Swords N A, Tracy P B, Mann K G
Department of Biochemistry, College of Medicine, University of Vermont, Burlington 05405.
Arterioscler Thromb. 1993 Nov;13(11):1613-22. doi: 10.1161/01.atv.13.11.1613.
The possibility that platelets release microvesicles on adherence to either von Willebrand factor (vWf) or collagen was examined by flow cytometry analysis of the supernatant above layers of adherent platelets. No microvesicle release was detected as a result of adherence to vWf or to collagen, a known platelet agonist. Approximately 8% of the total platelet mass was released as microvesicles after thrombin stimulation of the vWf- or collagen-adherent platelets. A larger portion of the vWf-adherent platelet membranes (approximately 21%) was released as microvesicles subsequent to platelet stimulation with the nonphysiological agonist calcium ionophore A23187. Calpeptin, a calpain inhibitor, had no effect on microvesicle release, suggesting that calpain proteolysis of platelet cytoskeletal proteins was not responsible for microvesicle shedding under the conditions studied. Examination of the vWf-adherent platelets by scanning electron microscopy showed that virtually no microvesicles bound to exposed vWf multimers. No microvesicle binding to the adherent platelets was observed, indicating that the majority of the microvesicles were shed from the platelet and vWf surface on platelet activation. The ability of the microvesicle population to support procoagulant activity was measured with a prothrombinase activity assay and was compared with the activity supported by the adherent platelet membranes. More than 85% of the total prothrombinase activity remained associated with the adherent platelet membranes, both for unstimulated platelets and platelets stimulated with physiological agonists. Furthermore, the residual activity found in the buffer fraction containing detached platelets and any released microvesicles could be attributed to the detached platelets. No activity could be attributed to the microvesicles, as thrombin stimulation of either vWf-or collagen-adherent platelets did not promote increased procoagulant activity relative to the unstimulated adherent platelets, even though microvesicle release was detected as a result of agonist addition. Neither full platelet activation nor microvesicle shedding played an essential role in generating procoagulant activity in the adherent platelet system.
通过对粘附血小板层上方的上清液进行流式细胞术分析，研究了血小板在粘附于血管性血友病因子（vWf）或胶原蛋白时释放微泡的可能性。未检测到因粘附于vWf或胶原蛋白（一种已知的血小板激动剂）而导致的微泡释放。在用凝血酶刺激vWf或胶原蛋白粘附的血小板后，约8%的血小板总质量以微泡形式释放。在用非生理性激动剂钙离子载体A23187刺激血小板后，较大比例（约21%）的vWf粘附血小板膜以微泡形式释放。钙蛋白酶抑制剂钙肽素对微泡释放没有影响，这表明在研究的条件下，血小板细胞骨架蛋白的钙蛋白酶水解与微泡脱落无关。通过扫描电子显微镜检查vWf粘附的血小板发现，实际上没有微泡与暴露的vWf多聚体结合。未观察到微泡与粘附血小板的结合，这表明大多数微泡是在血小板激活时从血小板和vWf表面脱落的。用凝血酶原酶活性测定法测量了微泡群体支持促凝血活性的能力，并与粘附血小板膜支持的活性进行了比较。对于未刺激的血小板和用生理性激动剂刺激的血小板，超过85%的总凝血酶原酶活性仍与粘附血小板膜相关。此外，在含有游离血小板和任何释放微泡的缓冲液部分中发现的残余活性可归因于游离血小板。微泡没有活性，因为尽管添加激动剂后检测到微泡释放，但凝血酶刺激vWf或胶原蛋白粘附的血小板相对于未刺激的粘附血小板并没有促进促凝血活性的增加。在粘附血小板系统中，完全的血小板激活和微泡脱落都没有在产生促凝血活性中起关键作用。
