Heterogeneous mechanisms of endothelium-dependent relaxation for thrombin and peptide activators of protease-activated receptor-1 in porcine isolated coronary artery

1. Mechanisms of protease-activated receptor-1 (PAR1)- and PAR2-induced relaxation were investigated in pre-contracted porcine coronary artery ring preparations. 2. Thrombin (0.01 - 0.3 u ml(-1)) and the PAR1-activating peptide SFLLRN (0.1 - 10 microM) caused concentration- and endothelium-dependent relaxation. pEC(50)s (-log u ml(-1) for enzymes, -log M for peptides) and maximum relaxations (R(max), %) for thrombin were 1.8+/-0.1 and 93.5+/-2.8% respectively, and for SFLLRN 6.8+/-0.1 and 90.8+/-1.3%. Similar concentration- and endothelium-dependent relaxations occurred with trypsin (pEC(50) 2.3+/-0.2; R(max) 94.1+/-1.9%) and the PAR2-activating peptide SLIGRL (pEC(50) 6.5+/-0.2; R(max) 92.4+/-1.6%). 3. Relaxations to thrombin, SFLLRN, trypsin and SLIGRL were significantly inhibited (P<0.05) to similar extents by the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine (L-NOARG; 100 microM) and the NO scavenger oxyhaemoglobin (20 microM), both separately and in combination. 4. In the presence of the L-type voltage-operated calcium channel (L-VOCC) inhibitor nifedipine (0.3 microM), K(+) (67 mM) abolished the L-NOARG-resistant relaxations to thrombin, SFLLRN, trypsin and SLIGRL. However, nifedipine alone significantly (P<0.05) reduced the pEC(50) (1.5+/-0.1) and R(max) (77.5+/-7.0%) for thrombin but had no effect on relaxations to SFLLRN, trypsin or SLIGRL. Furthermore, L-NOARG-resistant relaxations to thrombin were abolished by nifedipine, whereas relaxations to SFLLRN, trypsin or SLIGRL were not further inhibited by combined treatment with nifedipine and L-NOARG, than they were with L-NOARG treatment alone. 5. Similar selective inhibition of the L-NOARG-resistant relaxation to thrombin, but not SFLLRN, occurred with verapamil (1 microM) and diltiazem (3 microM). 6. Our results suggest heterogeneous mechanisms in the NO-independent relaxation to thrombin and peptide activators of PAR1 in the porcine coronary artery.     

Inhibition of cellular action of thrombin by N3-cyclopropyl-7-[[4-(1-methylethyl)phenyl]methyl]-7H-pyrrolo[3, 2-f]quinazoline-1,3-diamine (SCH 79797), a nonpeptide thrombin receptor antagonist

A growing body of evidence suggests an important contribution of the cellular actions of thrombin to thrombosis and restenosis following angioplasty. Recently we reported on SCH 79797 (N3-cyclopropyl-7-?[4-(1-methylethyl)phenyl]methyl?-7H-pyrrolo[3, 2-f]quinazoline-1,3-diamine) and its analogs as new potent, nonpeptide thrombin receptor antagonists. This study further characterizes the biochemical and pharmacological actions of pyrroloquinazoline inhibitors of protease activated receptor-1 (PAR-1) in human platelets and coronary artery smooth muscle cells (hCASMC). SCH 79797 and its N-methyl analog (SCH 203099) inhibited binding of a high-affinity thrombin receptor-activating peptide ([(3)H]haTRAP, Ala-Phe(p-F)-Arg-ChA-HArg-[(3)H]Tyr-NH(2)) to PAR-1 with IC(50) values of 70 and 45 nM, respectively. SCH 79797 inhibited [(3)H]haTRAP binding in a competitive manner. SCH 79797 and SCH 203099 inhibited alpha-thrombin- and haTRAP-induced aggregation of human platelets, but did not inhibit human platelet aggregation induced by the tethered ligand agonist for protease-activated receptor-4 (PAR-4), gamma-thrombin, ADP, or collagen. SCH 203099 inhibited surface expression of P-selectin induced by haTRAP and thrombin, and it did not increase P-selectin expression or prevent thrombin cleavage of the receptor. Thrombin and TFLLRNPNDK-NH(2) (TK), a PAR-1-selective agonist, produced transient increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in hCASMC. This increase in [Ca(2+)](i) was inhibited effectively by SCH 79797. However, the Ca(2+) transients induced by SLIGKV-NH(2,) a PAR-2-selective agonist, were not inhibited by SCH 79797. Thrombin- and TK-stimulated [(3)H]thymidine incorporation also was inhibited completely by SCH 79797. The results of this study demonstrate that SCH 79797 and SCH 203099 are potent, selective antagonists of PAR-1 in human platelets and hCASMC. These data also suggest that the thrombin stimulation of Ca(2+) transients and mitogenesis in hCASMC is mediated primarily through activation of PAR-1.     

Proteinase-activated receptor-1 agonists attenuate nociception in response to noxious stimuli

Proteinase-activated receptor-1 (PAR-1) is activated by thrombin and can be selectively activated by synthetic peptides (PAR-1-activating peptide: PAR-1-AP) corresponding to the receptor's tethered ligand. PAR-1 being expressed by afferent neurons, we investigated the effects of PAR-1 agonists on nociceptive responses to mechanical and thermal noxious stimuli. Intraplantar injection of selective PAR-1-AP increased nociceptive threshold and withdrawal latency, leading to mechanical and thermal analgesia, while control peptide had no effect. Intraplantar injection of thrombin also showed analgesic properties in response to mechanical, but not to thermal stimulus. Co-injection of PAR-1-AP with carrageenan significantly reduced carrageenan-induced mechanical and thermal hyperalgesia, while thrombin reduced carrageenan-induced mechanical but not thermal hyperalgesia. The fact that thrombin is not a selective agonist for PAR-1 may explain the different effects of thrombin and PAR-1-AP. These results identified analgesic properties for selective PAR-1 agonists that can modulate nociceptive response to noxious stimuli in normal and inflammatory conditions.     

Human thrombin receptor-activating peptide-induced proliferation of cultured vascular smooth muscle cells exhibits species specificity

Thrombin receptor stimulation in vitro signals many cellular events that are associated with the response to vascular injury in vivo. Indeed, we have previously shown that human α-thrombin and the 14-amino acid human thrombin receptor-activating peptide (huTRAP-14) stimulate proliferation of cultured rat aortic smooth muscle cells (SMC). In the present studies, the mitogenic response of rabbit vascular SMC to thrombin and huTRAP-14 was assessed using [³H]thymidine incorporation and cell number. Results demonstrated that thrombin stimulated mitogenesis of rabbit vascular SMC in culture and that the thrombin response was dependent on proteolytic activity. However, huTRAP-14 was not mitogenic for rabbit vascular SMC. Thus, there are species differences in huTRAP-14 responsiveness. As rat and rabbit models continue to be used extensively to evaluate mechanisms and potential therapies for human restenosis, it is important to identify any species differences in the mechanism whereby thrombin exerts its biological effects. © 1995 Wiley-Liss, Inc.     

Evidence that PAR-1 and PAR-2 mediate prostanoid-dependent contraction in isolated guinea-pig gallbladder

We have investigated the ability of protease-activated receptor-1 (PAR-1), PAR-2, PAR-3 and PAR-4 agonists to induce contractile responses in isolated guinea-pig gallbladder. Thrombin, trypsin, mouse PAR-1 activating (SFLLRN-NH(2)) peptide, and mouse PAR-2 activating (SLIGRL-NH(2)) and human PAR-2 activating (SLIGKV-NH(2)) peptides produced a concentration-dependent contractile response. Mouse PAR-4 activating (GYPGKF-NH(2)) peptide, the mouse PAR-1 reverse (NRLLFS-NH(2)) peptide, the mouse PAR-2 reverse (LRGILS-NH(2)) and human PAR-2 reverse (VKGILS-NH(2)) peptides caused negligible contractile responses at the highest concentrations tested. An additive effect was observed following the contractile response induced by either trypsin or thrombin, with the addition of a different PAR agonist (SFLLRN-NH(2) and SLIGRL-NH(2), respectively). Desensitization to PAR-2 activating peptide attenuated the response to trypsin but failed to attenuate the response to PAR-1 agonists, and conversely desensitization to PAR-1 attenuated the response to thrombin but failed to alter contractile responses to PAR-2 agonists. The contractile responses produced by thrombin, trypsin, SFLLRN-NH(2) and SLIGRL-NH(2) were markedly reduced in the presence of the cyclo-oxygenase inhibitor, indomethacin, whilst the small contractile response produced by NRLLFS-NH(2) and LRGILS-NH(2) were insensitive to indomethacin. The contractile responses to thrombin, trypsin, SFLLRN-NH(2) and SLIGRL-NH(2) were unaffected by the presence of: the non-selective muscarinic antagonist, atropine; the nitric oxide synthase inhibitor, L-NAME; the sodium channel blocker, tetrodotoxin; the combination of selective tachykinin NK(1) and NK(2) receptor antagonists, (S)-1-[2-[3-(3,4-dichlorphenyl)-1 (3-isopropoxyphenylacetyl) piperidin-3-yl] ethyl]-4-phenyl-1 azaniabicyclo [2.2.2] octane chloride (SR140333) and (S)-N-methyl-N-[4-acetylamino-4-phenylpiperidino-2-(3, 4-dichlorophenyl)-butyl] benzamide (SR48968), respectively. The results indicate that PAR-1 and PAR-2 activation causes contractile responses in the guinea-pig gallbladder, an effect that is mediated principally by prostanoid release, and is independent of neural mechanisms.     

Activity of recombinant trypsin isoforms on human proteinase-activated receptors (PAR): mesotrypsin cannot activate epithelial PAR-1, -2, but weakly activates brain PAR-1

Trypsin-like serine proteinases trigger signal transduction pathways through proteolytic cleavage of proteinase-activated receptors (PARs) in many tissues. Three members, PAR-1, PAR-2 and PAR-4, are trypsin substrates, as trypsinolytic cleavage of the extracellular N terminus produces receptor activation. Here, the ability of the three human pancreatic trypsin isoforms (cationic trypsin, anionic trypsin and mesotrypsin (trypsin IV)) as recombinant proteins was tested on PARs.Using fura 2 [Ca(2+)](i) measurements, we analyzed three human epithelial cell lines, HBE (human bronchial epithelial), A549 (human pulmonary epithelial) and HEK (human embryonic kidney)-293 cells, which express functional PAR-1 and PAR-2. Human mesotrypsin failed to induce a PAR-mediated Ca(2+) response in human epithelial cells even at high concentrations. In addition, mesotrypsin did not affect the magnitude of PAR activation by subsequently added bovine trypsin. In HBE cells, which like A549 cells express high PAR-2 levels with negligible PAR-1 levels (<11%), half-maximal responses were seen for both cationic and anionic trypsins at about 5 nM. In the epithelial cells, mesotrypsin did not activate PAR-2 or PAR-1, whereas both anionic and cationic trypsins were comparable activators.We also investigated human astrocytoma 1321N1cells, which express PAR-1 and some PAR-3, but no PAR-2. High concentrations (>100 nM) of mesotrypsin produced a relatively weak Ca(2+) signal, apparently through PAR-1 activation. Half-maximal responses were observed at 60 nM mesotrypsin, and at 10-20 nM cationic and anionic trypsins.Using a desensitization assay with PAR-2-AP, we confirmed that both cationic and anionic trypsin isoforms cause [Ca(2+)](i) elevation in HBE cells mainly through PAR-2 activation. Desensitization of PAR-1 with thrombin receptor agonist peptide in 1321N1 cells demonstrated that all three recombinant trypsin isoforms act through PAR-1.Thus, the activity of human cationic and anionic trypsins on PARs was comparable to that of bovine pancreatic trypsin. Mesotrypsin (trypsin IV), in contrast to cationic and anionic trypsin, cannot activate or disable PARs in human epithelial cells, demonstrating that the receptors are no substrates for this isoenzyme. On the other hand, mesotrypsin activates PAR-1 in human astrocytoma cells. This might play a role in protection/degeneration or plasticity processes in the human brain.     

Suppression by protease-activated receptor-2 activation of gastric acid secretion in rats

Activation of protease-activated receptor-2 (PAR-2), a receptor activated by trypsin/tryptase, induces neurally mediated gastric mucus secretion accompanied by mucosal cytoprotection. In the present study, we investigated whether PAR-2 could modulate gastric acid secretion in rats. Messenger RNAs for PAR-2 and PAR-1 were detected in the gastric mucosa and smooth muscle. The PAR-2-activating peptide SLIGRL-NH(2), but not the inactive control peptide, when administered i.v., strongly suppressed gastric acid secretion in response to carbachol, pentagastrin or 2-deoxy-D-glucose in the rats with a pylorus ligation. The PAR-2-mediated suppression of acid secretion was resistant to cyclooxygenase inhibition or ablation of sensory neurons by capsaicin. Our results provide novel evidence that in addition to stimulating neurally mediated mucus secretion, activation of PAR-2 suppresses gastric acid secretion independently of prostanoid production or sensory neurons. These dual actions of PAR-2 would result in gastric mucosal cytoprotection.     

Unexpected anti-hypertrophic responses to low-level stimulation of protease-activated receptors in adult rat cardiomyocytes

Activators of protease-activated receptors PAR-1 and PAR-2 such as thrombin and synthetic hexapeptides promote hypertrophy of isolated neonatal cardiomyocytes at pathological concentrations. Since PAR-activating proteases often show dual actions at low vs. high concentrations, the potential hypertrophic effects of low-level PAR activation were examined. In H9c2 cardiomyoblasts, messenger RNA (mRNA) expression of the hypertrophic marker atrial natriuretic peptide (ANP) was significantly increased only by higher concentrations of thrombin, trypsin or the synthetic PAR-2 agonist SLIGRL. The dual PAR-1/PAR-2 agonist SFLLRN did not influence basal ANP mRNA expression in H9c2 cells. Low concentration of thrombin or trypsin (up to 0.1 U/mL) or of the synthetic ligands SFLLRN and SLIGRL (1 μM); however, all suppressed ANP mRNA expression stimulated by angiotensin II (Ang II). The PAR-1 selective ligand TFLLRN exerted a comparable effect as SFLLRN. In adult rat cardiomyocytes, protein synthesis determined by [³H]phenylalanine incorporation was not increased by various PAR agonists at concentrations tenfold lower than conventionally used to study PAR function in vitro (10 μM for SFLLRN or SLIGRL, 0.1 U/mL for thrombin or trypsin). The positive control endothelin-1 (ET-1, 60 nM) however significantly increased protein synthesis in adult rat cardiomyocytes. Addition of low concentrations of PAR agonists to cardiomyocytes treated with ET-1 or Ang II suppressed [³H]phenylalanine incorporation induced by the hypertrophic stimuli. The inhibitory effect of SFLLRN effect was partially reversed by the PAR-1 antagonist RWJ56110. These findings suggest that physiological concentrations of PAR activators may suppress hypertrophy, in contrast to the pro-hypertrophic effects evident at high concentrations. PAR-1 and PAR-2 may dynamically control cardiomyocyte growth, with the net effect critically dependent upon local agonist concentrations. The precise significance of proposed concept of bimodal PAR function in cardiomyocytes remains to be defined, particularly in vivo where hemodynamic and other regulatory factors may counteract or mask the direct cellular actions described here.     

Endothelium-dependent and -independent responses to protease-activated receptor-2 (PAR-2) activation in mouse isolated renal arteries

Protease-activated receptors (PARs) are receptors which require proteolytic cleavage to be self-activated by newly exposed N-terminal `tethered ligands'', and hence serve as sensors for protelytic enzymes. While both the thrombin receptor (PAR-1) and PAR-2 (activated by tryptic enzymes) have been shown to mediate endothelium-dependent vasorelaxation, only PAR-1 has been shown to cause direct vascular smooth muscle contraction. In this study, we report that trypsin and the PAR-2 selective peptide ligand SLIGRL-NH₂ not only caused endothelium-dependent relaxation of mouse renal arteries but also direct smooth muscle contraction if endothelial nitric oxide synthase was inhibited or if the endothelium was removed.     

Roles of protease-activated receptor-2 (PAR-2), a G protein-coupled receptor, in modulation of exocrine gland functions

Protease-activated receptor-2 (PAR-2), a G protein-coupled receptor, is activated by proteolytic unmasking of the N-terminal extracellular tethered ligand that presumably binds to the extracellular loop 2 of the receptor itself. PAR-2 is widely distributed in the mammalian body and plays various roles in biological events in the cardiovascular, respiratory, alimentary, and central neurons systems. PAR-2-activating peptides administered systemically to mice and rats trigger prompt salivation in vivo. In an in vitro study, PAR-2 agonists including the endogenous PAR-2 activator trypsin induce secretion of amylase and mucin from isolated rat parotid glands and sublingual glands, respectively. PAR-2-activating peptides administered systemically also modulate pancreatic exocrine secretion in vivo as well as in vitro. In the gastric mucosa, PAR-2 stimulation enhances secretion of mucus and pepsinogen and suppresses acid secretion. Tear secretion can also be caused by PAR-2-related peptides in PAR-2-dependent and -independent manners. PAR-2 thus plays a general or key role in the regulation of exocrine secretion. This review focuses on the physiologic and/or pathophysiologic roles of PAR-2 in glandular exocrine secretion. The possibility of PAR-2 as a target for drug development is also discussed.     

B2 kinin receptor activation is the predominant mechanism by which trypsin mediates endothelium-dependent relaxation in bovine coronary arteries

The roles of kinin and protease-activated receptors (PAR) in endothelium-dependent relaxations to the serine protease, trypsin, were examined in rings of bovine left anterior descending coronary artery (LAD). Trypsin (0.01-30 U/ml) caused biphasic, endothelium-dependent relaxations-a high potency (0.01-0.3 U/ml), low efficacy relaxation [maximum relaxation (R (max)), 9.0 +/- 5.1%] followed by a lower potency (1-30 U/ml) but high efficacy (R (max), 90.4 +/- 5.5%) relaxation, which was abolished by aprotinin. Captopril (10 muM) caused an ~10-fold leftward shift of the second phase response such that the first phase was masked. The second phase relaxation to trypsin was inhibited in a concentration-dependent, non-surmountable manner by the B(2) antagonist, HOE-140. At 3 nM HOE-140, the second phase response to trypsin was abolished unmasking the first phase. Kallikrein (0.0003-0.3 U/ml) caused monophasic, endothelium-dependent relaxations (R (max), 33.7 +/- 14.6%), which were potentiated by captopril (R (max), 94.2 +/- 1.0%) and abolished by HOE-140. In the presence of captopril, the second phase relaxation to trypsin was only minimally inhibited by either N(G)-nitro-L: -arginine (100 muM) or 67 mM [K(+)](o) alone but markedly reduced when these two treatments were combined (R (max), 26.1 +/- 11.6% versus 98.6 +/- 2.9% in controls). The PAR1-activating peptide, SFLLRN (0.1-30 muM), but not the PAR2-activating peptide, SLIGRL, caused concentration-dependent relaxations (pEC(50), 5.9 +/- 0.0%; R (max), 43.3 +/- 8.3%). In conclusion, trypsin causes endothelium-dependent relaxations in the bovine LAD predominantly via release of endogenous BK, which in turn activates endothelial B(2) receptors. Only a minor role for PAR1-like receptors was evident in this tissue.     

Unproductive cleavage and the inactivation of protease-activated receptor-1 by trypsin in vascular endothelial cells

1 Using fura-2 fluorometry of [Ca(2+)](i) in response to thrombin, trypsin and protease-activated receptor activating peptides (PAR-APs), we determined whether trypsin cleaves protease-activated receptor 1 (PAR1) and activates it in the endothelial cells of the porcine aortic valves and human umbilical vein. 2 Once stimulated with thrombin, the subsequent application of trypsin induced a [Ca(2+)](i) elevation similar to that obtained without the preceding stimulation with thrombin in the valvular endothelial cells. However, the preceding stimulation with trypsin abolished the subsequent response to thrombin, but not to bradykinin or substance P. 3 The response to PAR1-AP (SFLLRNP) was significantly (P<0.05) reduced by the preceding stimulation with thrombin and PAR1-AP in the valvular endothelial cells, while, importantly, it remained unaffected by the preceding stimulation with either trypsin or PAR2-AP (SLIGRL). The response to PAR2-AP was reduced by the preceding stimulation with trypsin and PAP2-AP. PAR1-AP attenuated the subsequent responses not only to thrombin and PAR1-AP but also to trypsin and PAR2-AP, while PAR2-AP specifically attenuated the subsequent responses to trypsin and PAR2-AP. 4 In human umbilical vein endothelial cells, a higher affinity PAR1-AP (haPAR1-AP) (Ala-pF-Arg-Cha-HArg-Tyr-NH(2)) specifically attenuated the responses to thrombin but not trypsin. On the other hand, the response to haPAR1-AP was significantly (P<0.05) attenuated by the preceding stimulation with thrombin but not trypsin. 5 In conclusion, trypsin cleaved PAR1 but did not activate it in the endothelial cells. Moreover, the trypsin-cleaved PAR1 was no longer responsive to thrombin.     

Physiology of protease-activated receptors (PARs): involvement of PARs in digestive functions

The protease-activated receptor (PAR), a G protein-coupled receptor present on cell surface, mediates cellular actions of extracellular proteases. Proteases cleave the extracellular N-terminal of PAR molecules at a specific site, unmasking and exposing a novel N-terminal, a tethered ligand, that binds to the body of receptor molecules resulting in receptor activation. Amongst four distinct PARs that have been cloned, PARs 1, 3 and 4 are activated by thrombin, but PAR-2 is activated by trypsin or mast cell tryptase. Human platelets express two distinct thrombin receptors, PAR-1 and PAR-4, while murine platelets express PAR-3 and PAR-4. Apart from roles of PARs in platelet activation, PARs are distributed to a number of organs in various species, predicting their physiological importance. We have been evaluating agonists specific for each PAR, using multiple procedures including a HEK cell calcium signal receptor desensitization assay. Using specific agonists that we developed, we found the following: 1) the salivary glands express PAR-2 mRNA and secret saliva in response to PAR-2 activation; 2) pancreatic juice secretion occurs following in vivo PAR-2 activation; 3) PAR-1 and PAR-2 modulate duodenal motility. Collectively, PAR plays various physiological and/or pathophysiological roles, especially in the digestive systems, and could be a novel target for drug development.     

Modulation of capsaicin-evoked visceral pain and referred hyperalgesia by protease-activated receptors 1 and 2

Protease-activated receptors (PARs) 1 and 2 are expressed in capsaicin-sensitive sensory neurons, being anti- and pro-nociceptive, respectively. Given the possible cross talk between PAR-2 and capsaicin receptors, we investigated if PAR-2 activation could facilitate capsaicin-evoked visceral pain and referred hyperalgesia in the mouse and also examined the effect of PAR-1 activation in this model. Intracolonic (i.col.) administration of capsaicin triggered visceral pain-related nociceptive behavior, followed by referred hyperalgesia. The capsaicin-evoked visceral nociception was suppressed by intraperitoneal (i.p.) TFLLR-NH2, a PAR-1-activating peptide, but not FTLLR-NH2, a control peptide, and unaffected by i.col. TFLLR-NH2. SLIGRL-NH2, a PAR-2-activating peptide, but not LRGILS-NH2, a control peptide, administered i.col., facilitated the capsaicin-evoked visceral nociception 6-18 h after administration, while i.p. SLIGRL-NH2 had no effect. The capsaicin-evoked referred hyperalgesia was augmented by i.col. SLIGRL-NH2, but not LRGILS-NH2, 6-18 h after administration, and unaffected by i.p. SLIGRL-NH2, and i.p. or i.col. TFLLR-NH2. Our data suggest that PAR-1 is antinociceptive in processing of visceral pain, whereas PAR-2 expressed in the colonic luminal surface, upon activation, produces delayed sensitization of capsaicin receptors, resulting in facilitation of visceral pain and referred hyperalgesia.     

Oral administration of the thrombin receptor antagonist E5555 (atopaxar) attenuates intimal thickening following balloon injury in rats

Thrombin is a powerful agonist for a variety of cellular responses including platelet aggregation and vascular smooth muscle cell (SMC) proliferation. These actions are mediated by a thrombin receptor known as protease-activated receptor-1 (PAR-1). Recently we discovered that 1-(3-tert-butyl-4-methoxy-5-morpholinophenyl)-2-(5,6-diethoxy-7-fluoro-1-imino-1,3-dihydro-2H-isoindol-2-yl)ethanone hydrobromide (E5555, atopaxar) is a potent and selective thrombin receptor antagonist. This study characterized the pharmacological effects of E5555 on SMC proliferation in vitro and in a rat model of intimal thickening after balloon injury in vivo. E5555 selectively inhibited rat aortic SMC proliferation induced by thrombin and thrombin receptor-activating peptide (TRAP) with half maximal inhibitory concentration (IC(50)) values of 0.16 and 0.038 μM, respectively. E5555 did not inhibit rat SMC proliferation induced by basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) at concentrations up to 1μM. In addition, E5555 inhibited human aortic SMC proliferation induced by thrombin at concentrations of 0.3 and 3units/ml with IC(50) values of 0.028 and 0.079 μM, respectively, whereas it did not affect bFGF-induced proliferation at concentrations up to 1μM. Repeated oral administration of 30 mg/kg E5555 (once daily for 16 days) significantly reduced neointimal formation in the balloon-injured rat arterial model. These results suggested that a PAR-1 antagonist could be effective for treating restenosis following vascular intervention in addition to preventing thrombus formation. E5555 could thus have therapeutic potential for restenosis and chronic atherothrombotic disease.