Role of phosphatidylserine in the activation of blood coagulation factor VIII.

众所周知，在存在特定的膜磷脂（磷脂酰丝氨酸）的情况下，由tenase和凝血酶原酶复合物催化的凝血级联的两个连续反应被大大加速。本课题的目的是阐明PS特异性激活凝血级联反应的分子机制。我们首先建立了一个单克隆抗体（mAb），PS4 A7，它特异性结合PS和确定的重链和轻链可变区的氨基酸序列。结果发现，PS4 A7重链CDR-3区的12个氨基酸残基的合成肽与PS特异性结合，说明CDR-3区在与PS的结合中起主导作用，并来源于负责与PS特异性结合的肽基序。我们发现一种名为Id 8 F7的抗独特型mAb与蛋白激酶C（一种需要PS进行酶促活化的酶）以及凝血因子VIII（FVIII）和因子V（FV）广泛交叉反应。这一发现表明，PS特异性mAb和凝血因子共享的共同结构，这是负责与PS的特异性相互作用。为了确定PS特异性结合位点，我们映射的Id 8 F7结合位点FV使用的各种重组蛋白片段。结果表明，Id 8 F7结合位点位于FV轻链的C2区，是推测的PS特异性结合位点。

It is well known that two consecutive reactions of blood coagulation cascade catalyzed by the tenase and prothrombinase complex are accelerated greatly in the presence of a particular membrane phospholipid, phosphatidylserine. The aim of this project is to elucidate the molecular mechanism underlying the specific activation of blood coagulation cascade by PS.We have employed a series of immunochemical approach to identify the PS-specific binding sites on the blood coagulation factors. We first established a monoclonal antibody (mAb) , PS4A7, which binds specifically to PS and determine the amino acid sequences of the heavy-and light-chain variable regions. We found that the 12 amino acid residue synthetic peptide derived from the CDR-3 region of heavy chain of PS4A7 bound specifically to PS,indicating that the CDR-3 region paly a dominant role in the interaction with PS and from the peptide motif which is responsible for the specific interaction with PS.We then raised a series of the anti-idiotypic mAbs against the combining site of the PS-specific mAb. We found that one anti-idiotypic mAb, named Id8F7, cross-reacts extensively with proteins kinase C,a enzyme which requires PS for its enzymatic activation, and blood coagulation factor VIII (FVIII) and factor V (FV) . This finding indicates that the PS-specific mAb and the blood coagulation factors share the common structure, which is responsible for the specific interaction with PS.To identify the PS-specific binding site, we mapped the Id8F7-binding site on FV using the various recombinant fragments of the protein. The anlaysis showed that the Id8F7 binding site, which is a putative PS-specific binding site of the protein, locates in the C2-region of the FV light chain.