Bajzar L, Kalafatis M, Simioni P, Tracy P B
Department of Biochemistry, College of Medicine, University of Vermont, Burlington, Vermont 05405, USA.
J Biol Chem. 1996 Sep 20;271(38):22949-52. doi: 10.1074/jbc.271.38.22949.
Factor Va is the essential cofactor in prothrombinase-dependent activation of prothrombin. Resistance of Factor VaLeiden to inactivation by activated protein C (APC) contributes to thrombotic tendencies in subjects with the variant due, in part, to the inability to terminate thrombin production which increases both fibrin accretion and the frequency of thrombus formation. A reduced ability to inhibit thrombin generation, however, may lead to the stabilization of a clot through the activation of thrombin activatable fibrinolysis inhibitor (TAFI). This hypothesis was tested by determining the profibrinolytic effect of APC on lysis time using clots formed with plasma from either homozygous normal (n = 4) or homozygous factor VLeiden (n = 4) subjects. Clots were formed in the presence of tissue-type plasminogen activator, thrombin, phosphatidylcholine/phosphatidylserine vesicles, Ca2+, and various concentrations of APC. Approximately 10-fold more APC was required to reduce lysis time from 140 to 50 min in clots containing factor VLeiden compared to normal factor V. This effect was specific to the form of factor V present in plasma since identical results were obtained in an appropriately reconstituted purified system, which included both TAFI and either form of factor V purified from pooled plasma. In the absence of TAFI, APC did not affect clot lysis in experiments with either normal factor V or factor VLeiden. During the various lysis assays performed with purified components, clots were solubilized and the proteolytic alterations in factor V/Va were assessed by Western blotting using a specific factor Va heavy chain monoclonal antibody. The heavy chain of factor VaLeiden persisted for as long as 60 min, in the presence of 6.3 n APC indicating sustained activity of factor VaLeiden during the lysis assay. In contrast, no factor Va heavy chain was present after the first 5.0 min in clots formed in the presence of normal factor V and 6.3 n APC. These combined data indicate that factor VaLeiden specifically attenuates the profibrinolytic effect of APC. Thus, an impaired TAFI-dependent profibrinolytic response to APC in APC-resistant individuals appears to be an additional factor contributing to the prothrombotic tendencies in subjects with factor VLeiden.
因子Va是凝血酶原酶依赖性激活凝血酶原过程中必不可少的辅因子。因子V莱顿对活化蛋白C（APC）灭活具有抗性，这导致携带该变体的个体出现血栓形成倾向，部分原因是无法终止凝血酶的产生，从而增加了纤维蛋白的沉积和血栓形成的频率。然而，抑制凝血酶生成的能力降低可能会通过激活凝血酶激活的纤维蛋白溶解抑制剂（TAFI）导致血凝块稳定。通过使用来自纯合正常个体（n = 4）或纯合因子V莱顿个体（n = 4）的血浆形成的血凝块，测定APC对溶解时间的促纤溶作用，对这一假设进行了检验。在组织型纤溶酶原激活剂、凝血酶、磷脂酰胆碱/磷脂酰丝氨酸囊泡、Ca2+和不同浓度的APC存在的情况下形成血凝块。与正常因子V相比，含有因子V莱顿的血凝块中，将溶解时间从140分钟缩短至50分钟所需的APC大约多10倍。这种效应对于血浆中存在的因子V形式具有特异性，因为在适当重构的纯化系统中获得了相同的结果，该系统包括TAFI和从混合血浆中纯化的两种形式的因子V。在没有TAFI的情况下，在使用正常因子V或因子V莱顿进行的实验中，APC不影响血凝块溶解。在用纯化成分进行的各种溶解试验中，血凝块溶解，并使用特异性因子Va重链单克隆抗体通过蛋白质印迹法评估因子V/Va的蛋白水解变化。在存在浓度为6.3 n的APC时，因子V莱顿的重链持续长达60分钟，表明在溶解试验期间因子V莱顿具有持续活性。相比之下，在存在正常因子V和浓度为6.3 n的APC的情况下形成的血凝块中，在最初5.0分钟后不存在因子Va重链。这些综合数据表明，因子V莱顿特异性减弱了APC的促纤溶作用。因此，在APC抵抗个体中，TAFI依赖性对APC的促纤溶反应受损似乎是导致因子V莱顿个体血栓形成倾向的另一个因素。
