In vitro antiplatelet profile of FR171113, a novel non-peptide thrombin receptor antagonist

Synthetic peptides (5 to 14 amino acids), identical in sequence to the new amino-terminus of the thrombin receptor generated following cleavage by thrombin, act as thrombin receptor agonist peptides. Whilst thrombin receptor antagonist peptides are known, non-peptide thrombin receptor antagonists have yet to be described. In the present study, we compared the antiplatelet effects of 3-(4-chlorophenyl)-2-(2,4-dichlorobenzoylimino)-5-(methycarbonyl methylene)-1,3-thiazolidin-4-one (FR171113), a novel non-peptide thrombin receptor antagonist, with the known thrombin receptor antagonist 3-mercapto-propionyl-Phe-Cha-Cha-Arg-Asn-Asp-Lys-Tyr-OH (C186-65), and argatroban, a specific protease inhibitor of thrombin. FR171113 and C186-65 inhibited thrombin-induced platelet aggregation (IC50 = 0.29 mu M and 15 mu M, respectively) and Ser-Phe-Leu-Leu-Arg-Asn-NH2 [a synthetic thrombin receptor agonist peptide (TRAP-6)] induced platelet aggregation (0.15 mu M and 20 mu M, respectively) in human washed platelets. Argatroban potently inhibited thrombin-induced platelet aggregation (IC50 = 3.5 nM), but did not inhibit TRAP-6-induced aggregation even at 100 mu M. In contrast, these compounds did not show inhibitory effects on ADP- and collagen-induced aggregation in human platelet-rich plasma even at 100 mu M. FR171113 caused a parallel shift to the right of the concentration-response curve describing aggregation induced by TRAP-6. The Schild plot of the data had a slope of - 0.840 (r = 0.98) and the pA(2) was 7.29. In protease activity studies using a chromogenic substrate, argatroban inhibited thrombin protease activity in a dose-dependent manner, whereas FR171113 and C186-65 were inactive, even at 100 mu M Additionally, only argatroban displayed dose-dependent prolongation of thrombin time, activated partial thromboplastin time and prothrombin time. FR171113 and C186-65 showed no effects, even at a concentration of 100 mu M These results suggest that FR171113 has a similar mode of action to C186-65, but with more potent antiplatelet activity. In conclusion, FR171113 is suggested to be the first example of a non-peptide thrombin receptor antagonist. (C) 1999 Elsevier Science B.V. All rights reserved.