Yang T L, Cui J, Rehumtulla A, Yang A, Moussalli M, Kaufman R J, Ginsburg D
Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109-0650, USA.
Blood. 1998 Jun 15;91(12):4593-9.
Factor V (FV) is a central regulator of hemostasis, serving both as a critical cofactor for the prothrombinase activity of factor Xa and the target for proteolytic inactivation by the anticoagulant, activated protein C (APC). To examine the evolutionary conservation of FV procoagulant activity and functional inactivation by APC, we cloned and sequenced the coding region of murine FV cDNA and generated recombinant wild-type and mutant murine FV proteins. The murine FV cDNA encodes a 2,183-amino acid protein. Sequence comparison shows that the A1-A3 and C1-C2 domains of FV are highly conserved, demonstrating greater than 84% sequence identity between murine and human, and 60% overall amino acid identity among human, bovine, and murine FV sequences. In contrast, only 35% identity among all three species is observed for the poorly conserved B domain. The arginines at all thrombin cleavage sites and the R305 and R504 APC cleavage sites (corresponding to amino acid residues R306 and R506 in human FV) are invariant in all three species. Point mutants were generated to substitute glutamine at R305, R504, or both (R305/R504). Wild-type and all three mutant FV recombinant proteins show equivalent FV procoagulant activity. Single mutations at R305 or R504 result in partial resistance of FV to APC inactivation, whereas recombinant murine FV carrying both mutations (R305Q/R504Q) is nearly completely APC resistant. Thus, the structure and function of FV and its interaction with APC are highly conserved across mammalian species.
凝血因子V（FV）是止血过程的核心调节因子，它既是因子Xa凝血酶原酶活性的关键辅助因子，也是抗凝剂活化蛋白C（APC）进行蛋白水解失活作用的靶点。为了研究FV促凝血活性的进化保守性以及APC对其功能的失活作用，我们克隆并测序了小鼠FV cDNA的编码区，并制备了重组野生型和突变型小鼠FV蛋白。小鼠FV cDNA编码一种含2183个氨基酸的蛋白质。序列比较显示，FV的A1 - A3和C1 - C2结构域高度保守，小鼠和人类之间的序列同一性大于84%，人类、牛和小鼠FV序列的总体氨基酸同一性为60%。相比之下，保守性较差的B结构域在所有三个物种中的同一性仅为35%。所有三个物种中，所有凝血酶切割位点以及R305和R504 APC切割位点（对应于人类FV中的氨基酸残基R306和R506）的精氨酸都是不变的。我们构建了点突变体，将R305、R504或两者（R305/R504）处的精氨酸替换为谷氨酰胺。野生型和所有三种突变型FV重组蛋白均表现出相当的FV促凝血活性。R305或R504处的单突变导致FV对APC失活产生部分抗性，而携带双突变（R305Q/R504Q）的重组小鼠FV几乎完全抗APC。因此，FV的结构和功能及其与APC的相互作用在哺乳动物物种中高度保守。
