Role of procoagulant lipids in human prothrombin activation. 1. Prothrombin activation by factor Xa in the absence of factor Va and in the absence and presence of membranes
Role of procoagulant lipids in human prothrombin activation. 1. Prothrombin activation by factor Xa in the absence of factor Va and in the absence and presence of membranes
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DOI：
10.1021/bi0116893
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发表时间：
2002-01-22
期刊：
影响因子：
2.9
通讯作者：
Lentz, BR
中科院分区：
文献类型：
--
作者：
Wu, JR;Zhou, CM;Lentz, BR
凝血因子X-a激活凝血酶原需要两个键的蛋白分解，通常认为通过两个平行的、连续的途径发生。Arg(322)-Ile(323)的水解物可产生甲硫凝血酶(MzII(A))作为中间体，而Arg(273)-Thr(274)的水解物可产生凝血酶前2-片段1.2(Pre2-F1.2)。在没有凝血因子V-a和牛磷脂酰丝氨酸(PS)/棕榈酰油酰磷脂酰胆碱(25：75)膜存在和不存在的情况下，检测了人凝血因子X-a对人凝血酶原的激活。收集了四组数据：活性部位探针的荧光对凝血酶、MzII(A)和Pre2-F1.2敏感；合成底物(S-2238)检测凝血酶或MzII(A)活性部位的形成；十二烷基硫酸钠-PAGE检测中间体和凝血酶。荧光数据提供了活性部位的内部检查和SDS-PAGE测量。在没有膜的情况下，直接测量了中间体转化为凝血酶的动力学常数。在膜存在的情况下，MzII(A)和Pre2-F1.2都被迅速消耗，因此这些反应的动力学常数必须通过将三个数据集(凝血酶和MzII(A)活性部位的形成和Pre2的出现)同时拟合到平行序列模型来估计为可调参数。在没有膜的情况下，这个模型成功地描述了数据，并得到了MzII(A)形成速率的速率常数44M-1 S(-1)。相比之下，如果没有假设凝血酶原直接转化为凝血酶而不从膜-酶复合体释放出来的途径存在，平行-顺序模型不能描述在含有PS的膜存在的最佳浓度下的凝血酶原激活。结果表明，PS膜(1)调节凝血因子X-a，(2)改变凝血因子X-a的底物专一性，有利于甲硫唑凝血酶中间体，(3)通道中间体(MzII(A)或Pre2-F1.2)回到凝血因子X-a的活性部位，快速转化为凝血酶。
Activation of prothrombin by factor X-a requires proteolysis of two bonds and is commonly assumed to occur via by two parallel, sequential pathways. Hydrolysis of Arg(322)-Ile(323) produces meizothrombin (MzII(a)) as an intermediate, while hydrolysis of Arg(273)-Thr(274) produces prethrombin 2-fragment 1.2 (Pre2-F1.2). Activation by human factor X-a of human prothrombin was examined in the absence of factor V-a and in the absence and presence of bovine phosphatidylserine (PS)/palmitoyloleoylphosphatidylcholine (25:75) membranes. Four sets of data were collected: fluorescence of an active site probe (DAPA) was sensitive to thrombin, MzII(a), and Pre2-F1.2; a synthetic substrate (S-2238) detected thrombin or MzII(a) active site formation; and SDS-PAGE detected both intermediates and thrombin. The fluorescence data provided an internal check on the active site and SDS-PAGE measurements. Kinetic constants for conversion of intermediates to thrombin were measured directly in the absence of membranes. Both MzII(a) and Pre2-F1.2 were consumed rapidly in the presence of membranes, so kinetic constants for these reactions had to be estimated as adjustable parameters by fitting three data sets (thrombin and MzII(a) active site formation and Pre2 appearance) simultaneously to the parallel-sequential model. In the absence of membranes, this model successfully described the data and yielded a rate constant, 44 M-1 s(-1), for the rate Of MzII(a) formation. By contrast, the parallel-sequential model could not describe prothrombin activation in the presence of optimal concentrations of PS-containing membranes without assuming that a pathway existed for converting prothrombin directly to thrombin without release from the membrane-enzyme complex. The data suggest that PS membranes (1) regulate factor X-a, (2) alter the substrate specificity of factor X-a to favor the meizothrombin intermediate, and (3) "channel" intermediate (MzII(a) or Pre2-F1.2) back to the active site of factor X-a for rapid conversion to thrombin.