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Shaun R. Coughlin - One of the best experts on this subject based on the ideXlab platform.
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protease activated Receptor 3 is a second Thrombin Receptor in humans
Nature, 1997Co-Authors: Hiroaki Ishihara, Andrew J Connolly, Dewan Zeng, Mark L Kahn, Yaowu Zheng, Courtney Timmons, Tracy Tram, Shaun R. CoughlinAbstract:Thrombin is a coagulation protease that activates platelets, leukocytes, endothelial and mesenchymal cells at sites of vascular injury, acting partly through an unusual proteolytically activated G-protein-coupled Receptor1–3. Knockout of the gene encoding this Receptor provided definitive evidence for a second Thrombin Receptor in mouse platelets and for tissue-specific roles for different Thrombin Receptors4. We now report the cloning and characterization of a new human Thrombin Receptor, designated protease-activated Receptor 3 (PAR3). PAR3 can mediate throm-bin-triggered phosphoinositide hydrolysis and is expressed in a variety of tissues, including human bone marrow and mouse megakaryocytes, making it a candidate for the sought-after second platelet Thrombin Receptor. PAR3 provides a new tool for understanding Thrombin signalling and a possible target for therapeutics designed selectively to block thrombotic, inflammatory and proliferative responses to Thrombin.
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The Platelet Thrombin Receptor
The Platelet, 1997Co-Authors: Shaun R. CoughlinAbstract:Publisher Summary This chapter discusses the structure and function of Thrombin Receptor and their biological roles of the cloned Thrombin Receptor. The Thrombin Receptor's deduced amino acid sequence revealed it to be a member of the seven transmembrane domain Receptor family. Its primary sequence is most closely related to the Receptors for neuropeptides, glycoprotein hormones, and proinflammatory mediators such as C5a and IL8. The Receptor sequence reveals five consensus N-linked glycosylation sites in regions predicted to be extracellular. Recent unpublished studies from our group demonstrated that the Thrombin Receptor is rapidly phosphorylated upon activation, probably as part of the mechanisms for terminating Receptor signaling. Mapping of specific phosphorylation sites remains to be accomplished. Synthetic peptides mimicking the Thrombin Receptor's agonist peptide domain and the Receptor cDNA itself have provided new tools for defining the role of the cloned Receptor both in intracellular signaling events and in various Thrombin-induced cellular functions. For the purpose of this chapter, discussion of the Thrombin Receptor's role in cellular events will be largely confined to platelets.
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role of the Thrombin Receptor in development and evidence for a second Receptor
Nature, 1996Co-Authors: Andrew J Connolly, Hiroaki Ishihara, Mark L Kahn, Robert V Farese, Shaun R. CoughlinAbstract:Thrombin, a coagulation protease generated at sites of vascular injury, activates platelets, endothelial cells, leukocytes and mesenchymal cells. A G-protein-coupled Receptor that is proteolytically activated by Thrombin is a target for drug development aimed at blocking thrombosis, inflammation and proliferation. Here we show that although disruption of the Thrombin Receptor (tr) gene in mice causes about half of the tr-/- embryos to die at embryonic day 9-10, half survive to become grossly normal adult mice with no bleeding diathesis. Strikingly, tr-/- platelets respond strongly to Thrombin, whereas tr-/- fibroblasts lose their ability to respond to Thrombin. We conclude that the Thrombin Receptor plays an unexpected role in embryonic development, suggesting a possible new function for the 'coagulation' proteases themselves. Moreover, a second platelet Thrombin Receptor exists, and different Thrombin Receptors have tissue-specific roles. This may allow development of therapeutics that will selectively block Thrombin's different cellular actions.
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Determinants of Thrombin Receptor cleavage. Receptor domains involved, specificity, and role of the P3 aspartate.
The Journal of biological chemistry, 1995Co-Authors: Kenji Ishii, Yaowu Zheng, Robert E. Gerszten, John B. Welsh, Christoph W. Turck, Shaun R. CoughlinAbstract:Abstract Thrombin Receptor cleavage at the Arg--Ser peptide bond in the Receptor's amino-terminal exodomain is necessary and sufficient for Receptor activation. The rate of Receptor cleavage at this site is a critical determinant of the magnitude of the cellular response to Thrombin. These observations underscore the importance of defining the molecular basis for Thrombin-Receptor interaction and cleavage. We report that chimeric proteins bearing only Thrombin Receptor amino-terminal exodomain residues 36-60 are cleaved at rates similar to the wild-type Thrombin Receptor when expressed on the cell surface. A soluble amino-terminal exodomain protein was also cleaved efficiently by Thrombin with a K of 15-30 μM and k of approximately 50 s, with cleavage occurring only at the Arg--Ser peptide bond. In the context of previous studies, these data suggest that the Receptor's LDPR cleavage recognition sequence and DKYEPF hirudin-like domain account for Thrombin-Receptor interaction. Because a P3 aspartate in protein C's cleavage site inhibits cleavage by free Thrombin, we investigated the role of the P3 aspartate in the Receptor's LDPR sequence. Studies with mutant Receptors revealed an inhibitory role for this residue only in the absence of the Receptor's hirudin-like domain. These and other data suggest that the Receptor's hirudin-like domain causes a conformational change in Thrombin's active center to accommodate the LDPR sequence and promote efficient Receptor cleavage. Taken together, these studies imply that the Thrombin Receptor's amino-terminal exodomain contains all the machinery needed for efficient recognition and cleavage by Thrombin. Thrombin appears to bind and cleave this domain independently of the rest of the Receptor, with one Thrombin molecule probably activating multiple Receptors.
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Specificity of the Thrombin Receptor for agonist peptide is defined by its extracellular surface
Nature, 1994Co-Authors: Robert E. Gerszten, Ji Chen, Christoph W. Turck, Maki Ishli, Kenji Ishil, Ling Wang, Tania Nanevicz, Shaun R. CoughlinAbstract:G-PROTEIN-COUPLED Receptors for catecholamines and some other small ligands are activated when agonists bind to the transmem-brane region of the Receptor1. The docking interactions through which peptide agonists activate their Receptors are less well characterized2–7. The Thrombin Receptor is a specialized peptide Receptor. It is activated by binding its tethered ligand domain, which is unmasked upon Receptor cleavage by Thrombin8,9. Human and Xenopus Thrombin Receptor homologues are each selectively activated by the agonist peptide representing their respective tethered ligand domains. Here we identify Receptor domains that confer this agonist specificity by replacing the Xenopus Receptor's amino-terminal exodomain and three extracellular loops with the corresponding human structures. This switches Receptor specificity from Xenopus to human. The specificity of these Thrombin Receptors for their respective peptide agonists is thus determined by their extracellular surfaces. Our results indicate that agonist interaction with extracellular domains is important for Thrombin Receptor activation.
S M Seiler - One of the best experts on this subject based on the ideXlab platform.
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Development of potent Thrombin Receptor antagonist peptides
Journal of medicinal chemistry, 1996Co-Authors: Michael S Bernatowicz, Clifford E Klimas, Karen S Hartl, Marianne Peluso, Nick J Allegretto, S M SeilerAbstract:A peptide-based structure−activity study is reported leading to the discovery of novel potent Thrombin Receptor antagonists. Systematic substitution of nonproteogenic amino acids for the second and third residues of the human Thrombin Receptor “tethered ligand” sequence (SFLLR) led to a series of agonists with enhanced potency. The most potent pentapeptide agonist identified was Ser-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-NH2, 9 (EC50 ∼ 0.04 μM for stimulation of human platelet aggregation, ∼10-fold more potent than the natural pentapeptide). Systematic substitution of the NH2-terminal Ser in 9 with neutral hydrophobic NH2-acyl groups led to partial agonists and eventually antagonists with unprecedented potency (greater than 1000-fold increase over the previously reported antagonist 3-mercaptopropionyl-Phe-Cha-Cha-Arg-Lys-Pro-Asn-Asp-Lys-NH2). In the series of NH2-acyl tetrapeptide antagonists, N-trans-cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-NH2, 41 (BMS-197525), was identified as the tightest binding (IC...
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development of potent Thrombin Receptor antagonist peptides
Journal of Medicinal Chemistry, 1996Co-Authors: Michael S Bernatowicz, Clifford E Klimas, Karen S Hartl, Marianne Peluso, Nick J Allegretto, S M SeilerAbstract:A peptide-based structure−activity study is reported leading to the discovery of novel potent Thrombin Receptor antagonists. Systematic substitution of nonproteogenic amino acids for the second and...
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Thrombin Receptor antagonists.
Seminars in thrombosis and hemostasis, 1996Co-Authors: S M SeilerAbstract:Thrombin's proteolytically activated "tethered-ligand" Receptor is widely expressed and mediates many of Thrombin's actions on cells. Its central role in Thrombin-stimulated human platelet activation and vascular smooth muscle proliferation as well as location in atherosclerotic plaques suggests Receptor involvement in arterial thrombosis and atherosclerosis. Thrombin Receptor antagonists, should they be effective, could be more selective than Thrombin active site inhibitors in antithrombotic therapy as well as other indications. Blocking antibodies to peptides derived from the Thrombin Receptor have been used as prototypical Thrombin Receptor antagonists in vitro and have been useful in implicating this Receptor in Thrombin's actions on a variety of cell types. These antibodies have also shown the involvement of the Receptor in arterial thrombosis models in nonhuman primates. Amino acid substitution studies have shown the structural requirements for Receptor activation of peptides homologous to the new NH2-terminus. Peptide-based partial agonists and antagonists have been synthesized by NH2-terminal replacements of the serine in the Receptor activating peptides. Current Thrombin Receptor antagonists lack potency and some are partial agonists; however, it is expected that more potent compounds will result from further investigation. The potency limitations are important to overcome before serious evaluation of their efficacy can be determined.
Claude R. Benedict - One of the best experts on this subject based on the ideXlab platform.
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A peptide analogue of Thrombin Receptor-activating peptide inhibits Thrombin and Thrombin-Receptor-activating peptide-induced vascular smooth muscle cell proliferation.
Journal of cardiovascular pharmacology, 2001Co-Authors: Rajbabu Pakala, Chyou T. Liang, Claude R. BenedictAbstract:The serine protease Thrombin, in addition to its pivotal role in the coagulation cascade, plays an important role in the development of atherosclerosis and restenosis by inducing smooth cell proliferation. Thrombin exerts its cellular effects mainly by cleaving its own Receptor, leaving a new NH 2 -terminus that can act as a tethered ligand to activate the Thrombin Receptor. Peptides derived from the new NH 2 -terminus are able to fully activate Thrombin Receptor and mimic cellular effects of Thrombin. Peptides with structural similarities to the tethered ligand have been tested for their ability to prevent Thrombin- and tethered ligand-induced platelet aggregation and thrombus formation. We synthesized a peptide with multiple alanine substitutions in both critical and noncritical residues of tethered ligand that specifically inhibited platelet aggregation induced by Thrombin and Thrombin Receptor-activating peptide and prevented thrombus formation in a rabbit thrombosis model. In the present study we demonstrate that this peptide inhibited only Thrombin- and tethered ligand-induced human vascular smooth muscle cell proliferation as determined by ( 3 H)-thymidine incorporation and has no effect on platelet-derived growth factor and serum-induced smooth muscle cell proliferation. The inhibitory effect of this peptide is dependent on the concentration of the antagonist used and length of preincubation time. The possible mechanism by which this peptide exerts its inhibitory effect may by desensitizing the Thrombin Receptor. The results of the present study suggest that apart from being antithrombotic, tethered ligand antagonist peptides can also act as antiatherosclerotic or antirestenotic agents.
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Inhibition of arterial thrombosis by a peptide ligand of the Thrombin Receptor.
Thrombosis research, 2000Co-Authors: Rajbabu Pakala, Chyou T. Liang, Claude R. BenedictAbstract:Abstract Thrombin plays an important role in promoting arterial thrombosis by platelet activation and by catalyzing fibrin formation. Use of Thrombin inhibitors that block both the platelet-activating and fibrin formation properties of Thrombin are associated with hemostasis. This problem might be overcome by developing agents that block only the platelet-activating property of Thrombin. Because the platelet-activating property of Thrombin is mediated by the Thrombin Receptor, antagonists of the Thrombin Receptor might be efficacious and potentially safer with regard to bleeding complications. We investigated whether a peptide ligand (AFLARAA) of the Thrombin Receptor that blocked α-Thrombin and Thrombin Receptor activating peptide-induced platelet aggregation could inhibit thrombosis. A partially occlusive thrombus was generated by application of electric current in rabbit carotid artery. In control animals, the artery was completely occluded within 42±12 min after the current was discontinued. When the Thrombin Receptor activating peptide antagonist was given (100 μmol/kg as an IV bolus followed by 900 μmol/kg infusion for a period of 180 min) starting at the time the current was stopped, blood flow remained patent throughout the infusion period and for an additional 60 min after the infusion was stopped. The antithrombotic effect of the antagonist peptide was not associated with increased bleeding tendency, as judged by the amount of blood adsorbed by a gauze pad placed in a surgical incision extending to the muscle tissue and by a standard template bleeding time. These results indicate that Thrombin Receptor antagonist peptides can be used as antithrombotic agents.
Kenji Ishii - One of the best experts on this subject based on the ideXlab platform.
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Determinants of Thrombin Receptor cleavage. Receptor domains involved, specificity, and role of the P3 aspartate.
The Journal of biological chemistry, 1995Co-Authors: Kenji Ishii, Yaowu Zheng, Robert E. Gerszten, John B. Welsh, Christoph W. Turck, Shaun R. CoughlinAbstract:Abstract Thrombin Receptor cleavage at the Arg--Ser peptide bond in the Receptor's amino-terminal exodomain is necessary and sufficient for Receptor activation. The rate of Receptor cleavage at this site is a critical determinant of the magnitude of the cellular response to Thrombin. These observations underscore the importance of defining the molecular basis for Thrombin-Receptor interaction and cleavage. We report that chimeric proteins bearing only Thrombin Receptor amino-terminal exodomain residues 36-60 are cleaved at rates similar to the wild-type Thrombin Receptor when expressed on the cell surface. A soluble amino-terminal exodomain protein was also cleaved efficiently by Thrombin with a K of 15-30 μM and k of approximately 50 s, with cleavage occurring only at the Arg--Ser peptide bond. In the context of previous studies, these data suggest that the Receptor's LDPR cleavage recognition sequence and DKYEPF hirudin-like domain account for Thrombin-Receptor interaction. Because a P3 aspartate in protein C's cleavage site inhibits cleavage by free Thrombin, we investigated the role of the P3 aspartate in the Receptor's LDPR sequence. Studies with mutant Receptors revealed an inhibitory role for this residue only in the absence of the Receptor's hirudin-like domain. These and other data suggest that the Receptor's hirudin-like domain causes a conformational change in Thrombin's active center to accommodate the LDPR sequence and promote efficient Receptor cleavage. Taken together, these studies imply that the Thrombin Receptor's amino-terminal exodomain contains all the machinery needed for efficient recognition and cleavage by Thrombin. Thrombin appears to bind and cleave this domain independently of the rest of the Receptor, with one Thrombin molecule probably activating multiple Receptors.
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Thrombin Receptor activation confirmation of the intramolecular tethered liganding hypothesis and discovery of an alternative intermolecular liganding mode
Journal of Biological Chemistry, 1994Co-Authors: Ji Chen, Maki Ishii, Kenji Ishii, Ling Wang, S. R. CoughlinAbstract:Cleavage of the Thrombin Receptor's amino-terminal exodomain at the Arg41/Ser42 peptide bond within the sequence ... LDPR41/S42FLLRN ... is necessary and sufficient for Receptor activation by proteases. The synthetic peptide SFLLRN activates the Receptor independent of proteolysis. We proposed that the SFLLRN sequence is a tethered peptide ligand; Receptor cleavage unmasks this agonist which then binds intramolecularly to effect Receptor activation. The alternative hypothesis that Receptor cleavage or exogenous SFLLRN effect Receptor activation by disrupting tonic inhibitory interactions exerted by the Receptor's amino-terminal exodomain has not been excluded. We report that delta AMINO, a mutant Thrombin Receptor lacking the amino-terminal exodomain, was not constitutively active and responded to SFLLRN but not Thrombin when expressed in Xenopus oocytes or mammalian cells. Thrombin signaling was restored when delta AMINO was co-expressed with ATE-CD8 which encoded the Receptor's amino-terminal exodomain fused to the transmembrane domain of CD8. Co-expression of a Thrombin Receptor lacking a functional tethered ligand domain ("F43A") with a non-signaling Receptor mutant bearing an intact tethered ligand domain ("YYY") also reconstituted Thrombin signaling. However, the EC50 for Thrombin activation of cells co-expressing F43A and YYY was > 1000-fold that for cells expressing comparable levels of wild type Receptor, while EC50s for activation by SFLLRN were similar. These and other data refute the release from inhibition hypothesis and suggest that while intermolecular liganding between two Thrombin Receptor molecules can occur, the intramolecular tethered liganding mechanism is the predominant mode of Thrombin Receptor activation.
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Inhibition of Thrombin Receptor signaling by a G-protein coupled Receptor kinase. Functional specificity among G-protein coupled Receptor kinases.
Journal of Biological Chemistry, 1994Co-Authors: Kenji Ishii, Neil J. Freedman, Maki Ishii, Walter J Koch, Robert J Lefkowitz, Ji Chen, S. R. CoughlinAbstract:Abstract The Thrombin Receptor, a member of the seven membrane-spanning superfamily of G-protein coupled Receptors, is activated by an irreversible proteolytic mechanism, but signaling by activated Thrombin Receptors shuts off soon after Receptor activation. This shut-off mechanism is thought to be required for concentration-dependent responses to Thrombin and an important determinant of the cell's sensitivity to Thrombin. We report that the Thrombin Receptor is rapidly phosphorylated upon activation, consistent with the action of a G-protein-coupled Receptor kinase. Moreover, the G-protein coupled Receptor kinase BARK2 (beta-adrenergic Receptor kinase 2) blocked signaling by Thrombin Receptors coexpressed in Xenopus oocytes. In this system, rhodopsin kinase was inactive and BARK1 was markedly less effective than BARK2. Thrombin Receptor mutants which lacked potential serine and threonine phosphorylation sites in the Receptor's cytoplasmic tail were insensitive to inhibition by exogenous BARK2 but did confer concentration-dependent responses to Thrombin. Our studies demonstrate that a G-protein coupled Receptor kinase can shut off Thrombin Receptor signaling but that additional mechanism(s) for terminating signaling exist. These studies also reveal functional specificity among G-protein coupled Receptor kinases in a novel in vivo reconstitution system and show that heterologous expression of these kinases can be used to manipulate cellular responsiveness.
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Kinetics of Thrombin Receptor cleavage on intact cells. Relation to signaling.
The Journal of biological chemistry, 1993Co-Authors: Kenji Ishii, Lutz Hein, Brian K. Kobilka, Shaun R. CoughlinAbstract:Thrombin, a protease generated at sites of vascular injury, signals cellular responses vital for hemostasis and thrombosis. How Thrombin, an enzyme rather than a classical ligand, effects graded and concentration-dependent responses in its target cells has been a long-standing question. Thrombin activates its Receptor by cleaving off an activation peptide to unmask a tethered peptide ligand. We utilized a Thrombin Receptor with an epitope-tagged activation peptide to directly demonstrate Thrombin Receptor cleavage and to examine the kinetics of Receptor activation on intact cells. The rate of Thrombin Receptor cleavage was proportional to Thrombin concentration over the physiologic range, but low Thrombin concentrations ultimately cleaved and activated all Receptors. Cumulative phosphoinositide hydrolysis in response to Thrombin correlated precisely with cumulative Receptor cleavage. These data strongly suggest that each cleaved and activated Thrombin Receptor produces a "quantum" of phosphatidylinositol hydrolysis, then shuts off. Surprisingly, this shut off occurred despite the continued presence of cleaved and "activated" Receptors on the cell surface and at a time when the cells were refractory to Thrombin but sensitive to agonist peptide, suggesting that a novel shut off mechanism may have evolved to deal with the tethered ligand. Unlike the case with classical ligands, cells thus cannot detect differences in Thrombin concentrations as differences in fractional occupancy but rather must sense different rates of Receptor activation. Because each cleaved Thrombin Receptor generates a quantum of second messenger, the magnitude of the cell's response to Thrombin must be determined by the balance between rates of Receptor activation and second messenger clearance.
Marschall S. Runge - One of the best experts on this subject based on the ideXlab platform.
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Growth-related Responses in Arterial Smooth Muscle Cells Are Arrested by Thrombin Receptor Antisense Sequences
The Journal of biological chemistry, 1995Co-Authors: Elliot L. Chaikof, Rafael Caban, Chang-ning Yan, Gadiparthi N. Rao, Marschall S. RungeAbstract:Abstract The capacity of antisense sequences to the Thrombin Receptor to selectively inhibit Thrombin Receptor expression and limit mitogenic responses in vascular wall cells was investigated in vitro. Eight phosphorothioate oligodeoxynucleotides based on the sequences of the rat Thrombin Receptor (including sense, antisense, scrambled, and missense controls) were synthesized, characterized, and purified by high performance liquid chromatography. The antisense oligodeoxynucleotide (ODN 4) inhibitory effect was sequence-specific and both time- and concentration-dependent. A reduction in serum or α-Thrombin-induced smooth muscle cell (SMC) proliferation was noted as early as 3 days at 30 μM (82%; 6.17 ± 1.01 versus 34.08 ± 3.89 × 104 cells/well; p < 0.05) and at a dose as low as 15 μM after 4 days in culture (19%; p < 0.05). Nonspecific effects were enhanced after prolonged exposure of SMC to the antisense oligodeoxynucleotide (≥6 days). A reduction of inositol phosphate generation greater than 50% (p < 0.05) was detected after exposure of SMC to antisense but not to sense or scrambled nucleotide sequences. This was observed after stimulation with both Thrombin and SFFLRN (Thrombin Receptor peptide agonist). Northern blot analysis and enzyme-linked immunosorbent assays revealed 50 and 22% decreases, respectively, in Thrombin Receptor mRNA and protein (cell surface) levels in antisense oligonucleotide-treated (72 h) SMC as compared to untreated cells, suggesting that Thrombin Receptor down-regulation occurred at the pretranslational level. Thus, Thrombin Receptor-specific antisense sequences inhibit growthrelated effects both of serum and Thrombin on smooth muscle cells, potentially providing a new strategy for selective inhibition of Receptor-mediated arterial injury responses.
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Role of the Thrombin Receptor in restenosis and atherosclerosis
The American journal of cardiology, 1995Co-Authors: Demir Baykal, John F. Schmedtje, Marschall S. RungeAbstract:Abstract Thrombus generation is central to thrombosis at vascular lesion sites, including post-PCTA acute reocclusion and chronic restenosis. Thrombin stimulates platelet activation, monocyte and neutrophil chemotaxis, and endothelial production of prothrombotic factors. The varied physiologic effects of Thrombin are due to the widespread presence of Thrombin Receptors in many cell types. The Receptor is uniquely activated: Thrombin binds to the Receptor at the Thrombin anion-binding exosite, the Receptor ligand (“tethered ligand”) apparently being a sequence of 6 amino acids (SFLLRN). Thus, peptides corresponding to the sequence of the tethered ligand can stimulate almost all functions of native Thrombin itself. Several intracellular signaling pathways have been identified as important in the restenosis process: the G protein-related pathway, cyclic adenosine monophosphate (cAMP) mediator pathway, and tyrosine kinase activation pathway. In situ hybridization has demonstrated an increase in Thrombin Receptor mRNA throughout the period of neointimal and vascular lesion development. The mechanism of this increase is unknown, but may be mediated by multiple inflammatory modulators. Several strategies have been tested in animal models for inhibiting Thrombin: (1) Hirudin not only prevents Thrombin from cleaving fibrinogen, but also prevents Thrombin Receptor activation. (2) Thrombin Receptor antagonist peptides block platelet aggregation effects of Thrombin. (3) Mono- and polyclonal antibodies inhibit Thrombin Receptor activation. (4) Antisense oligonucleotides block Thrombin Receptor expression.
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Molecular cloning of the rat vascular smooth muscle Thrombin Receptor. Evidence for in vitro regulation by basic fibroblast growth factor.
The Journal of biological chemistry, 1992Co-Authors: Chizheng Zhong, David J. Hayzer, Marshall A. Corson, Marschall S. RungeAbstract:To study Thrombin's Receptor-mediated effects on vascular cells, we cloned and characterized a cDNA encoding a rat smooth muscle cell Thrombin Receptor. A rat aortic smooth muscle (RASM) cell cDNA library was screened with a 500-base pair (bp) sequence from the human Thrombin Receptor, obtained by polymerase chain reaction (PCR) amplification of cDNA synthesized from human erythropoietic leukemia (HEL) cell mRNA with PCR primers based on the published human Thrombin Receptor sequence. Clone pRTHR17 contains a 3418-bp insert that includes 50 bp of the 5'-untranslated region and the entire coding and 3'-untranslated regions of the RASM cell Thrombin Receptor. The sequence of pRTHR17 is 85% similar, at the nucleotide level, and 78% similar, at the deduced amino acid level, to the human Thrombin Receptor. Although the putative Thrombin cleavage and binding sites are present, there are significant differences between the rat and human Receptors in their amino-terminal sequences. Detectable signals (consisting of a single band of 3.45 kb) are present by Northern analysis of mRNA from RASM cells, and rat lung, kidney, and testes, but not in aorta or other tissues probed. The results of Southern analysis of rat genomic DNA are consistent with the existence of a single copy of the gene encoding this Receptor. The steady state Thrombin Receptor mRNA level is low in cultured growth-arrested RASM cells and not detectable in rat aorta. To determine whether regulation of the RASM cell Thrombin Receptor occurs under growth-stimulating conditions, growth-arrested RASM cells were treated with basic fibroblast growth factor (bFGF, recently proposed to be a major mitogen controlling vascular smooth muscle cell growth following injury (Lindner, V., and Reidy, M. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3739-3743)). There was a significant increase in Thrombin Receptor mRNA following the addition of bFGF. These data demonstrate that: 1) mRNA for a Thrombin Receptor similar to that reported from human megakaryocyte and hamster fibroblast cell lines is present in proliferating primary culture rat smooth muscle cells, 2) the most significant sequence differences are present in the amino-terminal tail of the Thrombin Receptor, and 3) the mRNA level for this Receptor is regulated under growth-stimulating conditions in vitro.