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）从Crotalus ruberwe纯化的C型凝集素的结构和功能已从C.Ruber的蛇毒中纯化了C型凝集素（CRL），并确定了CRL的主要结构。 CRL与C. aTrox（Cal）的凝集素显示出很高的相似性。 CRL和CAL显示出相似的糖特异性和分子质量，但显示出不同的二价阳离子征用。同源性建模表明，CRL中发现的氨基酸取代不影响凝集素的糖识别，但可能会改变构象并影响金属离子结合引起的糖结合口袋。2）克隆和botrocetinbotrocetin cdnas的克隆和表达，从pcr的venom gland的cdna库中克隆。将cDNA亚克隆到表达载体中，并用含有botrocetin cDNA的表达载体转染293T细胞。将细胞培养基浓缩并进行SDS-PAGE。观察到与肉毒杆菌相对应的条带，并在存在Von Willebrand因子（VWF）的存在下表现出血小板凝集活性。蛋白质还原后，蛋白质的VWF结合活性减少了。蛋白质抑制了ristocetin钙离子依赖性诱导的血小板凝集。4）通过凝集素印迹分析了adamtsi3人Adamts13sugar链部分的碳水化合物分析。 SSA和MAM显示与ADAMTS13结合，唾液酸酶消化后减少了。唾液酸酶消化后与ADAMTS13结合的RCA120和PNA表明在非还原末端存在N连接和0连接的糖链，并具有唾液酸残基。人Adamtsi3没有ABO（H）血型抗原性。