A phosphatidylserine binding site in factor Va C1 domain regulates both assembly and activity of the prothrombinase complex.
A phosphatidylserine binding site in factor Va C1 domain regulates both assembly and activity of the prothrombinase complex.
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DOI：
10.1182/blood-2008-02-138941
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发表时间：
2008-10
期刊：
影响因子：
20.3
通讯作者：
Rinku Majumder;M. Quinn-Allen;W. Kane;B. Lentz
中科院分区：
文献类型：
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作者：
Rinku Majumder;M. Quinn-Allen;W. Kane;B. Lentz
紧密相关的因子V(a) （FVa）和因子X(a) （FXa）作为必需的凝血酶原激活复合物，在凝血过程中聚集在含磷脂酰丝氨酸（PS）的血小板膜上。我们之前已经证明：(1)可溶性形式的PS （C6PS）触发溶液中完全活性的FVa- fxa复合物的组装；(2)2个C6PS分子与FVa轻链结合，其中一个占据C2结构域的一个位点。我们表达了人因子V(a) （rFVa）在C1结构域（Y1956,L1957） a、C2结构域（W2063,W2064） a或两个C结构域（Y1956,L1957,W2063,W2064） a中的突变。与野生型或C2突变因子rFVa相比，rFVa的C1和C1-C2结构域的突变使400 muM C6PS存在时FXa将凝血酶原活化为凝血酶的速率降低了14000至15000倍。FXa与rFVa（野生型、C2突变型、C1突变型和C1-C2突变型）结合的K(d)s分别为3、4、564和624 nM。平衡透析实验检测到4、3、2个C6PS分子分别与野生型rFVa、C1突变的rFVa和C1、c2突变的rFVa结合。由于FVa重链结合2个C6PS分子，我们得出结论，C2和C1结构域结合一个C6PS，与C1结构域结合调节凝血酶原复合物的组装。
Tightly associated factor V(a) (FVa) and factor X(a) (FXa) serve as the essential prothrombin-activating complex that assembles on phosphatidylserine (PS)-containing platelet membranes during blood coagulation. We have previously shown that (1) a soluble form of PS (C6PS) triggers assembly of a fully active FVa-FXa complex in solution and (2) that 2 molecules of C6PS bind to FVa light chain with one occupying a site in the C2 domain. We expressed human factor V(a) (rFVa) with mutations in either the C1 domain (Y1956,L1957)A, the C2 domain (W2063,W2064)A, or both C domains (Y1956,L1957,W2063,W2064)A. Mutations in the C1 and C1-C2 domains of rFVa reduced the rate of activation of prothrombin to thrombin by FXa in the presence of 400 muM C6PS by 14 000- to 15 000-fold relative to either wild-type or C2 mutant factor rFVa. The K(d')s of FXa binding with rFVa (wild-type, C2 mutant, C1 mutant, and C1-C2 mutant) were 3, 4, 564, and 624 nM, respectively. Equilibrium dialysis experiments detected binding of 4, 3, and 2 molecules of C6PS to wild-type rFVa, C1-mutated, and C1,C2-mutated rFVa, respectively. Because FVa heavy chain binds 2 molecules of C6PS, we conclude that both C2 and C1 domains bind one C6PS, with binding to the C1 domain regulating prothrombinase complex assembly.