Participation of thiolprotease in platelet activation was investigated. When platelets were treated with EST (L-trans-epoxysuccinyl-leucylamide (3-methyl)butane-ethyl ester, a membrane-permeable thiolprotease inhibitor) for 30 min, thrombin-induced aggregation and secretion were inhibited, and remained so even when the platelets were washed and resuspended in EST-free medium after the pretreatment. The inhibitory action of EST on thrombin-induced platelet aggregation and secretion was both dose-dependent and incubation-time-dependent. The inhibitory action of EST on platelet aggregation and secretion was shown not only in response to thrombin but also to platelet activating factor (PAF). Pretreatment of platelets with 1 mM EST for 30 min inhibited the 65% of calpain (thiolprotease) activity in platelets. The phosphorylation of 40 kDa and 20 kDa proteins of platelets caused by stimulation with thrombin was blocked by the pretreatment with 1 mM EST. Phosphatidylinositol hydrolysis and inositol-1-phosphate production, which appear after stimulation of platelets with thrombin, were also inhibited by the pretreatment with 1 mM EST. The results suggest that EST was incorporated to inside of platelets, and inhibited activation of platelet through inhibition of thiolprotease.