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Regulation of platelet plug formation by von Willebrand factor and its modulator proteins

冯·维勒布兰德因子及其调节蛋白对血小板栓子形成的调节

基本信息

批准号：
16510167
负责人：
TAEI Matsui
金额：
$ 2.18万
依托单位：
Fujita Health University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

1) Structure and function of C-type lectin purified from snake venom of Crotalus ruberWe have purified the C-type lectin(CRL) from the snake venom of C.ruber and determined the primary structure of CRL. CRL showed high similarity with the lectin from C. atrox (CAL). CRL and CAL showed similar sugar specificity and molecular mass, but showed different divalent cation-requirement. Homology modeling suggested that the amino acid substitution found in CRL does not affect sugar recognition of the lectin but might alter the conformation and influence the sugar binding pocket induced by the metal-ion binding.2)Cloning and expression of botrocetinBotrocetin cDNAs were cloned from cDNA library of venom gland by PCR. cDNAs were subcloned into the expression vector and 293T cells were transfected with the expression vector containing botrocetin cDNAs. The cell culture medium was concentrated and subjected to SDS-PAGE. The band corresponding to botrocetin was observed and the concentrates showed the platelet agglutination activity in the presence of von Willebrand factor (VWF).3)Isolation and characterization of VWF-binding protein from the venom of king cobraVWF-binding protein was isolated from the venom of king cobra (O.hannah) by several steps of chromatographies. The VWF-binding activity of the protein was diminished after reduction of the protein. The protein inhibited the platelet agglutination induced by ristocetin calcium ion-dependently.4)Carbohydrate analysis of human ADAMTS13Sugar chain moiety of ADAMTSI3 was analyzed by lectin blotting. SSA and MAM showed binding to ADAMTS13, which was diminished after sialidase-digestion. RCA120 and PNA bound to ADAMTS13 after sialidase digestion suggesting the presence of N-linked and 0-linked sugar chain with sialic acid residues at the non- reducing terminal. Human ADAMTSI3 did not have ABO(H) blood group antigenicity.

1)乌鸦蛇毒C型凝集素的结构与功能我们从乌鸦蛇毒中提纯了C型凝集素，并对其一级结构进行了测定。CRL与华支睾吸虫凝集素有很高的相似性。CRL和CAL表现出相似的糖专一性和相对分子质量，但表现出不同的二价阳离子需求。同源模拟表明，CRL中发现的氨基酸取代不会影响凝集素对糖的识别，但可能会改变其构象，并影响金属离子结合所诱导的糖结合口袋。2)克隆和表达Botrocetin Botrocetin。将cDNAs亚克隆到表达载体中，并将含有牛磺草素cDNA的表达载体导入293T细胞。细胞培养液浓缩后进行SDS-PAGE分析。3)眼镜王蛇毒VWF结合蛋白的分离与鉴定通过几步层析从眼镜王蛇蛇毒中分离出VWF结合蛋白。蛋白质还原后，其VWF结合活性降低。4)人ADAMTS13糖链的糖链部分用凝集素印迹法分析。SSA和MAM与ADAMTS13结合，唾液酸酶消化后结合减弱。RCA120和PNA经唾液酸酶切后与ADAMTS13结合，提示在非还原末端存在含唾液酸残基的N-连接和0-连接糖链。人ADAMTSI3不具有ABO(H)血型抗原性。