Department of Biochemistry, CARIM, Maastricht University, Maastricht, The Netherlands.
PLoS One. 2010 Apr 29;5(4):e10415. doi: 10.1371/journal.pone.0010415.
The generation of thrombin is a critical process in the formation of venous thrombi. In isolated plasma under static conditions, phosphatidylserine (PS)-exposing platelets support coagulation factor activation and thrombin generation; however, their role in supporting coagulation factor binding under shear conditions remains unclear. We sought to determine where activated factor X (FXa), (pro)thrombin, and fibrin(ogen) are localized in thrombi formed under venous shear.
METHODOLOGY/PRINCIPAL FINDINGS: Fluorescence microscopy was used to study the accumulation of platelets, FXa, (pro)thrombin, and fibrin(ogen) in thrombi formed in vitro and in vivo. Co-perfusion of human blood with tissue factor resulted in formation of visible fibrin at low, but not at high shear rate. At low shear, platelets demonstrated increased Ca(2+) signaling and PS exposure, and supported binding of FXa and prothrombin. However, once cleaved, (pro)thrombin was observed on fibrin fibers, covering the whole thrombus. In vivo, wild-type mice were injected with fluorescently labeled coagulation factors and venous thrombus formation was monitored in mesenteric veins treated with FeCl(3). Thrombi formed in vivo consisted of platelet aggregates, focal spots of platelets binding FXa, and large areas binding (pro)thrombin and fibrin(ogen).
CONCLUSIONS/SIGNIFICANCE: FXa bound in a punctate manner to thrombi under shear, while thrombin and fibrin(ogen) distributed ubiquitously over platelet-fibrin thrombi. During thrombus formation under venous shear, thrombin may relocate from focal sites of formation (on FXa-binding platelets) to dispersed sites of action (on fibrin fibers).
凝血酶的生成是静脉血栓形成过程中的一个关键步骤。在静态条件下的分离血浆中，暴露磷脂酰丝氨酸（PS）的血小板支持凝血因子激活和凝血酶生成；然而，它们在剪切条件下支持凝血因子结合的作用仍不清楚。我们试图确定在静脉剪切下形成的血栓中，激活的因子 X（FXa）、（原）凝血酶和纤维蛋白原（ogen）在何处定位。
方法/主要发现：荧光显微镜用于研究体外和体内形成的血栓中血小板、FXa、（原）凝血酶和纤维蛋白原（ogen）的积累。人体血液与组织因子共同灌注导致在低剪切速率下形成可见的纤维蛋白，但在高剪切速率下则不行。在低剪切下，血小板表现出增加的 Ca(2+)信号和 PS 暴露，并支持 FXa 和凝血酶原的结合。然而，一旦被切割，（原）凝血酶就会在纤维蛋白纤维上被观察到，覆盖整个血栓。在体内，用荧光标记的凝血因子对野生型小鼠进行注射，并在用 FeCl(3)处理的肠系膜静脉中监测静脉血栓形成。体内形成的血栓由血小板聚集物、血小板结合 FXa 的局灶性斑点和结合（原）凝血酶和纤维蛋白原（ogen）的大片区域组成。
结论/意义：FXa 在剪切下以点状方式结合到血栓中，而凝血酶和纤维蛋白原（ogen）在血小板-纤维蛋白血栓中广泛分布。在静脉剪切下形成血栓时，凝血酶可能从形成的局灶性部位（在结合 FXa 的血小板上）转移到弥散性作用部位（在纤维蛋白纤维上）。
