Atherogenesis may be conceptualized as a response to vascular cell injury or stress. The endothelium constitutes a thromboresistant surface that is intimately associated with flowing blood. However, perturbation of the endothelial cell by any of several stimuli confers a more prothrombotic, adhesive phenotype that predisposes to influx of cellular elements, release of mitogens, and lipid accumulation. We recently identified the calcium-regulated phospholipid-binding protein, annexin II, as an endothelial cell surface receptor for two fibrinolytic proteins, plasminogen and tissue plasminogen activator. Since annexin II appears to possess distinct binding domains for these two ligands, we have hypothesized that it mediates their assembly as well as generation of the multi-functional serine protease, plasmin. Our further studies have shown that purified native annexin II enhances the catalytic efficiency of t-PA- dependent plasminogen activation by 60-fold. Although several studies have demonstrated the presence of annexin II on endothelial cells, and other cells, the mechanism by which it is translocated to the cell surface is unknown since it lacks a typical hydrophobic signal sequence. Our preliminary data suggest that annexin II translocation is a slow process in resting cells, but that it is significantly more rapid in response to specific forms of cellular """"""""stress"""""""" such as heat shock or viral infection. Additional studies indicate that full-length annexin II must be proteolytically modified before it can function fully as fibrinolytic receptor, and that urokinase and t-PA represent candidate annexin II activators.
The specific aims of this proposal are to (1) determine the mechanism by which the endothelial cell translocates annexin II to the cell surface under resting conditions, (2) identify modifications of this process that occur in response to cellular injury or stress, and (3) delineate the mechanism by which annexin II is activated to become a fully functional catalytic receptor. The success of the proposed studies will depend upon critical interactions with other members of the Program Project. Specifically, Dr. Roy Silverstein will assist in preparation of stably transfected 293 cell lines, and Dr. Suman F. A. Pearce will conduct structural analyses of mutant annexins. Drs. David Hajjar and Robert Kaner will collaborate on studies involving processing of annexin II in the setting of virus-induced stress. We hypothesize that transport and processing of annexin II may be significantly perturbed under conditions that lead to cellular proliferation and lipid accumulation, the hallmarks of atherosclerosis.
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