PROTEASE-ACTIVATED RECEPTOR 3 AND USES THEREOF

Abstract: Disclosed are cDNAs and genomic DNAs encoding protease-activated receptor 3 (PAR3) from mouse and human, and the recombinant polypeptides expressed from such cDNAs. The recombinant receptor polypeptides, receptor fragments and analogs expressed on the surface of cells are used in methods of screening candidate compounds for their ability to act as agonists or antagonists to the effects of interaction between thrombin and PAR3. Agonists are used as the therapeutics to treat wounds, thrombosis, atherosclerosis, restenosis, inflammation, and other thrombin-activated disorders. Antagonists are used as therapeutics to control blood coagulation and thereby treating heart attack and stroke. Antagonists mediate inflammatory and proliferative responses to injury as occur in normal wound healing and variety of diseases including atherosclerosis, restenosis, pulmonary inflammation (ARDS) and glomerulosclerosis. Antibodies specific for a protease-activated receptor 3 (or receptor fragment or analog) and their use as a therapeutic are also disclosed.

Description: PROTEASE-ACTIVATED RECEPTOR 3 AND USES THEREOF Field of the Invention This invention relates to nucleic acids, their encoded protease-activated receptor 3 proteins, and screening assays for agonists and antagonists of the protease activated receptor 3 proteins. Background of the Invention Thrombin, a coagulation protease generated at sites of vascular injury, activates 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 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 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; USPN 5,256,766). This receptor revealed a novel proteolytic mechanism of activation and is referred to as PARl (protease-activated receptor 1) . PARl is activated by the binding of thrombin to and cleavage of PARl ' s amino terminal 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 PARl agonists and activate it independent of thrombin and receptor cleavage (Vu, T.-K.H. et al. (1991) Cell 64=1057-1068). To identify which of thrombin' s known cellular actions are mediated by PARl, a PARl knockout mouse was recently generated (Connolly, A. et al . (1996) Nature 381 : 516-519) . Analysis of mice in which both alleles of the PARl gene were disrupted provided definitive evidence for a second platelet thrombin receptor and for tissue specific roles of distinct thrombin receptors. Specifically, in mice, PARl is not important for platelet responses but is critical for fibroblast responses. 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. SUMMARY OF THE INVENTION The protease-activated receptor (PAR3) disclosed herein is useful in assaying libraries of compounds for their activity as thrombin agonists and antagonists. DNA encoding PAR3 is placed in a functional expression vector, expressed in a cell line, and used to assay compounds for activity as an agonist or antagonist of thrombin' s affect on PAR3. The invention features substantially pure DNA (cDNA or genomic DNA) encoding a protease-activated receptor 3 (PAR3) from vertebrate tissues (SEQ ID NO : 1 , SEQ ID N0:2, SEQ ID NO : 4 and SEQ ID NO: 5) and degenerate sequences thereof; substantially pure protease-activated receptor 3 polypeptides encoded thereby; as well as amino acid sequences substantially identical to the amino acid sequences SEQ ID NO : 3 and SEQ ID NO : 6 from mouse and human, respectively. The invention further comprises fragments of the PAR3 receptor which are activated by thrombin. Such fragments may have the same amino acid sequence as SEQ ID NOs : 3 and 6 or be at least 80% identical to the amino acid sequences SEQ ID NO : 3 and SEQ ID NO: 6. In various embodiments, the DNA, receptor or receptor fragment is derived from a 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. Another object is to provide a cell line genetically engineered to express the nucleotide sequence . Another object is to provide a method whereby a compound or library of compounds can be assayed as thrombin agonists or antagonists for their ability to activate or block the receptor expressed by the nucleotide sequence. An advantage of the present invention is that a novel thrombin receptor PAR3 is disclosed making it possible to identify novel thrombin agonists and antagonists which may not be identifiable via PARl or PAR2 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. 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 Fig. 1 is the complete nucleotide and amino acid sequences (SEQ ID NO : 1 and SEQ ID NO: 3, respectively) of the mouse protease-activated receptor 3 gene coding region cDNA. The deduced amino acid sequence of the receptor is provided below the nucleotide sequence and contains 369 amino acids. The deduced amino acid sequence begins at nucleotides 51-53 (ATG = Met) and ends at nucleotides 1158-1160 (TAG = stop) . Fig. 2 is the genomic sequence (containing exon 2) of the mouse protease-activated receptor 3 (SEQ ID NO : 2 ) . Fig. 3 is the nucleotide and deduced amino acid sequences (SEQ ID NO: 4 and SEQ ID NO: 6, respectively) of the human protease-activated gene coding region cDNA. The deduced amino acid sequence is provided below the nucleotide sequence and contains 374 amino acids. The coding region of the cDNA sequence begins at nucleotides 58-60 (ATG = Met) and ends at nucleotides 1180-1182 (TAG = stop) . Fig. 4 is the genomic sequence (containing exon 2) of the human protease-activated receptor 3 (SEQ ID NO: 5) . Fig. 5A shows the alignment of the deduced amino acid sequences (SEQ ID NO: 3, 6, 7, 8, 9) of the mouse PAR3 , human PAR3 , human PARl , and human PAR2. To indicate homology, gaps (represented by blank spaces) have been introduced into the five sequences. Transmembrane domains are overlined (TM1-7) . Fig. 5B shows the alignment of the hirudin-like portion of human PARl, PAR2 , and PAR3 amino acid sequences. Fig. 6 is a bar graph showing cell surface binding of Ml monoclonal antibody to Ml epitope on Cos 7 cells expressing hPAR3 or hPAR3 T39P in the presence and absence of a-thrombin. Fig. 7 is a bar graph of hPAR3 signaling in Cos 7 cells in the presence and absence of Gα:16 and the presence and absence of α-thrombin. Signaling is measured by phosphoinositide hydrolysis. Fig. 8 is a graph of phosphoinositide hydrolysis in response to PAR3 signaling as a function of increasing α-thrombin concentration, and in the presence and absence of Gαl6 protein. Fig. 9 is a graph of phosphoinositide hydrolysis in response to PAR3 signaling as a function of increasing γ-thrombin concentration, and in the presence and absence of Gαl6 protein. Fig. 10 is a graph comparing the specificity of PARl and PAR3 for thrombin. DESCRIPTION OF THE PREFERRED EMBODIMENTS 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 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 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. 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 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 3", "PAR3", "PAR3 receptor" and the like, is meant all or part of a vertebrate cell surface protein which is specifically activated by thrombin or a thrombin agonist thereby activating PAR3-mediated signalling events (e.g., phosphoinositide hydrolysis, Ca 2+ efflux, platelet aggregation) . The polypeptide is characterized as having the ligand activating properties (including the agonist activating and antagonist inhibiting properties) and tissue distribution described herein. Specifically, PAR3 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-translational modification (e.g., glycosylation). By "substantially pure" is meant that the protease-activated receptor 3 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, PAR3 polypeptide. A substantially pure PAR3 polypeptide may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding a PAR3 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 . 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 receptor. Such equivalent receptors can be isolated by extraction from the tissues or cells of any animal which naturally produce 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 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 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 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 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. CR 1651; and the Xenopus 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 PAR3 (or DNA encoding a biologically 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 PAR3 protein, or fragment or analog, thereof) . By "specifically activates", as used herein, is meant an agent, such as thrombin, a thrombin analog, a PAR3 agonist or other chemical agent including polypeptides such as an antibody, which activates protease-activated receptor 3, receptor polypeptide or a fragment or analog thereof to initiate PAR3 -mediated biological events as described herein, but which does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally includes a protease- activated receptor 3 polypeptide. By "specifically inhibits", as used herein, is meant an agent, such as a thrombin analog, a PAR3 antagonist or other chemical agent including polypeptides such as an antibody, which inhibits activation of protease-activated receptor 3, receptor polypeptide or a fragment or analog thereof, such as by inhibiting thrombin or by blocking activation of PAR3 by thrombin or other PAR3 activator. Preferably, the agent activates or inhibits the biological activity in vivo or in vi tro of the protein to which it binds. By "biological activity" is meant the ability of the protease-activated receptor 3 to bind thrombin or a PAR3 agonist and signal the appropriate cascade of biological events (e.g., phosphoinositide hydrolysis, Ca 2+ 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 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 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 of a PAR3 ligand. The method involves: a) contacting a candidate agonist compound with a recombinant protease-activated receptor 3 (or PAR3 agonist-binding fragment or analog) ; 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 PAR3 activation. Interaction may be cleavage of the receptor 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 agonist which will block responses by other thrombin substrates. By an "agonist" is meant a molecule which mimics a particular activity, in this case, interacting with a PAR3 ligand in a manner which activates thereby triggering the biological events which normally result from the interaction (e.g., phosphoinositide hydrolysis, Ca + efflux, and platelet aggregation) . Preferably, an agonist initiates a substantial 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 PAR3 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-occurring activating ligand or an agonist and the PAR3. The method involves: a) contacting a candidate antagonist compound with a first compound which includes a recombinant PAR3 (or agonist -binding fragment or analog) on the one hand and with a second compound which includes thrombin or a PAR3 agonist on the other hand; b) determining whether the first and second compounds interact or are prevented from interaction by the candidate compound; and c) identifying antagonistic compounds as those which interfere with the interaction of the first compound (PAR3 receptor) to the second compound (PAR3 agonist) and which thereby substantially reduce thrombin or PAR3 agonist-activated biological events (e.g., phosphoinositide hydrolysis, Ca 2+ efflux, and platelet aggregation) . By an "antagonist" is meant a molecule which blocks activation of a PAR3 receptor. This can be done by inhibiting a particular activity such as the ability of thrombin, for example, to interact with a protease- activated receptor 3 thereby triggering the biological events resulting from such an interaction (e.g., phosphoinositide hydrolysis, Ca 2+ efflux, and platelet secretion, or platelet aggregation) . An antagonist may bind to and thereby block activation of a PAR3 receptor. The terms "treatment", "treating", "treat" and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect . The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, particular a human, and includes : (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease. PREFERRED EMBODIMENTS In preferred embodiments of both screening methods, the recombinant PAR3 is stably expressed by a vertebrate cell which normally presents substantially no PAR3 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 agonist is a thrombin analog, PAR3 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 signalling in embryonic development. Such proteins and in particular PAR3 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, PAR3 peptide fragments or analogs thereof, or other compounds which mimic the action of thrombin upon interaction with the protease-activated receptor 3 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 3 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 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. 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 PAR3 gene (as cDNA or genomic DNA) allows its expression in a cell type which does not normally bear PAR3 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 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 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 3 from mouse and human. Expression vectors containing and capable of expressing the PAR3 DNA, as well as transformed cells containing and expressing the DNA of the invention are also described. Also described are possible PAR3 agonists and antagonists as well as screening assays for receptor agonists and receptor antagonists. EXAMPLE 1 Isolation of the Mouse Protease-Activated Receptor 3 Rat platelets were used as a source of RNA in the search for and cloning of PAR3 because rat platelets are more abundant than mouse platelets and, like mouse platelets, they do not respond to PARl agonist peptides (Connolly, A. et al . (1996) Nature 38JL: 516-519; and Connolly, T.M. et al (1994) Thromb Haemost 72: 627-33) . Total RNA was prepared from rat platelets using Trizol reagent (Gibco BRL) . cDNA was then prepared using random hexamer primers and the Superscript reverse transcriptase system (Gibco, BRL) . cDNA was then used as template for PCR amplification using a Robocycler Gradient 96 ® (Stratagene) and the primers 5'- GTITACATGCTI (A/C) AC (C/T) TIGCI (A/C/G/T) TIGC (A/C/G/T) GA-3 ' (SEQ ID NO: 10) and 5' - GGATAIACIACIGCIA(A/G/T) (A/G) (A/T) AIC (G/T) (A/C/G/T) TC-3 ' (SEQ ID NO:ll) at 5μM in 20μM Tris-HCl (pH 8.4), 50μM KC1, 1.5μM MgC12, 0.2μM dNTP, and 50U/μl Taq polymerase . Polymerase chain reaction temperature was varied as follows: 94°C for 4 min; 30 cycles of 94°C for 45 sec, 39°C for 60 sec, and 72°C for 90 sec; then 72°C for 7 min. PCR products were subcloned using the TA cloning kit (InVitrogen, San Diego, CA) . Rat cDNA clones with inserts of approximately 200 bp were analyzed by nucleic acid sequencing. One sequence predicted a novel G-protein coupled receptor related to PARl and PAR2. This 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 . (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) . The nucleotide sequences are shown in Figures 1-4. The rat PCR product was then used to clone the full length mouse cDNA and genomic DNA clones. The nucleotide sequences and deduced amino acid sequence of the mouse PAR3 are shown in Figs . 1 and 2. The human PAR3 cDNA used for the functional studies presented below was cloned from a Lamda gt 10 intestinal cDNA library (Clonetech) . Features of human PAR3 ' s amino acid sequence are shown in Figs . 5A and 5B by alignment of the deduced amino acid sequence of PAR3 with those of PARl and PAR2. Predicted transmembrane (TM) domains are overlined and predicted Asn-linked glycosylation sites in PAR3 are underlined in the figure. The amino terminal exodomains are compared in Fig. 5b, including the cleavage site ( A ) , the tethered ligand domains of PARl and PAR2 , and the predicted tethered ligand domain of PAR3 (underlined) . Also underlined is PAR3's hirudin-like domain (FEEFP) . The similar FEEIP and YEPFW sequences in hirudin and PARl, respectively are known to bind thrombin' s fibrinogen-binding exosite. The human PAR3 cDNA contained an open reading frame encoding a 374 amino acid putative G protein- coupled receptor (Fig. 3) . BLAST search of the Genbank and EST databases revealed this protein to be novel with 28% and 30% amino acid sequence identity to human PARl and PAR2 (Fig. 5a, Table I) . Its amino terminal exodomain revealed a possible thrombin cleavage site and a striking hirudin-like sequence (Fig. 5b). Like the carboxyl tail of hirudin itself, PARl's hirudin-like sequence is known to dock with thrombin' s fibrinogen binding exosite, an interaction important for efficient PARl cleavage by thrombin (Vu, T.-K.H. et al.(1991) Nature 353 . : 674-677 ; Liu, L. et al . (1991) J. Biol. Chem 266:16977-16980; Mathews , I.I. et al . (1994) Biochem 33 3266-79; Ishii, K. (1995) J. Biol. Chem 270:16435-16440, which references are herein incorporated by reference in their entirety) . These observations strongly suggested that this new receptor was a novel thrombin receptor. A comparison of PAR deduced amino acid sequences from human, mouse, and Xenopus is provided in Table I below. The % identity of the total sequence as well as the % identity of the transmembrane regions are shown. TABLE I h = human m = mouse x = Xenopus laevis EXAMPLE 2 Polypeptide Expression Polypeptides according to the invention may be produced by transformation of a 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 prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiae or mammalian cells, e.g., C0S-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, 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 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 PAR3 or an appropriate receptor fragment or analog (as described above) is inserted into the expression vector in an orientation designed to allow expression. Alternatively, the PAR3 (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 (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 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 PAR3 -encoding gene into the host cell chromosome is selected for by inclusion of 0.01-300 μM 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 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 . , supra) . Any of the 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. One particularly preferred stable expression system is a Rat 1 cell (ATCC) stably transfected with a pcDNAl/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 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 3 antibodies using, as an immunogen, the intact receptor or a peptide which includes a suitable protease-activate receptor 3 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 PAR3 protein (or fragment or analog, thereof) is expressed, it is isolated, e.g., using immunoaffinity chromatography. In one example, an anti-PAR3 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 . , supra) . 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 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) . EXAMPLE 3 Cleavage and Activation Studies of the Recombinant Protease-Activated Receptor 3 PAR3 was demonstrated to be a substrate for thrombin when expressed on the surface of Cos 7 cells (Fig. 6) . Human PARl or PAR3 cDNAs that were modified to encode receptors displaying a FLAG epitope (amino acid sequence DYKDDD (SEQ ID NO: 12) at a site amino to the thrombin cleavage site were transiently expressed in Cos7 cells. Epitope-tagged PARl has been previously described (Ishii, K. et al . (1993) J. Biol. Chem. 268:9780-9786) . The analogous epitope-tagged PAR3 cDNA was constructed so as to encode a new amino terminus with the sequence MDSKGSSQKGSRLLLLLWSNLLLCQGWS/DYKDDDDVE-TF (SEQ ID NO: 13) representing the prolactin signal peptide, putative signal peptidase site (/) , FLAG epitope DYKDDDD (SEQ ID NO: 12) and junction VE fused to amino acid 17 in PAR 3. cDNAs were subcloned into the mammalian expression vector pBJl . For receptor cleavage studies Cos 7 cells were transfected using DEAE-dextran and thrombin-mediated loss of Ml antibody (Kodak) binding to the FLAG epitope of the cell surface using a procedure described by Ishii et al. (Ishii, K. et al . (1993) supra) . Over 95% of Ml antibody binding was transfection-dependent in this system. Cells were incubated for 5 min. at 37°C in the presence (open columns) or absence (closed columns) of 20nM thrombin (Fig. 6) . For biochemical identification of the cleavage site, cleavage of soluble PAR3 amino terminal exodomain by thrombin was assayed as follows. A recombinant PAR3 soluble exodomain was prepared in which the amino terminal exodomain residues 21-94 were sandwiched between a translational start and hexahistidine tag (i.e. MG- [PAR3 21-94] -VEHHHHHH; where VEHHHHHH is SEQ ID NO: 18). The recombinant protein was expressed as a soluble polypeptide in E. coli , purified, and analyzed before and after thrombin cleavage as previously described for the analogous region of PARl (Ishii, K. (1995) J. Biol. Chem. 270:16435-16440) . Recombinant soluble amino terminal exodomain was cleaved in solution with 50nM thrombin for lh at 37°C, then analyzed by SDS-PAGE. Even prolonged incubation with a high concentration of thrombin yielded only one detectable cleavage event indicating that only one thrombin cleavage site exists in the PAR3 exodomain. Amino acid sequencing of the cleavage products revealed only a single new amino terminus with the sequence TFRG (see Fig. lb) . Thus, thrombin recognizes and cleaves PAR3 in the amino terminal exodomain between amino acids K38 and T39 with high specificity. EXAMPLE 4 PAR3 Signaling Activity The ability of PAR3 to mediate signaling by thrombin was tested. Xenopus oocytes were microinj ected with cRNA encoding epitope-tagged human PAR3 (hPAR3), hPAR3 bearing the T39P cleavage site mutation, or the F40A tethered ligand domain mutation. Thrombin-triggered 5 Ca release was measured as described in Vu et al . (Vu, T.-K. H. et al . (1991) supra) . Surface expression of wild type and mutant receptors was confirmed by Ml 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) . Microinj ection of Xenopus oocytes with human PAR3 cRNA conferred thrombin-dependent 45 Ca mobilization (Fig. 7) which reflects agonist-triggered phosphoinositide hydrolysis in this system. Mutation of PAR3 ' s thrombin cleavage site ablated thrombin signaling and thrombin rendered proteolytically inactive by the active site inhibitor PPACK failed to activate PAR3 even at concentrations as high as lμM. These data strongly suggest that cleavage of the K 38 -T 39 peptide bond is necessary for PAR3 activation by thrombin. The specificity of PAR3 and PARl signaling was also examined. Protease-triggered 45 Ca release was measured in Xenopus oocytes expressing human PARl or PAR3 stimulated with various concentrations of the arginine/lysine specific serine proteases trypsin, Factor Xa, Factor Vila, tissue plasminogen activator, or plasmin. Chymotrypsin, elastase, and cathepsin G were also tested. PAR3 was at least as specific for thrombin as thrombin receptor PARl (Fig. 10) . PAR3 signaling in Cos 7 cells was also examined. Cos 7 cells were transfected with human PARl or PAR3. Cells were then metabolically labelled with 3 H-inositol and phosphoinositide hydrolysis was measured in response to the indicated concentrations of a-thrombin (Fig. 8) or γ-thrombin (Fig. 9) as described by Ishii, et al . and Nanevicz et al . (Ishii, K. et al . (1993) supra ; and Nanevicz, T. et al . (1996) J. Biol. Chem. 271:702-706). Co-transfection with αl6, a G protein α-subunit expressed in hematopoietic cell lines (Amatruda III, T.T. et al. (1991) J. Biol. Chem. 266:5587-5591) caused a 50-150% increase in the maximal PAR3 -mediated response to thrombin in these cells in each of three separate experiments (Fig. 7) . The EC 50 for thrombin signaling through PAR3 in this system was approximately 0.2 nM, comparable to that seen with PARl and well within physiologically achievable thrombin concentrations (Fig 8) . γ-thrombin, which is defective in its anion-binding exosite (Rydel, T.J. et al . (1994) J. Biol. Chem. 269 : 22000-22006) , was two log units less potent than α-thrombin (EC 50 = 20nM; Fig. 9) . Similarly, incubation of α-thrombin with the fibrinogen binding exosite blocker hirugen (Skrzypczak, J.E. et al . (1991) J. Mol. Biol. 221:1379-1393) right-shifted the dose response curve two logs (not shown) . Alanine substitution at F 48 and E 49 in PAR3 ' s hirudin-like sequence, residues predicted to dock with thrombin' s fibrinogen-binding exosite by analogy with hirudin and PARl (Fig. 5B) also caused a decrease in thrombin signaling by PAR3. These data strongly suggest that PAR3 interacts with thrombin in a manner similar to PARl (Mathews, I. I., et al . (1994) Biochem. 33:3266-3279). Specifically, it is likely that PAR3 amino acids 48-52 (FEEFP, SEQ ID NO: 14) dock with thrombin' s fibrinogen- binding exosite while amino acids 35-38 (LTPK, SEQ ID NO: 15) dock with thrombin' s active center leading to cleavage of the K 38 - T 39 peptide bond. Synthetic peptides that mimic the new amino terminus unmasked by receptor proteolysis, the so called "tethered ligand domain", act as agonists for PARl and PAR2 (Vu, T. K.-H. et al . (1991) Cell 64:1057-1068; Nystedt, S. et al . (1994) PNAS USA 91:9208-9212; and USPN 5,256,766, which references are herein incorporated by reference in their entirety) . Peptides homologous to the tethered domain of PAR3 may be tested as potential agonists of PAR3 activity. Two peptides, TFRGAP (SEQ ID NO: 16) and TFRGAPPNS (SEQ ID NO: 17) were synthesized and tested for their ability to mimic the action of thrombin by causing PAR3 signaling as measured by phosphoinositide hydrolysis. Cos 7 cells expressing human PAR3 were incubated with the peptides at concentrations up to lOOμM. Phosphoinositide hydrolysis was not observed to be above control levels indicating that the synthetic peptides caused no detectable signaling by PAR3 under these conditions, whereas an EC 50 of 0.2 nM was determined for α-thrombin under the same assay conditions. These results demonstrate that monitoring phosphoinositide hydrolysis provides a useful means for assessing potential agonists for activity on PAR3 signaling for use as potential pharmaceutics. The tethered ligand domain of PAR3 was required for PAR3 activation by thrombin. Substitution of Ala for Phe 40 (the F40A PAR3 mutant) , which is analogous to the critical Phe 43 in PARl ' s tethered ligand (Scarborough, R.M. et al. (1992) J. Biol. Chem. 267:13146-13149) , ablated PAR3 signaling but not PAR3 cleavage by thrombin. The observation that cleavage of the Lys 38-Thr 39 peptide bond is necessary for PAR3 activation suggests that PAR3 is probably activated by the same tethered ligand mechanism utilized by PARl and 2. EXAMPLE 5 PAR3 Tissue Expression in Mouse and Human In situ hybridization of mouse tissue revealed the presence of PAR3 mRNA in megakaryocytes in mouse spleen. In the tissues examined (brain, eye, thymus , heart, lung, liver spleen, pancreas, stomach, small intestine, colon, kidneys, bladder, uterus, ovary, testis, skeletal muscle, peripheral nerve, and skin), megakaryocytes in the spleen were the only cells which displayed clearcut hybridization over background. Control samples in which hybridization was performed with a sense strand probe control were negative for all cells. Northern analysis of mouse tissues for PAR3 mRNA showed signals in spleen and lung, with low levels seen in brain, heart, and other tissues. Spleen is a hematopoietic organ in mouse, and megakaryocytes are sometimes seen trapped in the pulmonary microvasculature . Thus both Northern and in situ hybridization data suggest that PAR3 is most abundantly expressed in megakaryocytes in the mouse. The pharmacology of hPAR3 activation in Cos cells resembles that of mouse platelet activation. Both responses show subnanomolar EC 50 s for activation by α-thrombin and are thrombin active site- and fibrinogen- binding exosite-dependent . These observations support the concept that the mouse homolog of PAR3 is a thrombin receptor that mediates thrombin responses in mouse platelets. Whether human PAR3 function in human platelets remains to be determined. The in si tu hybridization studies were performed as follows. Anesthetized adult C57BL/6 mice were perfusion-fixed with 4% paraformaldehyde . Organs to be tested were dissected, trimmed, and immersion-fixed for 4 hours in 4% paraformaldehyde . Processed tissues were embedded in paraffin, and 5 mm sections were cut. Sense or antisense 35 S-riboprobe was transcribed in vi tro from mouse PAR2 cDNA subcloned into the EcoRl site of pBluescript II SK " (Stratagene, San Diego, CA) . Hybridization, wash, and development conditions were as reported for mouse PARl (Soifer, S.J. et al . (1993) Am. J. Pathol . 144 : 60-69) . To carry out Northern analysis a 32 P-labeled probe for the mouse message was 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 Clontech protocol for Northern analysis. Northern analysis of human tissues revealed that PAR3 mRNA is widely distributed with signals noted in small intestine, bone marrow, heart, pancreas, lung, liver, adrenal, trachea, lymph node, stomach, and peripheral blood leukocytes. The role of PAR3 in these various human tissues awaits definition; the finding of PAR3 in human bone marrow and leukocytes is consistent with PAR3 ' s playing a role in mediating activation of platelets and other hematopoietic cells by thrombin. EXAMPLE 6 Assays for Protease-Activated Receptor 3 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 vi tro functional assay method (e.g., the phosphoinositide hydrolysis assay, 45 Ca efflux assay, or platelet aggregation assay described herein) . This method includes, as components, thrombin and a recombinant protease-activated receptor 3 (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 3 component is produced by a cell that 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 1 cells or COS-7 cells. EXAMPLE 7 Screening For Protease-Activated Receptor 3 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 PAR3 activating compound) and the protease-activated receptor 3, thereby preventing or reducing the cascade of events that are mediated by that interaction. The elements of the screen are a PAR3 activator (such as thrombin) , a candidate antagonist, and recombinant PAR3 (or a suitable receptor fragment or analog, as outlined above) configured to permit detection of PAR3 activator, antagonist, and PAR3 function. An additional element may be a downstream substrate, such as phosphoinositide, the hydrolysis of which is used to measure thrombin activity (Ishii, K. et al . (1993) supra ; and Nanevicz, T. et al . (1996) supra) . Inhibition of thrombin-induced platelet aggregation may also be used as a means of monitoring an antagonist of PAR3 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 USPN 5,256,766). Alternately, platelets are incubated with a candidate PAR 3 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. Appropriate candidate thrombin antagonists include PAR3 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 3 antagonists include thrombin analogs as well as other peptide and non-peptide compounds and anti- PAR3 antibodies. AGONISTS Another aspect of the invention features screening for compounds that act as PAR3 ligand agonists. Activation of the PAR3 with thrombin or an agonist leads to a cascade of events (such as phosphoinositide hydrolysis, Ca 2+ efflux, and platelet aggregation), providing a convenient means for measuring thrombin or other agonist activity. The agonist screening assay of the invention utilizes recombinant cells expressing recombinant PAR3 (or a suitable receptor fragment or analog, as outlined herein) configured to permit detection of PAR3 function. Alternatively, a cell such as a leukocyte, a platelet, or a mesenchymal cell that naturally expresses PAR3 may be used. Other elements of the screen include a detectable downstream substrate of the PAR3 activation, such as radiolabelled phosphoinositide, the hydrolysis of which to a detectable product indicates PAR3 activation by the candidate agonist. 45 Ca efflux from a cell expressing PAR3 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 64=1057-1068; and USPN 5,256,766, which references are herein incorporated by reference in their entirety) . 5 Ca release by oocytes expressing cRNA encoding PAR3 are assessed as follows. Briefly, intracellular calcium pools are labeled by incubating groups of 30 oocytes in 300 μl calcium-free MBSH containing 50 μCi 45 CaCl 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 (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 45 Ca release per unit time is achieved. Two additional 10 -minute collections are obtained, then test 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 that the single electrode voltage-clamp technique is employed. Platelet aggregation may also be used as a means of monitoring PAR3 receptor activation (see, for example, Connolly, A.J. et al . Nature 381:516-519 (1996). In particular, mouse platelets may utilize only PAR 3 for thrombin signaling. Human platelets may use both PAR 1 and PAR 3. Thus both would be useful in deleting against function at PAR 3. An agonist useful in the invention is one which imitates the normal thrombin-mediated signal transduction pathway leading, e.g., to an increase in phosphoinositide hydrolysis. Appropriate candidate agonists include thrombin analogs or PAR3 tethered ligand domains or other agents which mimic the action of thrombin or the PAR 3 tethered ligand domain. Agonists would be useful for aiding discovery of antagonists. EXAMPLE 8 Anti-Protease-Activated Receptor 3 Antibodies Protease-activated receptor 3 (or immunogenic receptor fragments or analogs) may be used to raise antibodies useful in the invention. Receptor fragments preferred for the production of antibodies are those fragments deduced or shown experimentally to be extracellular . Antibodies directed to PAR3 peptides are produced as follows. Peptides corresponding to all or part of the PAR3 protein are produced using a peptide synthesizer by 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 PAR3 polypeptides described above and standard hybridoma technology (see, e.g., Kohler et al., Nature (1975) 256:495, 1975; Kohler et al . , 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 PAR3 by specific binding to the surface of PAR3 -transfected cells by Western blot or immunoprecipitation analysis (such as by the methods described in Ausubel et al . , supra) . Antibodies which specifically recognize PAR3 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 PAR3 (using the functional antagonist assays described herein) . Antibodies which antagonize thrombin: PAR3 binding or PAR3 function are considered to be useful antagonists in the invention. EXAMPLE 9 THERAPY Particularly suitable therapeutics for the treatment of wound healing, thrombosis, atherosclerosis, restenosis, inflammation, and other thrombin-mediated signalling 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 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-PAR3 antibodies produced as described 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 3 are useful antagonists which can be formulated as indicated above. Other therapeutically useful antagonists are peptides derived from PAR3 that bind to and block thrombin and include formulation comprising a pharmaceutically acceptable carrier and one or more of the following: (1) the isolated sequence LPIKTFRGAPPNSFEEFPFSALE ; (2) uncleavable thrombin inhibitor LPIKPFRGAPPNSFEEFPFSALE where the PAR 3 cleavage site PI' is mutated to block cleavage; (3) uncleavable thrombin inhibitor LPI (hR)TFRGAPPNSFEEFPFSALE where the PAR 3 cleavage site PI is mutated to block cleavage; hR is beta-homoarginine (the extra methylene group is in the main chain) ; (4) uncleavable thrombin inhibitor (dF) PRPFRGAPPNSFEEFPFSALE where the good active site binding sequence dFPR is substituted for LPIK; dF is D-Phenylalanine; (5) any of (l)-(4) above where all or part of the sequence TFRGAPPNS is replaced with spacer sequences such as GGG; (6) variations and combinations of (1) - (5) which 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 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, 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. PAR3 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. 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 signalling 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 PAR3 or receptor fragment or analog or the antibody employed is preferably specific for that species. OTHER EMBODIMENTS Polypeptides according to the invention include any protease-activated receptors (as described herein) . Such receptors may be derived from any source, but are preferably derived 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 PAR3 capable of interacting with thrombin. Such analogs and fragments may also be used to screen for PAR3 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 may be readily assayed using PAR3 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, 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) . Specific receptor fragments of interest include any portion of the PAR3 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 PAR3 in vivo (e.g., by interfering with the interaction between the receptor and thrombin) . The sequence of figure 5B is most likely to bind thrombin. Modification of the (K38/T39) cleavage site for example, substitution of proline for T39 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 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 hydrophobicity/hydrophilicity calculation such as the Chou-Fasman method (see, e.g., Chou and Fasman, Ann. Rev. Biochem. (1978) 47 . : 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. 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 PAR3 , using the assays described herein. Analogs of useful receptor fragments (as described above) may also be produced and tested for efficacy as screening components or antagonists (using the assays described herein) ; such analogs are also considered to be useful in the invention. 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 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 PAR3 agonists or antagonists. 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. SEQUENCE LISTING (1) GENERAL INFORMATION (i) APPLICANT: The Regents of the University of California (ii) TITLE OF THE INVENTION: Protease Activated Receptor 3 and Uses Thereof (iii) NUMBER OF SEQUENCES: 23 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Fulbright & Jaworski L.L.P. (B) STREET: 865 S. Figueroa Street, 29th Floor (C) CITY: Los Angeles (D) STATE: CA (E) COUNTRY: USA (F) ZIP: 90017-2571 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: DOS (D) SOFTWARE: FastSEQ for Windows Version 2.0 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Berliner, Robert (B) REGISTRATION NUMBER: 20,121 (C) REFERENCE/DOCKET NUMBER: 5555-462 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 213-892-9200 (B) TELEFAX: 213-680-4518 (C) TELEX: (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1224 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: TGACTTTGTA TACTTAACAA CATCCTGTAG CCGGGTCTCA GGACATCAAG ATGAAAATCC 60 TTATCTTGGT TGCAGCTGGG CTGCTGTTTC TGCCAGTCAC TGTTTGCCAA AGTGGCATAA 120 ATGTTTCAGA CAACTCAGCA AAGCCAACCT TAACTATTAA GAGTTTTAAT GGGGGTCCCC 180 AAAATACCTT TGAAGAATTC CCACTTTCTG ACATAGAGGG CTGGACAGGA GCCACCACAA 240 CTATAAAAGC GGAGTGTCCC GAGGACAGTA TTTCAACTCT CCACGTGAAT AATGCTACCA 300 TAGGATACCT GAGAAGTTCC TTAAGTACCC AAGTGATACC TGCCATCTAT ATCCTGCTGT 360 TTGTGGTTGG TGTACCATCC AACATCGTGA CCCTGTGGAA ACTCTCCTTA AGGACCAAAT 420 CCATCAGTCT GGTCATCTTT CACACCAACC TGGCCATCGC AGATCTCCTT TTCTGTGTCA 480 CACTGCCATT TAAGATCGCC TACCATCTCA ATGGCAACAA CTGGGTATTT GGCGAGGTCA 540 TGTGCCGGAT CACCACGGTC GTTTTCTACG GCAACATGTA CTGCGCTATC CTGATCCTCA 600 CTTGCATGGG CATCAACCGC TACCTGGCCA CGGCTCACCC TTTCACATAC CAGAAGCTGC 660 CCAAACGCAG CTTCTCCTTG CTCATGTGTG GCATAGTGTG GGTCATGGTT TTCTTATACA 720 TGCTGCCCTT TGTCATCCTG AAGCAGGAGT ACCACCTCGT CCACTCAGAG ATCACCACCT 780 GCCACGATGT CGTCGACGCG TGCGAGTCCC CATCATCCTT CCGATTCTAC TACTTCGTCT 840 CCTTAGCATT CTTTGGGTTC CTCATCCCGT TTGTGATCAT CATCTTCTGT TACACGACTC 900 TCATCCACAA ACTTAAATCA AAGGATCGGA TATGGCTGGG CTACATCAAG GCCGTCCTCC 960 TCATCCTTGT GATTTTCACA ATTTGCTTTG CCCCCACCAA CATCATACTC GTAATCCACC 1020 ATGCCAACTA CTACTACCAC AATACCGACA GCTTGTACTT TATGTATCTT ATTGCTCTGT 1080 GCCTGGGGAG CCTGAATAGC TGCCTAGATC CATTCCTTTA CTTTGTCATG TCGAAAGTTG 1140 TAGATCAGCT TAATCCTTAG TCGGCAATGG CAAGACCACT TTAGAGACCA AGGAGAGATA 1200 TCTGGGAAGA CATACATGCT TGGC 1224 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1124 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CCATATGCTA ATATTTCCTT TCAATTACAG GCATAAATGT TTCAGACAAC TCAGCAAAGC 60 CAACCTTAAC TATTAAGAGT TTTAATGGGG GTCCCCAAAA TACCTTTGAA GAATTCNNNN 120 NNNTACAACT CTCCATGTGA ATAATGCTAC CATGGGATAC CTGAGAAGTT CCTTAAGTAC 180 CAAAGTGATA CCTGCCATCT ACATCCTGGT GTTTGTGATT GGTGTACCAG CGAACATCGT 240 GACCCTGTGG AAACTCTCCT CAAGGACCAA ATCCATCTGT CTGGTCATCT TTCACACCAA 300 CCTGGCCATC GCGGATCTCC TTTTCTGTGT CACGCTGCCG TTTAAGATCN NCCTACCATC 360 TCAATGGCAA CAACTGGGTA TTTGGCGAGG TCATGTGCCG GATCACCACG GTCGTTTTCT 420 ACGGCAACAT GTACTGCGCT ANNNTCCTGA TCCTCACCTG CATGGGCATC AACCGCTACC 480 TGGCCACGGC TCACCCTTTC ACATACCAGA AGCTGCCCAA ACGCAGCTTC TCCATGCTCA 540 TGTGTGGCAT GGTGTGGGTC ATGGTTTTCT TATACATGCT GCCCTTTGTC ATCCNNNAAG . 600 CAGGAGTACC ACCTCGTCCA CTCCGAGATC ACCACCTGCC ACGATGTCGT CGACGCGTGC 660 GANTCCCCAT CATCCTTCCG ATTCTACTAC TTCGTCTCCT TAGCATTCTT TGGGTTCCTC 720 ATCCCGTTTG TGATCATCAT CTTCTGTTAC ACGACTCTCA TCCACAAACT TAAATCAAAA 780 GATCNGATAT GGCTGGGCTA CATCAAGGCC GTCCTCCTCA TCCTTGTGAA TTTCACCATC 840 TGCTTCCCCC CCACCAAGNN NNNNGATATC TGGGAAGACG TACATGCTTG GCTGACTTGT 900 GCATGGCACC ATCAGCTCAA TTTTTAATTT TTTAATTTTA ATTTAATTTA ATTTTATGTT 960 TTTGAGACAG AGCCTCACTG TGTAGTCCTG GCTGGCCTGG CTGGTTCTCT ATTTAGACCA 1020 GGTTAGCCTT GAACTCACAG AGATCTGCCT GCTTCTGCCT CCCAAGTGCT GGGTTCAACC 1080 AGGTCTGGCA AGCGCTCCAT TTTTCAGCTC CTCTGCAACA GTGC 1124 (2) INFORMATION FOR SEQ ID N0:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 407 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii MOLECULE TYPE: protein (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3: Thr Leu Tyr Thr Xaa Gin His Pro Val Ala Gly Ser Gin Asp He Lys 1 5 10 15 Met Lys lie Leu He Leu Val Ala Ala Gly Leu Leu Phe Leu Pro Val 20 25 30 Thr Val Cys Gin Ser Gly lie Asn Val Ser Asp Asn Ser Ala Lys Pro 35 40 45 Thr Leu Thr He Lys Ser Phe Asn Gly Gly Pro Gin Asn Thr Phe Glu 50 55 60 Glu Phe Pro Leu Ser Asp He Glu Gly Trp Thr Gly Ala Thr Thr Thr 65 70 75 80 He Lys Ala Glu Cys Pro Glu Asp Ser He Ser Thr Leu His Val Asn 85 90 95 Asn Ala Thr He Gly Tyr Leu Arg Ser Ser Leu Ser Thr Gin Val He 100 105 110 Pro Ala He Tyr He Leu Leu Phe Val Val Gly Val Pro Ser Asn He 115 120 125 Val Thr Leu Trp Lys Leu Ser Leu Arg Thr Lys Ser He Ser Leu Val 130 135 140 He Phe His Thr Asn Leu Ala He Ala Asp Leu Leu Phe Cys Val Thr 145 150 155 160 Leu Pro Phe Lys He Ala Tyr His Leu Asn Gly Asn Asn Trp Val Phe 165 170 175 Gly Glu Val Met Cys Arg He Thr Thr Val Val Phe Tyr Gly Asn Met 180 185 190 Tyr Cys Ala He Leu He Leu Thr Cys Met Gly He Asn Arg Tyr Leu 195 200 205 Ala Thr Ala His Pro Phe Thr Tyr Gin Lys Leu Pro Lys Arg Ser Phe 210 215 220 Ser Leu Leu Met Cys Gly He Val Trp Val Met Val Phe Leu Tyr Met 225 230 235 240 Leu Pro Phe Val He Leu Lys Gin Glu Tyr His Leu Val His Ser Glu 245 250 255 He Thr Thr Cys His Asp Val Val Asp Ala Cys Glu Ser Pro Ser Ser 260 265 270 Phe Arg Phe Tyr Tyr Phe Val Ser Leu Ala Phe Phe Gly Phe Leu He 275 280 285 Pro Phe Val He He He Phe Cys Tyr Thr Thr Leu He His Lys Leu 290 295 300 Lys Ser Lys Asp Arg He Trp Leu Gly Tyr He Lys Ala Val Leu Leu 305 310 315 320 He Leu Val He Phe Thr He Cys Phe Ala Pro Thr Asn He He Leu 325 330 335 Val He His His Ala Asn Tyr Tyr Tyr His Asn Thr Asp Ser Leu Tyr 340 345 350 Phe Met Tyr Leu He Ala Leu Cys Leu Gly Ser Leu Asn Ser Cys Leu 355 360 365 Asp Pro Phe Leu Tyr Phe Val Met Ser Lys Val Val Asp Gin Leu Asn 370 375 380 Pro Xaa Ser Ala Met Ala Arg Pro Leu Xaa Arg Pro Arg Arg Asp He 385 390 395 400 Trp Glu Asp He His Ala Trp 405 (2) INFORMATION FOR SEQ ID N0:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1224 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: TGCTCCATGA TTTTACAGAT TTCATAACGT TTAAGAGACG GGACTCAGGT CATCAAAATG 60 AAAGCCCTCA TCTTTGCAGC TGCTGGCCTC CTGCTTCTGT TGCCCACTTT TTGTCAGAGT 120 GGCATGGAAA ATGATACAAA CAACTTGGCA AAGCCAACCT TACCCATTAA GACCTTTCGT 180 GGAGCTCCCC CAAATTCTTT TGAAGAGTTC CCCTTTTCTG CCTTGGAAGG CTGGACAGGA 240 GCCACGATTA CTGTAAAAAT TAAGTGCCCT GAAGAAAGTG CTTCACATCT CCATGTGAAA 300 AATGCTACCA TGGGGTACCT GACCAGCTCC TTAAGTACTA AACTGATACC TGCCATCTAC 360 CTCCTGGTGT TTGTAGTTGG TGTCCCGGCC AATGCTGTGA CCCTGTGGAT GCTTTTCTTC 420 AGGACCAGAT CCATCTGTAC CACTGTATTC TACACCAACC TGGCCATTGC AGATTTTCTT 480 TTTTGTGTTA CATTGCCCTT TAAGATAGCT TATCATCTCA ATGGGAACAA CTGGGTATTT 540 GGAGAGGTCC TGTGCCGGGC CACCACAGTC ATCTTCTATG GCAACATGTA CTGCTCCATT 600 CTGCTCCTTG CCTGCATCAG CATCAACCGC TACCTGGCCA TCGTCCATCC TTTCACCTAC 660 CGGGGCCTGC CCAAGCACAC CTATGCCTTG GTAACATGTG GACTGGTGTG GGCAACAGTT 720 TTCTTATATA TGCTGCCATT TTTCATACTG AAGCAGGAAT ATTATCTTGT TCAGCCAGAC 780 ATCACCACCT GCCATGATGT TCACAACACT TGCGAGTCCT CATCTCCCTT CCAACTCTAT 840 TACTTCATCT CCTTGGCATT CTTTGGATTC TTAATTCCAT TTGTGCTTAT CATCTACTGC 900 TATGCAGCCA TCATCCGGAC ACTTAATGCA TACGATCATA GATGGTTGTG GTATGTTAAG 960 GCGAGTCTCC TCATCCTTGT GATTTTTACC ATTTGCTTTG CTCCAAGCAA TATTATTCTT 1020 ATTATTCACC ATGCTAACTA CTACTACAAC AACACTGATG GCTTATATTT TATATATCTC 1080 ATAGCTTTGT GCCTGGGTAG TCTTAATAGT TGCTTAGATC CATTCCTTTA TTTTCTCATG 1140 TCAAAAACCA GAAATCACTC CACTGCTTAC CTTACAAAAT AGTGAAATGA TCTTAGAGAA 1200 CAAGGACAGC CATCACAGAG AACG 1224 (2) INFORMATION FOR SEQ ID N0:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1102 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5: ACAGGCATGG AAAATGATAC AAACAACTTG GCAAAGCCAA CCTTACCCAT TAAGACCTTT 60 CGTGGAGCTC CCCCAAATTC TTTTGAAGAG TTCCCCTTTT CTGCCTTGGA AGGCTGGACA 120 GGAGCCACGA TTACTGTAAA AATTAAGTGC CCTGAAGAAA GTGCTTCACA TCTCCATGTG 180 AAAAATGCTA CCATGGGGTA CCTGACCAGC TCCTTAAGTA CTAAACTGAT ACCTGCCATC 240 TACCTCCTGG TGTTTGTAGT TGGTGTCCCG GCCAATGCTG TGACCCTGTG GATGCTTTTC 300 TTCAGGACCA GATCCATCTG TACCACTGTA TTCTACACCA ACCTGGCCAT TGCAGATTTT 360 CTTTTTTGTG TTACATTGCC CTTTAAGATA GCTTATCATC TCAATGGGAA CAACTGGGTA 420 TTTGGAGAGG TCCTGTGCCG GGCCACCACA GTCATCTTCT ATGGCAACAT GTACTGCTCC 480 ATTCTGCTCC TTGCCTGCAT CAGCATCAAC CGCTACCTGG CCATCGTCCA TCCTTTCACC 540 TACCGGGGCC TGCCCAAGCA CACCTATGCC TTGGTAACAT GTGGACTGGT GTGGGCAACA 600 GTTTTCTTAT ATATGCTGCC ATTTTTCATA CTGAAGCAGG AATATTATCT TGTTCAGCCA 660 GACATCACCA CCTGCCATGA TGTTCACAAC ACTTGCGAGT CCTCATCTCC CTTCCAACTC 720 TATTACTTCA TCTCCTTGGC ATTCTTTGGA TTCTTAATTC CATTTGTGCT TATCATCTAC 780 TGCTATGCAG CCATCATCCG GACACTTAAT GCATACGATC ATAGATGGTT GTGGTATGTT 840 AAGGCGAGTC TCCTCATCCT TGTGATTTTT ACCATTTGCT TTGCTCCAAG CAATATTATT 900 CTTATTATTC ACCATGCTAA CTACTACTAC AACAACACTG ATGGCTTATA TTTTATATAT 960 CTCATAGCTT TGTGCCTGGG TAGTCTTAAT AGTTGCTTAG ATCCATTCCT TTATTTTCTC 1020 ATGTCAAAAA CCAGAAATCA CTCCACTGCT TACCTTACAA AATAGTGAAA TGATCTTAGA 1080 GAACAAGGAC AGCCATCACA GA 1102 (2) INFORMATION FOR SEQ ID N0:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 408 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Cys Ser Met He Leu Gin He Ser Xaa Arg Leu Arg Asp Gly Thr Gin 1 5 10 15 Val He Lys Met Lys Ala Leu He Phe Ala Ala Ala Gly Leu Leu Leu 20 25 30 Leu Leu Pro Thr Phe Cys Gin Ser Gly Met Glu Asn Asp Thr Asn Asn 35 40 45 Leu Ala Lys Pro Thr Leu Pro He Lys Thr Phe Arg Gly Ala Pro Pro 50 55 60 Asn Ser Phe Glu Glu Phe Pro Phe Ser Ala Leu Glu Gly Trp Thr Gly 65 70 75 80 Ala Thr He Thr Val Lys He Lys Cys Pro Glu Glu Ser Ala Ser His 85 90 95 Leu His Val Lys Asn Ala Thr Met Gly Tyr Leu Thr Ser Ser Leu Ser 100 105 110 Thr Lys Leu He Pro Ala He Tyr Leu Leu Val Phe Val Val Gly Val 115 120 125 Pro Ala Asn Ala Val Thr Leu Trp Met Leu Phe Phe Arg Thr Arg Ser 130 135 140 He Cys Thr Thr Val Phe Tyr Thr Asn Leu Ala He Ala Asp Phe Leu 145 150 155 160 Phe Cys Val Thr Leu Pro Phe Lys He Ala Tyr His Leu Asn Gly Asn 165 170 175 Asn Trp Val Phe Gly Glu Val Leu Cys Arg Ala Thr Thr Val He Phe 180 185 190 Tyr Gly Asn Met Tyr Cys Ser He Leu Leu Leu Ala Cys He Ser He 195 200 205 Asn Arg Tyr Leu Ala He Val His Pro Phe Thr Tyr Arg Gly Leu Pro 210 215 220 Lys His Thr Tyr Ala Leu Val Thr Cys Gly Leu Val Trp Ala Thr Val 225 230 235 240 Phe Leu Tyr Met Leu Pro Phe Phe He Leu Lys Gin Glu Tyr Tyr Leu 245 250 255 Val Gin Pro Asp He Thr Thr Cys His Asp Val His Asn Thr Cys Glu 260 265 270 Ser Ser Ser Pro Phe Gin Leu Tyr Tyr Phe He Ser Leu Ala Phe Phe 275 280 285 Gly Phe Leu He Pro Phe Val Leu He He Tyr Cys Tyr Ala Ala He 290 295 300 He Arg Thr Leu Asn Ala Tyr Asp His Arg Trp Leu Trp Tyr Val Lys 305 310 315 320 Ala Ser Leu Leu He Leu Val He Phe Thr He Cys Phe Ala Pro Ser 325 330 335 Asn He He Leu He He His His Ala Asn Tyr Tyr Tyr Asn Asn Thr 340 345 350 Asp Gly Leu Tyr Phe He Tyr Leu He Ala Leu Cys Leu Gly Ser Leu 355 360 365 Asn Ser Cys Leu Asp Pro Phe Leu Tyr Phe Leu Met Ser Lys Thr Arg 370 375 380 Asn His Ser Thr Ala Tyr Leu Thr Lys Xaa Xaa Asn Asp Leu Arg Glu 385 390 395 400 Gin Gly Gin Pro Ser Gin Arg Thr 405 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 425 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7: Met Gly Pro Arg Arg Leu Leu Leu Val Ala Ala Cys Phe Ser Leu Cys 1 5 10 15 Gly Pro Leu Leu Ser Ala Arg Thr Arg Ala Arg Arg Pro Glu Ser Lys 20 25 30 Ala Thr Asn Ala Thr Leu Asp Pro Arg Ser Phe Leu Leu Arg Asn Pro 35 40 45 Asn Asp Lys Tyr Glu Pro Phe Trp Glu Asp Glu Glu Lys Asn Glu Ser 50 55 60 Gly Leu Thr Glu Tyr Arg Leu Val Ser He Asn Lys Ser Ser Pro Leu 65 70 75 80 Gin Lys Gin Leu Pro Ala Phe He Ser Glu Asp Ala Ser Gly Tyr Leu 85 90 95 Thr Ser Ser Trp Leu Thr Leu Phe Val Pro Ser Val Tyr Thr Gly Val 100 105 110 Phe Val Val Ser Leu Pro Leu Asn He Met Ala He Val Val Phe He 115 120 125 Leu Lys Met Lys Val Lys Lys Pro Ala Val Val Tyr Met Leu His Leu 130 135 140 Ala Thr Ala Asp Val Leu Phe Val Ser Val Leu Pro Phe Lys He Ser 145 150 155 160 Tyr Tyr Phe Ser Gly Ser Asp Trp Gin Phe Gly Ser Glu Leu Cys Arg 165 170 175 Phe Val Thr Ala Ala Phe Tyr Cys Asn Met Tyr Ala Ser He Leu Leu 180 185 190 Met Thr Val He Ser He Asp Arg Phe Leu Ala Val Val Tyr Pro Met 195 200 205 Gin Ser Leu Ser Trp Arg Thr Leu Gly Arg Ala Ser Phe Thr Cys Leu 210 215 220 Ala He Trp Ala Leu Ala He Ala Gly Val Val Pro Leu Val Leu Lys 225 230 235 240 Glu Gin Thr He Gin Val Pro Gly Leu Asn He Thr Thr Cys His Asp 245 250 255 Val Leu Asn Glu Thr Leu Leu Glu Gly Tyr Tyr Ala Tyr Tyr Phe Ser 260 265 270 Ala Phe Ser Ala Val Phe Phe Phe Val Pro Leu He He Ser Thr Val 275 280 285 Cys Tyr Val Ser He He Arg Cys Leu Ser Ser Ser Ala Val Ala Asn 290 295 300 Arg Ser Lys Lys Ser Arg Ala Leu Phe Leu Ser Ala Ala Val Phe Cys 305 310 315 320 He Phe He He Cys Phe Gly Pro Thr Asn Val Leu Leu He Ala His 325 330 335 Tyr Ser Phe Leu Ser His Thr Ser Thr Thr Glu Ala Ala Tyr Phe Ala 340 345 350 Tyr Leu Leu Cys Val Cys Val Ser Ser He Ser Ser Cys He Asp Pro 355 360 365 Leu He Tyr Tyr Tyr Ala Ser Ser Glu Cys Gin Arg Tyr Val Tyr Ser 370 375 380 He Leu Cys Cys Lys Glu Ser Ser Asp Pro Ser Ser Tyr Asn Ser Ser 385 390 395 400 Gly Gin Leu Met Ala Ser Lys Met Asp Thr Cys Ser Ser Asn Leu Asn 405 410 415 Asn Ser He Tyr Lys Lys Leu Leu Thr 420 425 (2) INFORMATION FOR SEQ ID N0:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 394 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8: Met Arg Ser Pro Ser Ala Ala Trp Leu Leu Gly Ala Ala He Leu Leu 1 5 10 15 Ala Ala Ser Leu Ser Cys Ser Gly Thr He Gin Gly Thr Asn Arg Ser 20 25 30 Ser Lys Gly Arg Ser Leu He Gly Lys Val Asp Gly Thr Ser His Val 35 40 45 Thr Gly Lys Gly Val Thr Val Glu Thr Val Phe Ser Val Asp Glu Phe 50 55 60 Ser Ala Ser Val Leu Thr Gly Lys Leu Thr Thr Val Phe Leu Pro He 65 70 75 80 Val Tyr Thr He Val Phe Val Val Gly Leu Pro Ser Asn Gly Met Ala 85 90 95 Leu Trp Val Phe Leu Phe Arg Thr Lys Lys Lys His Pro Ala Val He 100 105 110 Tyr Met Ala Asn Leu Ala Leu Ala Asp Leu Leu Ser Val He Trp Phe 115 120 125 Pro Leu Lys He Ala Tyr His He His Gly Asn Asn Trp He Tyr Gly 130 135 140 Glu Ala Leu Cys Asn Val Leu He Gly Phe Phe Tyr Gly Asn Met Tyr 145 150 155 160 Cys Ser He Leu Phe Met Thr Cys Leu Ser Val Gin Arg Tyr Trp Val 165 170 175 He Val Asn Pro Met Gly His Ser Arg Lys Lys Ala Asn He Ala He 180 185 190 Gly He Ser Leu Ala He Trp Leu Leu He Leu Leu Val Thr He Pro 195 200 205 Leu Tyr Val Val Lys Gin Thr He Phe He Pro Ala Leu Asn He Thr 210 215 220 Thr Cys His Asp Val Leu Pro Glu Gin Leu Leu Val Gly Asp Pro Phe 225 230 235 240 Leu Ser Leu Ala He Gly Val Phe Leu Phe Pro Ala Phe Leu Thr Ala 245 250 255 Ser Ala Tyr Val Leu Met He Arg Met Leu Arg Ser Ser Ala Met Asp 260 265 270 Glu Asn Ser Glu Lys Lys Arg Lys Arg Ala He Lys Leu He Val Thr 275 280 285 Val Leu Ala Met Tyr Leu He Cys Phe Thr Pro Ser Asn Leu Leu Leu 290 295 300 Val Val His Tyr Phe Leu He Lys Ser Gin Gly Gin Ser His Val Tyr 305 310 315 320 Ala Leu Tyr He Val Ala Leu Cys Leu Ser Thr Leu Asn Ser Cys He 325 330 335 Asp Pro Phe Val Tyr Tyr Phe Val Ser His Asp Phe Arg Asp His Ala 340 345 350 Lys Asn Ala Leu Leu Cys Arg Ser Val Arg Thr Val Lys Gin Met Gin 355 360 365 Val Ser Leu Thr Ser Lys Lys His Ser Arg Lys Ser Ser Ser Tyr Ser 370 375 380 Ser Ser Ser Thr Thr Val Lys Thr Ser Tyr 385 390 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9: Asp Phe Glu Glu He Pro Glu Glu Tyr Leu Gin 1 5 10 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...29 (D) OTHER INFORMATION: N=Inosine at residues 3, 12, 18, 21, and 24 (A) NAME/KEY: Other (B) LOCATION: 22...27 (D) OTHER INFORMATION: N=A or C or G or T at residues 22 and 27 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: GTNTACATGC TNMACYTNGC NNTNGCNGA 29 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 6...21 (D) OTHER INFORMATION: N=Inosine at residues 6, 9, 12, 15, and 21 (A) NAME/KEY: Other (B) LOCATION: 24 (D) OTHER INFORMATION: N=A or C or G or T at residue 24 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GGATANACNA CNGCNADRWA NCKNTC 26 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Asp Tyr Lys Asp Asp Asp 1 5 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Met Asp Ser Lys Gly Ser Ser Gin Lys Gly Ser Arg Leu Leu Leu Leu 1 5 10 15 Leu Val Val Ser Asn Leu Leu Leu Cys Gin Gly Val Val Ser Asp Tyr 20 25 30 Lys Asp Asp Asp Asp Val Glu 35 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:14: Phe Glu Glu Phe Pro 1 5 (2) INFORMATION FOR SEQ ID N0:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: Leu Thr Pro Lys 1 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Thr Phe Arg Gly Ala Pro 1 5 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:17: Thr Phe Arg Gly Ala Pro Pro Asn Ser 1 5 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:18: Leu Pro He Lys Thr Phe Arg Gly Ala Pro Pro Asn Ser Phe Glu Glu 1 5 10 15 Phe Pro Phe Ser Ala Leu Glu 20 (2) INFORMATION FOR SEQ ID N0:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: Leu Pro He Lys Pro Phe Arg G ly Ala Pro Pro Asn Ser Phe Glu Glu 1 5 10 15 Phe Pro Phe Ser Ala Leu G lu 20 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: Leu Pro He Xaa Thr Pro Phe Arg Gly Ala Pro Pro Asn Ser Phe Glu 1 5 10 15 G lu Phe Pro Phe Ser Ala Leu G lu 20 (2) INFORMATION FOR SEQ ID N0:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Xaa Pro Arg Pro Phe Arg Gly Ala Pro Pro Asn Ser Phe Glu Glu Phe 1 5 10 15 Pro Phe Ser Ala Leu Glu 20 (2) INFORMATION FOR SEQ ID N0:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Leu Pro I le Lys 1 (2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Thr Phe Arg Gly Ala Pro Pro Asn Ser 1 5

Claims: CLAIMS That which is claimed is: 1. Substantially pure DNA encoding a protease- activated receptor 3. 2. The DNA of claim 1, wherein the DNA is mammalian. 3. Substantially pure DNA having the nucleotide sequence selected from the group consisting of Fig. 1 (SEQ ID N0:1), or degenerate variants thereof, and encoding the amino acid sequence of Fig. 1 (SEQ ID NO : 3 ) ; Fig. 2 (SEQ ID NO : 2 ) , or degenerate variants thereof encoding an amino acid sequence comprising the amino acid sequence of Fig. 1 (SEQ ID NO:3); Fig. 3 (SEQ ID NO:4), or degenerate variants thereof encoding the amino acid sequence of Fig. 2 (SEQ ID NO:6); and Fig. 4 (SEQ ID NO: 5), or degenerate variants thereof encoding an amino acid sequence comprising the amino acid sequence of Fig. 3 (SEQ ID NO: 6) . 4. Substantially pure DNA having 50% or greater sequence identity to the DNA sequence of a sequence selected from the group consisting of SEQ ID NO : 1 , SEQ ID NO:2, SEQ ID NO : 4 and SEQ ID NO : 5 and which hybridizes to the DNA sequence SEQ ID NO:l, SEQ ID NO : 2 , SEQ ID NO : 3 , or SEQ ID NO : 5 , respectively. 5. An isolated protease-activated receptor 3 protein. 6. The substantially pure protein of claim 5 having an amino acid sequence selected from the group consisting of the sequence shown in Fig. 1 (SEQ ID NO: 3) . and the sequence shown in Fig. 2 (SEQ ID NO: 6) . 7. A substantially pure polypeptide having an amino acid sequence which is at least 80% identical to an amino acid sequence selected from the group consisting of the sequence shown in Fig. 1 (SEQ ID NO: 3) and the sequence shown in Fig. 2 (SEQ ID NO: 6), wherein a) said polypeptide is activated by thrombin; and b) said polypeptide mediates phosphoinositide hydrolysis in a cell expressing said polypeptide on its surface. 8. A substantially pure polypeptide which is a fragment or analog of a protease-activated receptor 3 comprising a domain capable of activation by thrombin and mediating phosphoinositide hydrolysis. 9. A vector comprising the DNA of claim 1. 10. A cell comprising the vector of claim 9. 11. An assay device, comprising: a support surface; and a cell of claim 10. 12. The assay device of claim 11, wherein the cell is bound to the support surface or present in a suspension on the support surface. 13. A method of testing a candidate compound for its ability to act as an agonist of a protease-activated receptor 3 ligand, the method comprising: a) contacting a candidate compound with a cell which expresses on its surface a recombinant protease- activated receptor 3 protein or biologically active fragment or analog thereof; b) measuring PAR3 -mediated response of the cell; and c) identifying the candidate compound as an agonist wherein the contacting causes a substantial increase in PAR3 -mediated response. 14. A method of testing a candidate compound for the ability to act as an antagonist of a protease- activated receptor 3 ligand, the method comprising: a) contacting in the presence of a protease- activated receptor agonist a candidate compound with a cell which expresses on its surface a recombinant protease-activated receptor 3 protein or biologically active fragment or analog thereof; b) measuring PAR3 -mediated response of the cell; and c) identifying the candidate compound as an antagonist wherein the contacting causes a substantial decrease in PAR3 -mediated response relative to PAR3- mediated response in the absence of the candidate antagonist . 15. The method of claim 14, wherein the cell is a mammalian cell which normally presents substantially no protease-activated receptor 3 on its surface, the PAR3 - mediated response measured in intracellular phosphoinositide hydrolysis in the cell. 16. The method of claim 14, further comprising: mixing thrombin with platelets and the identified candidate compound; and observing the effect of the candidate compound on mediating platelet aggregation. 17. A therapeutic composition, comprising: a protease-activated receptor 3 ligand agonist; and physiologically-acceptable carrier , 18. A therapeutic composition, comprising: a protease-activated receptor 3 ligand antagonist; and a physiologically-acceptable carrier. 19. The composition of claim 18, wherein the antagonist is selected from the group consisting of: (1) the isolated sequence LPIKTFRGAPPNSFEEFPFSALE ; (2) uncleavable thrombin inhibitor LPIKPFRGAPPNSFEEFPFSALE where the PAR 3 cleavage site PI' is muted to block cleavage; (3) uncleavable thrombin inhibitor LPl (hR) TFRGAPPNSFEEFPFSALE where the PAR 3 cleavage site PI is mutated to block cleavage; hR is beta-homoarginine (the extra methylene group is in the main chain) ; (4) uncleavable thrombin inhibitor (dF) PRPFRGAPPNSFEEFPFSALE where the good active site binding sequence dFPR is substituted for LPIK; dF is D-Phenylalanine; (5) any of (l)-(4) above where all or part of the sequence TFRGAPPNS is replaced with spacer sequences such as GGG; (6) variations and combinations of (1) - (5) which act as antagonists. 20. A method of treatment, comprising: administering to a patient a therapeutically effective amount of the composition of claim 18.

Inventors: CONNOLLY ANDREW, COUGHLIN SHAUN R, ISHIHARA HIROAKI, CONNOLLY, ANDREW, COUGHLIN, SHAUN, R., ISHIHARA, HIROAKI
Applicants: UNIV CALIFORNIA, THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Application year: 1997
Main classification: A61K31
IPC codes: A61K38, A61K45, A61P7, C07K14, C12N1, C12N5, C12N15, C12P21, C12Q1, G01N33