Dachary-Prigent J, Freyssinet J M, Pasquet J M, Carron J C, Nurden A T
URA CNRS 1464, Hôpital Cardiologique, Pessac, France.
Blood. 1993 May 15;81(10):2554-65.
Annexin V, a protein with a high affinity and a strict specificity for aminophospholipids at physiologic calcium concentrations, was used to probe platelet activation and the development of procoagulant activity. Platelet secretion was studied in parallel using VH10, a murine monoclonal antibody specific for GMP-140, an alpha-granule membrane glycoprotein. Both proteins were labeled with fluorescein isothiocyanate and platelet activation was assessed by flow cytometry. Microparticles, which are shed from the platelet surface and also support procoagulant activity, were distinguished from platelets according to their associated light scattering signal. The relative ability of different inducers to trigger exposure of the procoagulant surface and microparticle formation was: ionophore A23187 > thrombin plus collagen > collagen > thrombin. The density of aminophospholipid on microparticles was higher than on remnant platelets. Platelet activation by these agonists was accompanied by GMP-140 exposure, both on platelets and microparticles. Here, thrombin was the most efficient agonist. The mechanisms responsible for the above processes were investigated using E-64-d, a specific membrane-permeable inhibitor of Ca(2+)-activated protease (calpain); tetracaine, an activator of calpain; and N-ethylmaleimide and diamide, two sulfhydryl-reactive agents. These agents were added to platelets alone or before stimulation by agonists. Calpain activity was assessed by the hydrolysis of cytoskeletal proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results showed that calpain activity is not essential for aminophospholipid translocation or for secretion. In contrast, although sulfhydryl-reactive agents alone can trigger procoagulant activity, they inhibit microvesicle formation and platelet secretion induced by the above agonists, suggesting that different mechanisms account for these phenomena. The use of annexin V in flow cytometry is a rapid method to assess procoagulant activity in platelets and the loss of phospholipid asymmetry in cell membranes.
膜联蛋白V是一种在生理钙浓度下对氨基磷脂具有高亲和力和严格特异性的蛋白质，用于探测血小板活化和促凝血活性的发展。同时使用VH10研究血小板分泌，VH10是一种针对α-颗粒膜糖蛋白GMP-140的鼠单克隆抗体。两种蛋白质都用异硫氰酸荧光素标记，并通过流式细胞术评估血小板活化。从血小板表面脱落并也支持促凝血活性的微粒，根据其相关的光散射信号与血小板区分开来。不同诱导剂触发促凝表面暴露和微粒形成的相对能力为：离子载体A23187>凝血酶加胶原>胶原>凝血酶。微粒上氨基磷脂的密度高于残余血小板。这些激动剂引起的血小板活化伴随着血小板和微粒上GMP-140的暴露。在这里，凝血酶是最有效的激动剂。使用E-64-d（一种Ca(2+)激活蛋白酶（钙蛋白酶）的特异性膜通透性抑制剂）、丁卡因（一种钙蛋白酶激活剂）以及N-乙基马来酰亚胺和二酰胺（两种巯基反应剂）研究上述过程的机制。这些试剂单独添加到血小板中或在激动剂刺激之前添加。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定的细胞骨架蛋白水解来评估钙蛋白酶活性。结果表明，钙蛋白酶活性对于氨基磷脂易位或分泌不是必需的。相反，尽管单独的巯基反应剂可以触发促凝血活性，但它们抑制上述激动剂诱导的微泡形成和血小板分泌，这表明这些现象的机制不同。在流式细胞术中使用膜联蛋白V是一种评估血小板促凝血活性和细胞膜中磷脂不对称性丧失的快速方法。
