凝血酶原Arg541Trp突变导致静脉血栓发生的机制

The pathogenesis of hereditary venous thromboembolism (VTE) has not been fully elucidated. Investigation on the pathogenic mechanism of gain-of-function mutations helps to further elucidate the problem. In previous work, we found a prothrombin mutation Arg541Trp which had normal activity and antigen level but resulted in the occurrence of familial VTE. However, the mechanism by which it caused was unclear. Thrombin, which was the activated form of prothrombin, was involved in several coagulation processes including procoagulation, anticoagulation and anti-fibrinolysis. The imbalanced functions of these processes can eventually lead to the hypercoagulable state and the occurrence of VTE. According to preliminary results from patient’s plasma and structural analysis, we hypothesized that mutations impaired (1) thrombin inactivation by affecting the binding between antithrombin and thrombin, and (2) protein C (PC) activation by affecting the binding between thrombin and PC. The combined effects of them resulted in imbalanced functions of thrombin and led to the hypercoagulant state. We intended to study the pathogenic mechanism of Arg541Trp mutation by observing the effects of the mutation on the interaction between thrombin and antithrombin and protein C through enzyme kinetics and functional experiments, and evaluating the thrombotic risk of mutant protein in vivo using mouse mesenteric venous thrombosis model.

遗传性静脉血栓形成（venous thromboembolism, VTE）的致病机制尚未完全阐明。研究功能获得型突变的致栓机制有助于进一步阐明该问题。我们在前期工作中发现了一个促凝活性和含量均正常但导致了家族性VTE发生的凝血酶原新突变Arg541Trp，但其致栓机制不明。凝血酶原活化后形成的凝血酶具有促凝、抗凝、抗纤溶多重功能，各功能间的失衡可导致血液的高凝状态和VTE发生。根据血浆检测结果及蛋白结构分析结果，我们猜测突变可能通过影响（1）凝血酶与抗凝血酶结合导致凝血酶灭活受损，以及（2）凝血酶与蛋白C的结合导致蛋白C活化受损，最终导致了凝血酶各功能间的失衡而诱导了高凝状态。我们拟通过酶动力学及凝血功能实验观察突变对凝血酶与抗凝血酶、蛋白C相互作用的影响，并利用小鼠肠系膜静脉血栓模型评估突变蛋白在体内的致栓风险，从而阐明Arg541Trp突变导致VTE发生的机制。

静脉血栓的遗传病因并未完全阐明。前期研究在3个静脉血栓家系中发现了11名携带凝血酶原Arg541Trp突变的携带者，且有8名有静脉血栓发生史，高度提示凝血酶原Arg541Trp突变与静脉血栓发生密切相关，但其致栓机制不明确。为了明确突变的致病性，我们在体外表达了重组的凝血酶原Arg541Trp突变蛋白，利用酶动力学、凝血酶生成试验、各种功能实验对其促凝、抗凝等功能进行了全面研究。结果发现，突变的发生虽然导致凝血酶促凝活性的轻度下降，但也导致了抗凝血酶对其灭活的轻度受损；更为重要的是，突变凝血酶对蛋白C的活化能力严重受损，使得蛋白C系统的抗凝功能减弱。换言之，相较于促凝功能的轻微减弱，Arg541Trp突变凝血酶的抗凝功能受损更为严重，综合表现为高凝倾向，导致了静脉血栓的发生。综上所述，凝血酶原Arg541Trp突变通过影响蛋白C抗凝系统的功能间接导致高血栓风险，是一个新的致栓遗传性危险因素。