Contribution of the prothrombin fragment 2 domain to the function of factor Va in the prothrombinase complex

被引:39
作者
Krishnaswamy, S
Walker, RK
机构
[1] Division of Hematology/Oncology, Department of Medicine, Emory University, Atlanta
[2] Division ot Hematology/Oncology, Department of Medicine, Emory University, Atlanta, GA 30322
[3] Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston
关键词
D O I
10.1021/bi9623993
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The prothrombinase complex assembles through reversible interactions between factor Xa, factor Va and acidic phospholipid-containing membranes in the presence of calcium ions. This complex catalyses the conversion of prothrombin to thrombin through two proteolytic steps. We have used prethrombin 2 as a substrate analog for the first cleavage reaction of prothrombin activation (cleavage at Arg(323)-Ile(324)) catalyzed by the prothrombinase complex and have also relied on the known ability of prethrombin 2 to interact tightly but reversibly with fragment 2 or fragment 1.2. The kinetics of cleavage at Arg(323)-Ile(324) have been assessed with these substrate analogs to investigate the contribution of cofactor-substrate interactions mediated by the fragment 2 domain to the ability of factor Va to enhance the catalytic efficiency of factor Xa within the prothrombinase complex. Initial velocity measurements indicated that the rate of prethrombin 2 cleavage by the factor Xa-PCPS binary complex was increased by a factor of similar to 1300 upon the addition of saturating concentrations of factor Va to assemble prothrombinase. Although the measured initial velocity was higher when either fragment 2 or fragment 1.2 was present, the factor Va-dependent enhancement in initial rate (2600- and 1500-fold) was comparable in each case. Steady state kinetic constants were obtained using prethrombin 2, prethrombin 2 plus fragment 2, and prethrombin 2 plus fragment 1.2 as substrates. For each substrate, the addition of saturating concentrations of factor Va to the Xa-PCPS binary complex led to increases in catalytic efficiency of between 1000 and 9000-fold. The kcat/Km for prethrombin 2 cleavage by prothrombinase was essentially identical to that obtained for prethrombin 2 saturated with fragment 2. Thus, comparable accelerating effects of factor Va are observed independent of the presence of the fragment 2 domain in the substrate. The results indicate that interactions between factor Va and the substrate mediated by the fragment 2 domain do not contribute in a dominant way to the ability of factor Va to enhance the catalytic efficiency of factor Xa within the prothrombinase complex.
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页码:3319 / 3330
页数:12
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