Department of Biochemistry, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.
J Biol Chem. 2012 Nov 9;287(46):38647-55. doi: 10.1074/jbc.M112.407791. Epub 2012 Sep 18.
The protease α-thrombin is a key enzyme of the coagulation process as it is at the cross-roads of both the pro- and anti-coagulant pathways. The main source of α-thrombin in vivo is the activation of prothrombin by the prothrombinase complex assembled on either an activated cell membrane or cell fragment, the most relevant of which is the activated platelet surface. When prothrombinase is assembled on synthetic phospholipid vesicles, prothrombin activation proceeds with an initial cleavage at Arg-320 yielding the catalytically active, yet effectively anticoagulant intermediate meizothrombin, which is released from the enzyme complex ∼30-40% of the time. Prothrombinase assembled on the surface of activated platelets has been shown to proceed through the inactive intermediate prethrombin-2 via an initial cleavage at Arg-271 followed by cleavage at Arg-320. The current work tests whether or not platelet-associated prothrombinase proceeds via a concerted mechanism through a study of prothrombinase assembly and function on collagen-adhered, thrombin-activated, washed human platelets in a flow chamber. Prothrombinase assembly was demonstrated through visualization of bound factor Xa by confocal microscopy using a fluorophore-labeled anti-factor Xa antibody, which demonstrated the presence of distinct platelet subpopulations capable of binding factor Xa. When prothrombin activation was monitored at a typical venous shear rate over preassembled platelet-associated prothrombinase neither potential intermediate, meizothrombin or prethrombin-2, was observed in the effluent. Collectively, these findings suggest that platelet-associated prothrombinase activates prothrombin via an efficient concerted mechanism in which neither intermediate is released.
蛋白酶α-凝血酶是凝血过程中的关键酶，因为它处于促凝和抗凝途径的交汇点。体内α-凝血酶的主要来源是凝血酶原酶复合物在激活的细胞膜或细胞碎片上的组装，其中最相关的是激活的血小板表面。当凝血酶原酶在合成磷脂囊泡上组装时，凝血酶原的激活最初在 Arg-320 处进行切割，产生具有催化活性但实际上具有抗凝作用的中间产物 meizothrombin，该中间产物约有 30-40%的时间从酶复合物中释放出来。已表明在激活的血小板表面组装的凝血酶原酶通过在 Arg-271 处的初始切割，然后在 Arg-320 处的切割，通过无活性的中间产物 prethrombin-2 进行。目前的工作通过在流动室中研究胶原附着的、凝血酶激活的、已洗涤的人类血小板上的凝血酶原酶的组装和功能，检验血小板相关凝血酶原酶是否通过协同机制进行。通过使用荧光标记的抗因子 Xa 抗体对结合的因子 Xa 进行共聚焦显微镜可视化，证明了血小板相关凝血酶原酶的组装，这表明存在能够结合因子 Xa 的不同血小板亚群。当在典型的静脉剪切速率下监测预组装的血小板相关凝血酶原酶中的凝血酶原激活时，在流出物中未观察到潜在的中间产物 meizothrombin 或 prethrombin-2。总的来说，这些发现表明血小板相关凝血酶原酶通过一种有效的协同机制激活凝血酶原，其中没有中间产物释放。
