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是体内激活凝血（又称血栓形成）途径的主要起始分子。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。TF（tTF）的细胞表面结构域的三维结构和的tTF。VIIa二元复合物已被确定通过晶体学方法，并分别使用计算模型加上定点诱变。然而，双分子蛋白酶TF.VIIa通过与特异性底物酶原蛋白X、IX或Ixalpha的瞬时组装，随后蛋白水解转化为活性酶Xa和IXa来发挥功能。该项目的目标是三元复合物TF.VIIa.X的结构生物学及其功能。目的1将阐明X和TF.VIIa之间的结构和接触，以形成三元复合物并激活X。目标2将解决X底物的进入与Xa产物的退出的问题，作为复合物的功能速率的控制。这也可能是一个重要的元素，在敏感性的复合物的抑制，作为一个结果，协会的抑制剂与Xa还没有存在的三元复合物。目标3将继续在三元复合物的结晶方面取得进展。第二个主题是通过组合搜索利用现有的和新开发的噬菌体展示技术，以确定“探针”的内皮细胞表面结构的意义在体内的分子多样性。目的5将试图表征这些位点，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。这些研究提供了潜在的血栓形成级联的结构生物学和分子细胞生物学的理解，并确定在体内血管内皮表面的蛋白质组学。

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.