TISSUE FACTOR TERNARY COMPLEX AND THE VASCULAR SURFACE

有大量证据支持目前的结论，即细胞表面TF是体内凝血（即血栓形成）途径激活的主要启动分子。TF首先通过与血浆VIIa高亲和力结合形成双分子蛋白酶TF.VIIa发挥作用。三维结构的细胞表面区域的TF （tTF）和tTF的。通过晶体学方法和计算模型结合定点诱变分别确定了VIIa二元配合物。然而，双分子蛋白酶TF。VIIa通过与特定底物酶原蛋白X、IX或Ixalpha的短暂组装，然后蛋白水解转化为活性酶Xa和IXa发挥作用。研究三元配合物TF.VIIa.X的结构生物学及其功能是本课题的研究目标。目的1将讨论X和TF之间的结构和接触。通过形成三元配合物并激活X。Aim 2将解决X底物进入和Xa产物退出的问题，以控制配合物的功能速率。这也可能是三元配合物中不存在的Xa抑制剂联合抑制复合物的易感性的重要因素。目的3将继续研究三元配合物的结晶。第二个主题是内皮细胞在体内的分子多样性，通过组合搜索利用现有的和新开发的噬菌体展示技术来识别对内皮细胞表面结构有意义的“探针”。目标5将尝试表征这些位点，鉴定存在的分子，并克隆新分子的cDNA。这项研究的目标将集中在动脉粥样硬化和血管生成内皮和器官之间的差异。这些研究为进一步了解血栓形成级联的结构生物学和分子细胞生物学以及体内血管内皮表面的蛋白质组学提供了潜力。

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.