutic composition, comprising: a PAR4 ligand agonist and a physiologically-acceptable carrier.

16. A therapeutic composition, comprising: 15 a PAR4 ligand antagonist; and a physiologically-acceptable carrier. -29-