Structure-Function Relationships of Proteins Modulating Platelet Adhesion and Aggregation

调节血小板粘附和聚集的蛋白质的结构-功能关系

基本信息

批准号：
09480155
负责人：
TITANI Koiti
金额：
$ 4.74万
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 (B).
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 2000
项目状态：
已结题

项目摘要

1) Studies on the isolation and structure-function relationship of snake venom proteins modulating interaction between von Willebrand factor (vWF) and platelets. We have searched, identified and structurally characterized novel snake venom proteins that modulate platelet adhesion and aggregation by interacting with vWF.a) We screened. vWF-binding proteins from 19 species of snake venoms and discovered "bitiscetin", a novel vWF-modulating protein from the venom of Bitis arietans, and "kaouthiagin", a vWF-cleaving metalloproteinase from the venom of Naja kaouthia. We purified the two proteins and determined their complete amino acid sequences. Since the sequence of bitiscetin was not highly homologous to that of botrocetin with a similar activity, we expressed many mutants of A-1 loop of vWF, which binds to it, to identify residues interacting with bitiscetin. The results indicated that bitiscetin interacts with a site not identical but very close to that interacts with botrocetin. Kaout … More hiagin was shown to be composed of metalloproteinase, disintegrin-like and Cys-rich domains. Interestingly, the Cys-rich domain contained a RGD sequence. We demonstrated that both the disintegrin-like and Cys-rich domains have the disintegrin activity using synthetic cyclic peptides. b) We purified a protein termed "mamushigin" from the venom of Agkistrodon halys blomhoffii (Japanese "mamushi") which binds to platelet GPIb to induce the aggregation. We isolated the cDNA clones encoding the α and β chains from the library and predicted the complete amino acid sequences of both the chains. From the same venom, we also isolated and determined the complete amino acid sequence of a serine protease termed halystase which releases bradykinin from kininogen in human plasma.2) Origin of plasma vWF with ABO blood group antigens. vWF is a unique plasma protein with the blood group antigens. Since those antigens on human plasma vWF are not changed to those of the donors after ABO-mismatched bone marrow transplantation, we concluded that plasma vWF with the blood groups is derived from endothelial cells, but not from megakaryocytes (platelets). Less

1）研究蛇毒蛋白的分离和结构功能关系，调节von Willebrand因子（VWF）和血小板之间的相互作用。我们搜索，鉴定和结构表征的新型蛇毒蛋白通过与VWF.A相互作用来调节血小板的粘附和聚集。来自19种蛇毒的VWF结合蛋白，发现了“ Bitiscetin”，这是一种新型的VWF调节蛋白，从Bitis Arietans的毒液中调节蛋白，而“ Kaouthiagin”是Naja Kaouthia的毒液中的VWF裂解金属蛋白酶。我们纯化了两种蛋白质，并确定了它们的完整氨基酸序列。由于比特切尔的序列与具有相似活性的肉骨蛋白的序列并非高度同源，因此我们表达了许多与biticetin相互作用的VWF的A-1环的突变体。结果表明，比特汀与位点相互作用不完全相同，但与肉鸡蛋白相互作用非常接近。 Kaout…更多的Hiagin被证明是由金属蛋白酶，分解蛋白样和富含Cys的结构域组成的。有趣的是，富含Cys的域包含RGD序列。我们证明了裂解蛋白样和富含Cys的结构域都使用合成环状肽具有分解蛋白活性。 b）我们从Agkistrodon Halys Blomhoffii（日语“ Mamushi”）的毒液中纯化了一种称为“ mamushigin”的蛋白质，该蛋白质与血小板GPIB结合以诱导聚集。我们分离了从文库编码α和β链编码α和β链的cDNA克隆，并预测了这两个链的完整氨基酸序列。从同一毒液中，我们还分离并确定了称为Halystase的串行蛋白的完整氨基酸序列，该蛋白称为Halystase，该蛋白在人血浆中从吉尼蛋白中释放了肌蛋白。 VWF是具有血型抗原的独特血浆蛋白。由于在ABO不匹配的骨髓移植后，人血浆VWF上的那些抗原并未更改为供体的抗原，因此我们得出结论认为，带有血型的血浆VWF来自内皮细胞，但不是来自巨核细胞（血小板）。较少的