Ishii H, Suzuki Y, Horie S, Nakagawa M, Kazama M
Department of Clinical Biochemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan.
Biochim Biophys Acta. 1992 Dec 15;1175(1):37-43. doi: 10.1016/0167-4889(92)90007-x.
Proteolytic activation of calpain (calcium-dependent neutral protease) I in thrombin-stimulated platelets was determined by following the production of the 76- and 78-kDa forms from the 80-kDa subunit of calpain I as measured by immunoblotting using monospecific antibody to human calpain I, and the correlation between the extents of calpain I activation and ATP release was investigated. When platelets were stimulated with thrombin in the range from 0.01 to 0.5 U/ml, the maximal 60% activation of calpain I was achieved within 15 s after the stimulation, and ATP release began after the maximal activation had been reached. The extent of ATP release decreased in parallel with the decrease in activation ratio of calpain I on treatment of platelets with EGTA or EST, a membrane-permeable inhibitor of calpain. Although pretreatment of platelets with EST did not affect the thrombin-dependent elevation of the cytosolic Ca2+ concentration, both the inhibition of calpain I activation and the reduction of ATP release were observed as a function of EST concentration. These results suggest that calpain I participates in one of the processes leading to the ATP release reaction of platelets stimulated with thrombin.
通过使用抗人钙蛋白酶I的单特异性抗体进行免疫印迹法，追踪钙蛋白酶I 80 kDa亚基产生的76 kDa和78 kDa形式，来测定凝血酶刺激的血小板中钙蛋白酶（钙依赖性中性蛋白酶）I的蛋白水解激活，并研究钙蛋白酶I激活程度与ATP释放之间的相关性。当血小板用0.01至0.5 U/ml范围内的凝血酶刺激时，刺激后15秒内钙蛋白酶I达到最大60%的激活，并且在达到最大激活后开始ATP释放。在用EGTA或钙蛋白酶的膜通透性抑制剂EST处理血小板时，ATP释放程度与钙蛋白酶I激活率的降低平行下降。尽管用EST预处理血小板不影响凝血酶依赖性的胞质Ca2+浓度升高，但观察到钙蛋白酶I激活的抑制和ATP释放的减少是EST浓度的函数。这些结果表明，钙蛋白酶I参与了导致凝血酶刺激的血小板ATP释放反应的过程之一。
