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

磷脂酰丝氨酸在凝血因子 VIII 激活中的作用。

基本信息

批准号：
04671343
负责人：
UMEDA Masato
金额：
$ 1.34万
依托单位：
THE TOKYO METROPOLITAN INSTITUTE OF MEDICAL SCIENCE (1994)The University of Tokyo (1992-1993)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

项目摘要

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.

众所周知，在一种特殊的膜磷脂磷脂-磷脂酰丝氨酸的存在下，由张力酶和凝血酶原复合物催化的两个连续的凝血级联反应大大加速。本项目旨在阐明ps特异性激活凝血级联的分子机制，采用一系列免疫化学方法鉴定了ps在凝血因子上的特异性结合位点。我们首先建立了特异性结合PS的单克隆抗体PS4A7，并确定了重链和轻链可变区的氨基酸序列。我们发现从PS4A7重链CDR-3区衍生的12个氨基酸残基合成肽与PS特异性结合，表明CDR-3区在与PS的相互作用中起主导作用，并从与PS特异性相互作用的肽基序中提取了一系列针对PS特异性单抗结合位点的抗独特型单抗。我们发现一种名为Id8F7的抗独特型单抗与蛋白激酶C（一种需要PS才能激活的酶）和凝血因子VIII （FVIII）和因子V （FV）发生广泛的交叉反应。这一发现表明，ps特异性单克隆抗体与凝血因子具有共同的结构，该结构负责与ps特异性相互作用。为了确定ps特异性结合位点，我们利用该蛋白的各种重组片段在FV上绘制了id8f7结合位点。分析表明，Id8F7结合位点位于FV轻链的c2区，是该蛋白推测的ps特异性结合位点。