Extracellular protein disulfide isomerase regulates feedback activation of platelet thrombin generation via modulation of coagulation factor binding
Extracellular protein disulfide isomerase regulates feedback activation of platelet thrombin generation via modulation of coagulation factor binding
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DOI：
10.1111/j.1538-7836.2011.04509.x
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发表时间：
2011-11-01
影响因子：
10.4
通讯作者：
Kehrel, B. E.
中科院分区：
文献类型：
--
作者：
Jurk, K.;Lahav, J.;Kehrel, B. E.
背景：蛋白二硫异构酶（PDI）控制血小板整合素功能、组织因子（TF）激活，并集中在血管损伤的纤维蛋白和血栓形成部位。目的：探讨表面硫醇异构酶，尤其是PDI在凝血酶介导的血小板凝血酶扩增中的作用。方法/结果：使用新开发的凝血酶依赖性血小板凝血酶生成实验，我们观察到血小板表面凝血酶生成的反馈激活不依赖于TF，因为与载体处理的对照组相比，抗TF抗体抑制TF诱导的血小板耗尽血浆中的凝血酶形成没有作用。在血小板存在的情况下，凝血酶生成的反馈激活分别被膜外巯基阻滞剂或巯基异构酶抑制剂杆菌肽和抗pdi抗体RL90显著减弱。血小板凝血酶的形成依赖于凝血因子与血小板表面的结合。因此，研究了巯基异构酶在这种结合中的作用。共聚焦显微镜和流式细胞术显示，与未受刺激的血小板相比，凝血酶刺激的血小板表现出增加的表面相关PDI和细胞外二硫还原酶活性。流式细胞术分析显示，在刺激血小板和暴露于磷脂酰丝氨酸后加入巯基阻滞剂或PDI抑制剂，以排除其对原发性血小板活化的影响，可显著抑制所有凝血因子与凝血酶刺激血小板的结合。结论：因此，表面相关的PDI是凝血因子连接到凝血酶刺激的血小板以及随后血小板凝血酶生成的反馈激活的重要调节因子。因此，细胞表面硫醇异构酶可能是控制止血和血栓形成的有力靶点。
Background: Protein disulfide isomerase (PDI) controls platelet integrin function, tissue-factor (TF) activation, and concentrates at fibrin and thrombus formation sites of vascular injury. Objective: To investigate the involvement of surface thiol isomerases and especially PDI, in thrombin-mediated thrombin amplification on human platelets. Methods/results: Using a newly developed thrombin-dependent platelet thrombin generation assay, we observed that the feedback activation of thrombin generation on the platelet surface does not depend on TF, as anti-TF antibodies inhibiting TF-induced thrombin formation in platelet-depleted plasma had no effect compared with vehicle-treated controls. Feedback activation of thrombin generation in the presence of platelets was significantly diminished by membrane impermeant thiol blockers or by the thiol isomerase-inhibitors bacitracin and anti-PDI antibody RL90, respectively. Platelet thrombin formation depends on binding of coagulation factors to the platelet surface. Therefore, involvement of thiol isomerases in this binding was investigated. As shown by confocal microscopy and flow cytometry, thrombin-stimulated platelets exhibited increased surface-associated PDI as well as extracellular disulfide reductase activity compared with unstimulated platelets. Flow cytometric analysis revealed that membrane impermeant thiol blockers or PDI inhibitors, which had been added after platelet stimulation and after phosphatidylserine exposure to exclude their influence on primary platelet activation, significantly inhibited binding of all coagulation factors to thrombin-stimulated platelets. Conclusions: Thus, surface-associated PDI is an important regulator of coagulation factor ligation to thrombin-stimulated platelets and of subsequent feedback activation of platelet thrombin generation. Cell surface thiol isomerases might be therefore powerful targets to control hemostasis and thrombosis.