Role of the acidic hirudin-like COOH-terminal amino acid region of factor Va heavy chain in the enhanced function of prothrombinase.
Role of the acidic hirudin-like COOH-terminal amino acid region of factor Va heavy chain in the enhanced function of prothrombinase.
复制标题
Va 因子重链酸性水蛭素样 COOH 末端氨基酸区域在增强凝血酶原酶功能中的作用。
DOI：
10.1021/bi800593k
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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：
Hirbawi,Jamila;Bukys,MichaelA;Barhoover,MelissaA;Erdogan,Evrim;Kalafatis,Michael
凝血酶原酶通过在Arg 320处的初始切割，随后在Arg 271处的切割来活化凝血酶原。该途径的特征在于产生具有酶活性的瞬时中间体甲唑凝血酶，其具有增加的显色底物活性，但凝血活性差。因子Va的重链在COOH末端含有酸性区域（残基680−709）。我们已经表明，从这个区域（DYDYQ）的五肽抑制凝血酶原酶通过抑制meizothrobin生成抑制凝血酶原激活。为了确定这些区域的功能，我们创建了一个缺失重链最后30个氨基酸的突变重组因子V分子（因子VΔ680−709）和一个具有695 DYDY 698 → AAAA取代的突变分子（因子V4 A）。与野生型因子Va（因子VaWt）的凝血活性相比，两种重组突变因子Va分子的凝血活性受损。使用使用纯化试剂的测定，我们发现与因子VaΔ680− 709组装的凝血酶原酶显示出增加了39%的inkcat，而与因子Va 4A组装的凝血酶原酶显示出增加了20%的inkcat，与因子VaWt组装的凝血酶原酶相比，凝血酶原酶活化。凝胶电泳分析凝血酶原激活与突变体分子组装的凝血酶原酶显示了延迟凝血酶原激活与持久性meizothrobin。我们的数据表明，因子Va重链的COOH-末端区域确实是至关重要的协调凝血酶原激活凝血酶原酶，因为它调节美佐凝血酶裂解Arg 271，并表明，这部分因子Va是部分负责增强凝血功能的凝血酶原酶。
Prothrombinase activates prothrombin through initial cleavage at Arg320followed by cleavage at Arg271. This pathway is characterized by the generation of an enzymatically active, transient intermediate, meizothrombin, that has increased chromogenic substrate activity but poor clotting activity. The heavy chain of factor Va contains an acidic region at the COOH terminus (residues 680−709). We have shown that a pentapeptide from this region (DYDYQ) inhibits prothrombin activation by prothrombinase by inhibiting meizothrombin generation. To ascertain the function of these regions, we have created a mutant recombinant factor V molecule that is missing the last 30 amino acids from the heavy chain (factor VΔ680−709) and a mutant molecule with the695DYDY698→ AAAA substitutions (factor V4A). The clotting activities of both recombinant mutant factor Va molecules were impaired compared to the clotting activity of wild-type factor Va (factor VaWt). Using an assay employing purified reagents, we found that prothrombinase assembled with factor VaΔ680−709displayed an ∼39% increase inkcat, while prothrombinase assembled with factor Va4Aexhibited an ∼20% increase inkcatfor the activation of prothrombin as compared to prothrombinase assembled with factor VaWt. Gel electrophoresis analyzing prothrombin activation by prothrombinase assembled with the mutant molecules revealed a delay in prothrombin activation with persistence of meizothrombin. Our data demonstrate that the COOH-terminal region of factor Va heavy chain is indeed crucial for coordinated prothrombin activation by prothrombinase because it regulates meizothrombin cleavage at Arg271and suggest that this portion of factor Va is partially responsible for the enhanced procoagulant function of prothrombinase.