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] 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 ([H-3]haTRAP, Ala-Phe(p-F)-Arg-ChA-HArg-[H-3]Tyr-NK,) to PAR-1 with IC50 values of 70 and 45 nM, respectively. SCH 79797 inhibited [H-3]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-NH2 (TK), a PAR-l selective agonist, produced transient increases in cytosolic free Ca2+ concentration ([Ca2+](i)) in hCASMC. This increase in [Ca2+](i) was inhibited effectively by SCH 79797. However, the Ca2+ transients induced by SLIGKV-NH2, a PAR-2-selective agonist, were not inhibited by SCH 79797. Thrombin- and TK-stimulated [H-3]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 Ca2+ transients and mitogenesis in hCASMC is mediated primarily through activation of PAR-1. (C) 2000 Elsevier Science Inc.
