Ishii H, Kuboki M, Fujii J, Hiraishi S, Kazama M
Department of Clinical Biochemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan.
Thromb Res. 1990 Mar 15;57(6):847-61. doi: 10.1016/0049-3848(90)90152-3.
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.
研究了硫醇蛋白酶在血小板激活中的作用。当血小板用EST（L-反式环氧琥珀酰-L-亮氨酰胺（3-甲基）丁烷乙酯，一种可透过膜的硫醇蛋白酶抑制剂）处理30分钟时，凝血酶诱导的聚集和分泌受到抑制，并且即使在预处理后将血小板洗涤并重悬于不含EST的培养基中，这种抑制作用仍然存在。EST对凝血酶诱导的血小板聚集和分泌的抑制作用呈剂量依赖性和孵育时间依赖性。EST对血小板聚集和分泌的抑制作用不仅表现在对凝血酶的反应中，也表现在对血小板活化因子（PAF）的反应中。用1 mM EST预处理血小板30分钟可抑制血小板中65%的钙蛋白酶（硫醇蛋白酶）活性。用1 mM EST预处理可阻断凝血酶刺激引起的血小板40 kDa和20 kDa蛋白的磷酸化。凝血酶刺激血小板后出现的磷脂酰肌醇水解和肌醇-1-磷酸生成也受到1 mM EST预处理的抑制。结果表明，EST进入血小板内部，并通过抑制硫醇蛋白酶来抑制血小板的激活。
