TISSUE FACTOR TERNARY COMPLEX AND THE VASCULAR SURFACE

有大量证据支持目前的结论，即细胞表面转铁蛋白是体内激活凝血途径的主要启动分子，也称为血栓形成途径。Tf首先与血浆VIIa高亲和力结合，形成双分子蛋白水解酶TF.VIIa。用结晶学方法和结合定点突变的计算模型分别测定了Tf(TTf)和tTF.VIIa二元复合体的细胞表面域的三维结构。然而，双分子蛋白酶TF.VIIa的功能是通过与特定底物酶原蛋白X、IX或IxAlpha瞬时组装，然后将其转化为活性酶Xa和IXa。三元络合物TF.VIIa.X的结构生物学及其功能是本项目的目标。Aim 1将解决X和Tf之间的结构和接触，VIIa形成三元络合物并激活X。Aim 2将解决X底物的进入与Xa产物的退出的问题，作为复合体功能速率的控制。这也可能是该络合物对抑制的敏感性的一个重要因素，这是由于抑制剂与Xa的结合，而Xa是三元络合物中不存在的。目标3将继续在三元络合物的结晶方面取得进展。第二个主题是通过组合搜索活体内皮细胞的分子多样性，利用现有的和新开发的噬菌体展示技术来识别具有意义的内皮细胞表面结构的“探针”。目标5将尝试确定这些位点的特征，识别存在的分子，并克隆新分子的cDNA.这次搜索的目标将集中在动脉粥样硬化和血管生成的内皮细胞以及器官之间的差异。这些研究为进一步了解血栓形成级联反应的结构生物学和分子细胞生物学以及鉴定体内血管内皮细胞表面的蛋白质组学提供了可能。

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.