TISSUE FACTOR TERNARY COMPLEX AND THE VASCULAR SURFACE

组织因子三元复合物和血管表面

• 批准号: 6109402
• 负责人: THOMAS Scott EDGINGTON
• 金额: $ 31.49万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6109402
• 关键词: X ray crystallography; blood coagulation; clotting factor; coagulation factor IX; coagulation factor VII; coagulation factor X; combinatorial chemistry; enzyme activity; enzyme structure; laboratory mouse; laboratory rat; membrane structure; nuclear magnetic resonance spectroscopy; nucleic acid sequence; polymerase chain reaction; protease inhibitor; protein structure function; site directed mutagenesis; structural biology; thromboplastin; tissue /cell culture; vascular endothelium

There is substantial evidence to support the current conclusion that cell surface TF is the major initiating molecule for the in vivo activation of the coagulation, aka thrombogenic, pathways. TF functions first by high affinity association with plasma VIIa to form the bimolecular protease TF.VIIa. The three dimensional structure of the cell surface domain of TF (tTF) and of the tTF.VIIa binary complex have been determined by crystallographic methods and separately using computational models coupled with site directed mutagenesis. However, the bimolecular protease TF.VIIa functions by the transient assembly with specific substrate zymogen proteins X, IX, or Ixalpha followed by proteolytic conversion to the active enzymes Xa and IXa. The structural biology of the ternary complex TF.VIIa.X and its function is the goal of this project. Aim 1 will address the structure and contacts between X and TF.VIIa to form the ternary complex and activate X. Aim 2 will address the issue of access of X substrate versus exit of Xa product as controls of the functional rate of the complex. This may also be an important element in the susceptibility of the complex to inhibition as a result of association of inhibitors with Xa which has not existed from the ternary complex. Aim 3 will continue progress in crystallization of the ternary complex. The second topic is the molecular diversity of the endothelial in vivo by combinatorial search using existing and newly developed phage display technology to identify "probes" for endothelial surface structures of significance. Aim 5 will attempt to characterize such sites, identify the molecules present, and clone the cDNA for novel molecules. The targets for this search will focus on atherosclerotic and angiogenic endothelial and differences between organs. These studies provide the potential to advance understanding of the structural biology and molecular cell biology of the thrombogenic cascade and to identify proteomics of the vascular endothelial surface in vivo.

有大量证据支持当前的结论，即细胞表面TF是体内激活凝结（又称血栓形成途径）的主要启动分子。 TF首先由高亲和力与等离子体VIIA形成双分子蛋白酶TF.VIIA的功能。 TF（TTF）和TTF.VIIA二进制复合物的细胞表面结构域的三维结构已通过晶体学方法确定，并分别使用与位点的定向诱变相结合的计算模型。然而，瞬态组装的双分子蛋白酶TF.VIIA具有特定底物Zymogen蛋白X，IX或IXALPHA，然后将蛋白水解转化为活性酶XA和IXA。三元复合物tf.viia.x及其功能的结构生物学是该项目的目标。 AIM 1将解决X和TF.VIIA之间的结构和接触，以形成三元络合物并激活X。IAM2将解决X底物与XA产品退出的访问问题，作为该综合体功能速率的控制。由于抑制剂与Xa的关联，这也可能是复合物对抑制作用的敏感性的重要因素，而Xa尚未从三元复合物中存在。 AIM 3将继续在三元络合物的结晶方面继续前进。第二个主题是使用现有和新开发的噬菌体显示技术组合搜索内皮体内内皮的分子多样性，以识别重要性的内皮表面结构的“探针”。 AIM 5将尝试表征此类位点，识别存在的分子并克隆新分子的cDNA。此搜索的目标将集中于动脉粥样硬化和血管生成的内皮和器官之间的差异。这些研究提供了提高对血栓形成级联反应的结构生物学和分子细胞生物学的潜力，并鉴定体内血管内皮表面的蛋白质组学。