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

1) Crotalus ruber蛇毒c型凝集素的结构与功能我们从Crotalus ruber蛇毒中纯化了c型凝集素（CRL），并确定了CRL的一级结构。CRL与C. atrox （CAL）凝集素具有较高的相似性。CRL和CAL具有相似的糖特异性和分子质量，但具有不同的二价阳离子需要量。同源性模型表明，CRL中的氨基酸取代不影响凝集素的糖识别，但可能改变构象并影响金属离子结合诱导的糖结合袋。2)利用PCR技术从蛇毒腺cDNA文库中克隆出botrocetin cDNA。将cdna亚克隆到表达载体中，用含有botrocetin cdna的表达载体转染293T细胞。细胞培养基浓缩，SDS-PAGE检测。在血管性血液病因子（VWF）存在的情况下，浓缩物具有血小板凝集活性。通过层层析，从眼镜王蛇的毒液中分离出了vwf结合蛋白。蛋白还原后，蛋白的vwf结合活性降低。该蛋白以钙离子依赖性抑制利斯托司汀诱导的血小板凝集。4)人adamts13的碳水化合物分析采用凝集素印迹法分析ADAMTSI3的糖链片段。SSA和MAM与ADAMTS13结合，经唾液酸酶酶切后减少。RCA120和PNA在唾液酸酶消化后结合到ADAMTS13上，表明在非还原末端存在n-链和0-链的糖链和唾液酸残基。人ADAMTSI3不具有ABO(H)血型抗原性。

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.