Verhallen P F, Bevers E M, Comfurius P, Zwaal R F
Department of Biochemistry, University of Limburg, Maastricht, The Netherlands.
Biochim Biophys Acta. 1987 Sep 18;903(1):206-17. doi: 10.1016/0005-2736(87)90170-2.
The relationship between platelet calpain-activity and platelet procoagulant-activity was investigated by comparison of the time course of their generation after platelet stimulation by calcium ionophore A23187, or by the combined action of collagen and thrombin, or during exposure of platelets to the local anesthetics dibucaine or tetracaine. In addition, the Ca2+ dose-response curves of both activities in intact platelets, obtained by stimulation with A23187 in the presence of Ca2+/HEDTA-buffers, were compared. Platelet procoagulant activity was determined by assaying for prothrombinase activity in the presence of saturating concentrations of factors Xa, Va, and prothrombin. Platelet calpain activity was monitored by the degradation of its major substrates (filamin, talin, myosin) and the formation of their fragments as judged from protein patterns after gel electrophoresis. Platelet stimulation by A23187 resulted in a fast increase in prothrombinase activity, reaching its maximum level after about 20 seconds. Filamin and talin were completely hydrolysed within 15 s, and myosin was partly degraded between 15 and 30 s after platelet activation. When platelets were activated by collagen plus thrombin, prothrombinase activity was generated with a sigmoid time course, the steepest increase being observed between 1 and 2 min after platelet activation. Proteolysis of filamin and talin occurred between 0.5 and 1.5 min after platelet activation, while degradation of myosin became visible after 2 to 2.5 min. Dibucaine and tetracaine were both found to be potent stimulators of prothrombinase activity, with half-maximal activities obtained at 0.7 and 2.8 mM, respectively. Using suboptimal concentrations of both local anesthetics, it was found that the generation of prothrombinase activity closely paralleled that of calpain activity over a time course of 1 hour. Ca2+ titration of intact platelets using A23187 and Ca2+/HEDTA buffers, revealed half-maximal response at about 15 microM free Ca2+ for both calpain and prothrombinase activity. These findings strongly suggest a causal relationship between generation of a procoagulant platelet surface and calpain-mediated degradation of filamin, talin, and myosin. Since an increased procoagulant activity reflects an increased exposure of phosphatidylserine at the platelet outer surface, the present findings suggest that platelet cytoskeletal proteins are involved in the regulation of membrane lipid asymmetry.
通过比较钙离子载体A23187刺激血小板后、胶原蛋白和凝血酶联合作用后或血小板暴露于局部麻醉剂丁卡因或丁哌卡因期间，血小板钙蛋白酶活性和血小板促凝活性的产生时间进程，研究了它们之间的关系。此外，还比较了在Ca2+/HEDTA缓冲液存在下用A23187刺激完整血小板时，两种活性的Ca2+剂量反应曲线。通过在饱和浓度的因子Xa、Va和凝血酶原存在下测定凝血酶原酶活性来确定血小板促凝活性。通过凝胶电泳后蛋白质图谱判断其主要底物（细丝蛋白、踝蛋白、肌球蛋白）的降解及其片段的形成来监测血小板钙蛋白酶活性。A23187刺激血小板导致凝血酶原酶活性快速增加，约20秒后达到最高水平。细丝蛋白和踝蛋白在15秒内完全水解，肌球蛋白在血小板活化后15至30秒内部分降解。当血小板由胶原蛋白加凝血酶激活时，凝血酶原酶活性呈S形时间进程产生，在血小板活化后1至2分钟观察到最陡的增加。细丝蛋白和踝蛋白的蛋白水解发生在血小板活化后0.5至1.5分钟，而肌球蛋白的降解在2至2.5分钟后可见。发现丁卡因和丁哌卡因都是凝血酶原酶活性的有效刺激剂，分别在0.7和2.8 mM时获得半数最大活性。使用两种局部麻醉剂的次优浓度，发现在1小时的时间进程中，凝血酶原酶活性的产生与钙蛋白酶活性的产生密切平行。使用A23187和Ca2+/HEDTA缓冲液对完整血小板进行Ca2+滴定，发现钙蛋白酶和凝血酶原酶活性在约15 microM游离Ca2+时出现半数最大反应。这些发现强烈表明促凝血小板表面的产生与钙蛋白酶介导的细丝蛋白、踝蛋白和肌球蛋白降解之间存在因果关系。由于促凝活性增加反映了血小板外表面磷脂酰丝氨酸暴露增加，目前的发现表明血小板细胞骨架蛋白参与膜脂质不对称性的调节。
