YD-3, a novel inhibitor of protease-induced platelet activation

1 In the present study, the antiplatelet effects and mechanisms of a new sy nthetic compound YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole] were exam ined. 2 YD-3 inhibited the aggregation of washed rabbit platelets caused by throm bin (IC50 = 28.3 mu M), but had no or little inhibitory effect on that indu ced by arachidonic acid, collagen, platelet-activating factor (PAF) or U466 19. YD-3 also suppressed generation of inositol phosphates caused by thromb in. On the other hand, thrombin-induced fibrin formation was not affected b y YD-3, indicating YD-3 does not inhibit the proteolytic activity of thromb in. 3 In washed human platelets, however, YD-3 had only mild inhibitory effect on the low concentration (0.05 u ml(-1)) of thrombin-induced human platelet aggregation, and did not affect that induced by higher concentrations (gre ater than or equal to 0.1 u ml(-1)) of thrombin or SFLLRN, the protease-act ivated receptor 1 (PAR1) agonist peptide. By contrast, YD-3 inhibited both human and rabbit platelet aggregation elicited by trypsin with IC50 values of 38.1 mu M and 5.7 mu M, respectively. 4 YD-3, at 100 mu M, had no effect on ristocetin-induced glycoprotein Ib (G PIb)-dependent aggregation of human platelets. In addition, platelets treat ed with chymotrypsin, which cleaves GPIb, enhanced rather than attenuated t he inhibition of YD-3 on thrombin-induced human platelet aggregation. These data indicate that GPIb plays no role in the antiplatelet effect of YD-3. 5 In SFLLRN-desensitized human platelets, high concentration of thrombin (1 u ml (-1)) could still elicit intracellular Ca2+ mobilization, and the ris e of [Ca2+], was prevented by either leupeptin or YD-3. 6 Our results suggest that YD-3 inhibits a non-PAR1 thrombin receptor which mediates the major effect of thrombin in rabbit platelets, but in human pl atelets, this receptor function becomes significant only when the function of PAR1 has been blocked or attenuated.