Cooperative regulation of the activity of factor Xa within prothrombinase by discrete amino acid regions from factor Va heavy chain.
Cooperative regulation of the activity of factor Xa within prothrombinase by discrete amino acid regions from factor Va heavy chain.
复制标题
通过因子 Va 重链的离散氨基酸区域协同调节凝血酶原酶内因子 Xa 的活性。
DOI：
10.1021/bi801241r
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发表时间：
2008
期刊：
影响因子：
2.9
通讯作者：
Kalafatis,Michael
中科院分区：
文献类型：
--
作者：
Barhoover,MelissaA;Orban,Tivadar;Bukys,MichaelA;Kalafatis,Michael
凝血酶原酶复合物催化凝血酶原活化为α-凝血酶。我们已经反复证明，氨基酸区域695 DYDY 698从因子Va的重链的COOH末端调节凝血酶原酶的切割率的凝血酶原在Arg 271。我们最近还证明了氨基酸区域334 DY 335是凝血酶原酶最佳活性所必需的。为了评估这六个氨基酸残基对辅因子活性的影响，我们创建了重组因子Va分子，其在氨基酸区域334-335和695 - 698处组合突变，如下所示：因子V3 K（334 DY 335 → KF和695 DYDY 698 → KFKF）、因子VKF/4A（334 DY 335 → KF和695 DYDY 698 → AAAA）和因子V6 A（334 DY 335 → AA和695 DYDY 698 → AAAA）。表达重组因子V分子并纯化至均一。与野生型分子（因子VaWt）对酶的亲和力相比，因子Va 3 K、因子VaK 4/4A和因子Va 6A对因子Xa的亲和力降低。与用因子VaWt组装的凝血酶原酶获得的值相比，用饱和浓度的因子Va 3 K组装的凝血酶原酶具有6倍降低的凝血酶原活化的二级速率常数，而用饱和浓度的因子VaKF/4A和因子Va 6A组装的凝血酶原酶具有约1.5倍降低的二级速率常数。总之，数据表明，来自因子Va重链的氨基酸区域334-335与氨基酸区域695 - 698是凝血酶原酶内调节因子Xa对凝血酶原的切割和活化的协同机制的一部分。
The prothrombinase complex catalyzes the activation of prothrombin to α-thrombin. We have repetitively shown that amino acid region695DYDY698from the COOH terminus of the heavy chain of factor Va regulates the rate of cleavage of prothrombin at Arg271by prothrombinase. We have also recently demonstrated that amino acid region334DY335is required for the optimal activity of prothrombinase. To assess the effect of these six amino acid residues on cofactor activity, we created recombinant factor Va molecules combining mutations at amino acid regions 334–335 and 695−698 as follows: factor V3K(334DY335→ KF and695DYDY698→ KFKF), factor VKF/4A(334DY335→ KF and695DYDY698→ AAAA), and factor V6A(334DY335→ AA and695DYDY698→ AAAA). The recombinant factor V molecules were expressed and purified to homogeneity. Factor Va3K, factor VaK4/4A, and factor Va6Ahad reduced affinity for factor Xa, when compared to the affinity of the wild-type molecule (factor VaWt) for the enzyme. Prothrombinase assembled with saturating concentrations of factor Va3Khad a 6-fold reduced second-order rate constant for prothrombin activation compared to the value obtained with prothrombinase assembled with factor VaWt, while prothrombinase assembled with saturating concentrations of factor VaKF/4Aand factor Va6Ahad approximately 1.5-fold reduced second-order rate constants. Overall, the data demonstrate that amino acid region 334–335 together with amino acid region 695−698 from factor Va heavy chain are part of a cooperative mechanism within prothrombinase regulating cleavage and activation of prothrombin by factor Xa.