TISSUE FACTOR TERNARY COMPLEX AND THE VASCULAR SURFACE

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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.