Strategically situated between vessel wall and flowing blood, the endothelium plays a key role in thromboregulation. Indeed, the endothelial cell expresses several surface molecules that support the assembly and regulation of proteases involved in hemostasis and thrombosis. Recent data indicates that plasminogen and its activators can bind specifically to endothelial cells through mechanisms that preserve or enhance their activity. In this application, the Applicants propose to examine the hypothesis that endothelial cells support the generation of plasmin by virtue of a cell surface receptor for tissue plasminogen activator (t-PA) and its substrate, plasminogen (PLG). They have recently purified, cloned, and expressed a M ~40,000 endothelial cell surface protein, annexin II, which has the ability to bind both of these ligands in a specific and independent fashion. Although the annexins were originally identified as a family of calcium-dependent, phospholipid binding proteins, possibly involved in exocytosis, phospholipase regulation, or calcium flux, our recent studies suggest that annexin II can dramatically enhance t-PA- dependent plasmin generation in a purified system and on cell surfaces. Over the next five years, they will examine this hypothesis by [1] examining the molecular mechanisms by which annexin II interacts with the endothelial cell surface, [2] identifying specific sequences essential for plasminogen binding to the mature molecule, [3] defining the t-PA binding domain of annexin II, [4] delineating the mechanism of by which annexin II enhances t-PA-dependent plasminogen activation, and [5] elucidating mechanisms of transcriptional regulation of annexin II expression in endothelial can myeloid cells. These studies will involve a variety of cell and molecular biologic techniques. Chimeric and site-directed mutants, expressed in eukaryotic systems or mammalian transfection assays, will be used in addition to mimetic peptides to analyze functional domains. Studies of function will include radio ligand binding studies, cell binding experiments,and assays of plasminogen activation. Transcriptional regulation of the annexin II gene will be examined through nuclear run-on studies, gel shift assays, and expression of potential transactivating factors and promoter-based constructs. Hopefully, this work will provide a detailed understanding of the molecular interactions that define the role of annexin II in cell surface plasmin generation.
Showing the most recent 10 out of 58 publications
