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首页> 外文期刊>Biochemistry >Contribution of the prothrombin fragment 2 domain to the function of factor Va in the prothrombinase complex.
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Contribution of the prothrombin fragment 2 domain to the function of factor Va in the prothrombinase complex.

机译：凝血酶原片段2结构域对凝血酶原酶复合物中因子Va的功能的贡献。

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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 Arg323-Ile324) 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 Arg323-Ile324 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 approximately 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.
机译：在钙离子存在下，凝血酶原酶复合物通过因子Xa，因子Va和酸性含磷脂膜之间的可逆相互作用而组装。该复合物通过两个蛋白水解步骤催化凝血酶原向凝血酶的转化。我们已经使用凝血酶原2作为底物类似物，用于由凝血酶原酶复合物催化的凝血酶原激活的第一次裂解反应（在Arg323-Ile324处裂解），并且还依赖于凝血酶原2与片段2或片段紧密但可逆地相互作用的已知能力。 1.2。已经用这些底物类似物评估了在Arg323-Ile324处的切割动力学，以研究由片段2结构域介导的辅因子-底物相互作用对凝血因子Va增强凝血酶原酶复合物中的因子Xa的催化效率的能力的贡献。初始速度测量结果表明，添加饱和浓度的因子Va组装凝血酶原酶后，因子Xa-PCPS二元复合物对凝血酶原2的切割速率增加了约1300倍。尽管当存在片段2或片段1.2时，测得的初始速度较高，但在每种情况下，因子Va依赖的初始速率增强（2600和1500倍）是可比较的。使用凝血酶原2，凝血酶原2加片段2和凝血酶原2加片段1.2作为底物获得稳态动力学常数。对于每种底物，在Xa-PCPS二元络合物中添加饱和浓度的因子Va导致催化效率提高了1000到9000倍。通过凝血酶原酶裂解凝血酶原2的kcat / Km与用片段2饱和的凝血酶原2获得的kcat / Km基本相同。因此，观察到相当的因子Va加速作用，而与底物中片段2结构域的存在无关。结果表明，因子Va与由片段2结构域介导的底物之间的相互作用不以主要方式有助于因子Va增强凝血酶原酶复合物中因子Xa的催化效率的能力。

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