Human factor Va (hfVa) is the important regulatory subunit of prothrombinase. Recent modeling data have suggested a critical role for amino acid Arg⁷⁰¹ of hfVa for human prothrombin (hPro) activation by prothrombinase. Furthermore, it has also been demonstrated that hfVa has a different effect than that of bovine fVa on prethrombin-1 activation by prothrombinase. The difference between the two cofactor molecules was also found within the Asn⁷⁰⁰–Arg⁷⁰¹ dipeptide in the human factor V (hfV) molecule, which is replaced by the Asp–Glu sequence in bfV. As a consequence, we produced a recombinant hfV (rhfV) molecule with the substitution ⁷⁰⁰NR⁷⁰¹→DE. rhfV^NR→DE together with the wild-type molecule (rhfV^WT) were expressed in COS7 cells, purified, and tested for their capability to function within prothrombinase. Kinetic studies showed that the K_d of rhfVa^NR→DE for human fXa as well as the k_cat and K_m of prothrombinase made with rhfVa^NR→DE for hPro activation were similar to the values obtained following hPro activation by prothrombinase made with rhfVa^WT. Remarkably, sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses of hPro activation time courses demonstrated that the rate of cleavage of hPro by prothrombinase reconstituted with rhfVa^NR→DE was significantly delayed with substantial accumulation of meizothrombin, and delayed thrombin generation, when compared to activation of hPro by prothrombinase made with rhfVa^WT. These unanticipated results provide significant insights on the role of the carboxyl-terminal end of the heavy chain of hfVa for hPro cleavage and activation by prothrombinase and show that residues ⁷⁰⁰NR⁷⁰¹ regulate at least in part the enzyme–substrate/product interaction during fibrin clot formation.

中文翻译：

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因子Va重链的酸性COOH末端对凝血酶原活性和功能的抑制作用

人因子Va（hfVa）是凝血酶原的重要调节亚基。最近的建模数据表明，hfVa的Arg ⁷⁰¹氨基酸对凝血酶原酶激活人凝血酶原（hPro）的作用至关重要。此外，还证明了hfVa与牛fVa在凝血酶原酶激活凝血酶原1上具有不同的作用。在人因子V（hfV）分子的Asn ⁷⁰⁰ –Arg ⁷⁰¹二肽中也发现了两个辅助因子分子之间的差异，该差异被bfV中的Asp–Glu序列取代。结果，我们产生了具有替换⁷⁰⁰ NR ⁷⁰¹ →DE的重组hfV（rhfV）分子。rhfV ^NR→DE与野生型分子（rhfV ^WT）一起在COS7细胞中表达，纯化并测试了它们在凝血酶原中的功能。动力学研究表明，人fXa的rhfVa ^NR→DE的K _d以及用于hPro激活的rhfVa ^NR→DE制备的凝血酶原酶的k _cat和K _m与通过rhfVa ^WT的凝血酶原激活hPro所获得的值相似。。值得注意的是，hPro活化时间过程的十二烷基硫酸钠聚丙烯酰胺凝胶电泳分析表明，用rhfVa ^NR→DE重构的凝血酶原酶裂解hPro的速率与用rhfVa ^WT制成的凝血酶原酶激活hPro相比，Meizothrombin的大量积累显着延迟了凝血酶的生成，并延迟了凝血酶的生成。这些出乎意料的结果提供了关于hfVa重链羧基末端对凝血酶原酶裂解和激活hPro的作用的重要见解，并表明残基⁷⁰⁰ NR ⁷⁰¹至少部分调节了纤维蛋白凝块中酶-底物/产物的相互作用。编队。