Phosphatidylserine binding alters the conformation and specifically enhances the cofactor activity of bovine factor Va.
Phosphatidylserine binding alters the conformation and specifically enhances the cofactor activity of bovine factor Va.
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DOI：
10.1021/bi011844d
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发表时间：
2002-04
期刊：
影响因子：
2.9
通讯作者：
X. Zhai;A. Srivastava;D. Drummond;D. Daleke;B. Lentz
中科院分区：
文献类型：
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作者：
X. Zhai;A. Srivastava;D. Drummond;D. Daleke;B. Lentz
因子 V(a) 是丝氨酸蛋白酶因子 X(a) 的辅助因子，在 Ca(2+) 和血小板膜表面存在的情况下将凝血酶原激活为凝血酶。血小板膜脂质磷脂酰丝氨酸 (PS) 调节因子 X(a) 的蛋白水解活性以及凝血酶原的结构。在这里，我们询问 PS 是否还调节因子 V(a) 的结构和辅因子活性，因子 V(a) 是由一条重链（A1-A2 结构域）和一条轻链（A3-C1-C2 结构域）组成的异二聚体。我们使用荧光、圆二色性、平衡透析和活性测量来证明以下内容：（1）因子 V(a) 有四个二己酰基-sn-甘油-3-磷酸-L-丝氨酸（C(6)PS，PS 的可溶形式）位点；重链和轻链各结合两个 C(6)PS 分子。 (2) 在没有 Ca(2+) 的情况下，仅保留两个位点，一个位于重链，另一个位于轻链。 (3) 与这些位点的结合引起构象变化，这通过内在荧光和圆二色光谱的变化以及辅因子活性的变化来证明。 (4) 四个脂质结合位点中至少有一些对于可溶性脂质种类是非特异性的，但调节辅因子活性的位点对于 C(6)PS、磷脂酸或磷脂酰（高）丝氨酸具有特异性，并产生与含 PS 膜所见的响应相当的响应。 (5) 与 Ca(2+) 一样，C(6)PS 也介导因子 V(a) 重链 (V(a)-HC) 和轻链 (V(a)-LC) 之间的相互作用。我们得出结论，PS 调节凝血酶原激活复合物的辅因子和酶。
Factor V(a) is a cofactor for the serine protease factor X(a) that activates prothrombin to thrombin in the presence of Ca(2+) and a platelet membrane surface. A platelet membrane lipid, phosphatidylserine (PS), regulates the proteolytic activity of factor X(a) as well as the structure of prothrombin. Here we ask whether PS also regulates the structure and cofactor activity of factor V(a), which is a heterodimer composed of one heavy chain (A1-A2 domains) and one light chain (A3-C1-C2 domains). We use fluorescence, circular dichroism, equilibrium dialysis, and activity measurements to demonstrate the following: (1) Factor V(a) has four sites for dicaproyl-sn-glycero-3-phospho-L-serine (C(6)PS, a soluble form of PS); the heavy and light chains each bind two C(6)PS molecules. (2) In the absence of Ca(2+), only two sites remain, one in the heavy chain and another in the light chain. (3) Binding to these sites causes conformational changes evidenced by changes in intrinsic fluorescence and in CD spectra and changes in cofactor activity. (4) At least some of the four lipid binding sites are nonspecific with respect to soluble lipid species, but the site(s) that regulate(s) cofactor activity is (are) specific for C(6)PS, phosphatidic acid, or phosphatidyl(homo)serine and produce a response comparable to that seen with a PS-containing membrane. (5) Like Ca(2+), C(6)PS also mediates the interaction between factor V(a) heavy (V(a)-HC) and light (V(a)-LC) chains. We conclude that PS regulates both the cofactor and the enzyme of the prothrombin-activating complex.