CALCIUM INVOLVEMENT IN AMINOPHOSPHOLIPID EXPOSURE AND MICROPARTICLE FORMATION DURING PLATELET ACTIVATION - A STUDY USING CA2+-ATPASE INHIBITORS
CALCIUM INVOLVEMENT IN AMINOPHOSPHOLIPID EXPOSURE AND MICROPARTICLE FORMATION DURING PLATELET ACTIVATION - A STUDY USING CA2+-ATPASE INHIBITORS
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DOI：
10.1021/bi00036a039
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发表时间：
1995-09-12
期刊：
影响因子：
2.9
通讯作者：
NURDEN, AT
中科院分区：
文献类型：
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作者：
DACHARYPRIGENT, J;PASQUET, JM;NURDEN, AT
已知血小板活化过程中促凝血活性和微粒形成的发展取决于胞质Ca 2+水平的增加。我们已经研究了导致这些事件的机制，使用FITC标记的重组膜联蛋白V，一种蛋白质，具有高亲和力的氨基磷脂结合，在流式细胞术。特别是，我们表明，Ca 2 +-ATP酶抑制剂毒胡萝卜素和cyclopiazonic酸作为有效的诱导剂的氨基磷脂曝光和微粒形成的离子载体A23187。相比之下，2，5-二叔丁基-1，4-苯并氢醌诱导可忽略的微粒形成，虽然血小板大量结合膜联蛋白V-FITC。已经发生的血小板活化通过与VH 10（α-颗粒膜糖蛋白GMP-140的特异性单克隆抗体）的结合研究和凝血酶原酶活性测量来证实。这些结果表明，微泡形成不是对氨基磷脂暴露的自动反应。Ca ~（2+）-ATP酶抑制剂诱导不同的细胞内Ca ~（2+）水平，如使用Fluo-3作为钙染料测量的。毒胡萝卜素（3 μ M）为10 +/- 4 μ M（n = 11），环匹阿尼酸（100 μ M）为19.6 +/- 2.2 μ M（n = 8），2，5-二叔丁基-1，4-苯并氢醌（100 μ M）为0.619 +/- 0.137 μ M（n = 8）。钙蛋白酶活性，通过分析细胞骨架蛋白的降解在血小板中进行评估，仅观察到与刺激微粒形成的代理。通过测量血小板活化期间膜联蛋白V结合来研究磷脂跨双层运动。结果显示，由离子载体A23187诱导的氨基磷脂暴露（t（1/2）= 133 +/- 14 s）比由TG诱导的氨基磷脂暴露（t（1/2）= 280 +/- 30 s）更快，尽管测定中的限速步骤是膜联蛋白V与活化血小板的结合（t（1/2）= 70-80 s）。有趣的是，在激活过程中膜联蛋白V本身的存在抑制微粒的形成，虽然钙蛋白酶的血小板蛋白降解继续发生。我们的研究结果清楚地表明：（i）氨基磷脂暴露和血小板微泡形成是独立的，但密切调节的事件和（ii），而这两个过程都与细胞内Ca 2+的增加，微泡形成还需要Ca 2+诱导的钙蛋白酶激活和膜联蛋白V抑制的融合过程。
The development of procoagulant activity and microparticle formation during platelet activation is known to depend on an increase in cytosolic Ca2+ levels. We have studied the mechanisms leading to these events using FITC-labeled recombinant annexin V, a protein which binds with a high affinity to aminophospholipids, in flow cytometry. In particular, we show that the Ca2+-ATPase inhibitors thapsigargin and cyclopiazonic acid are as potent inducers of aminophospholipid exposure and microparticle formation as the ionophore A23187. In contrast, 2,5-di-tert-butyl-1,4-benzohydroquinone induced negligible microparticle formation, although platelets abundantly bound annexin V-FITC. That platelet activation had occurred was confirmed by binding studies with VH10, a monoclonal antibody specific for the alpha-granule membrane glycoprotein GMP-140, and by prothrombinase activity measurements. These results demonstrate that microvesiculation is not an automatic response to aminophospholipid exposure, The Ca2+-ATPase inhibitors induced different intracellular Ca2+ levels as measured using fluo-3 as a calcium dye. These were 10 +/- 4 mu M (n = 11) for thapsigargin (3 mu M), 19.6 +/- 2.2 mu M (n = 8) for cyclopiazonic acid (100 mu M), and 0.619 +/- 0.137 mu M (n = 8) for 2,5-di-tert-butyl-l,4-benzohydroquinone (100 mu M). Calpain activity, as assessed in platelets by analyzing the degradation of cytoskeletal proteins, was only observed with agents that stimulated microparticle formation. Phospholipid transbilayer movement was studied by measuring annexin V binding during platelet activation. Results showed that aminophospholipid exposure induced by ionophore A23187 (t(1/2) = 133 +/- 14 s) was more rapid than that induced by TG (t(1/2) = 280 +/- 30 s), although the rate-limiting step in the assay was the binding of annexin V to activated platelets (t(1/2) = 70-80 s). Interestingly, the presence of annexin V itself during the activation inhibited microparticle formation, although degradation of platelet proteins by calpain continued to occur. Our results clearly show (i) that aminophospholipid exposure and platelet microvesiculation are independent but closely regulated events and (ii) that while both processes are associated with an increase in intracellular Ca2+, microvesiculation additionally requires Ca2+-induced calpain activation and a fusion process inhibited by annexin V.