Traditionally, fibrinolysis has been conceptualized as a process oriented on a and confined to the surface of a fibrin clot. Recent published data generated by the applicant, however, demonstrate specific, high affinity, saturable binding for both glu-plasminogen (PLG) and tissue plasminogen activator (t-PA) to the surface of cultured human umbilical vein endothelial cells (HUVEC). Upon binding to HUVEC, PLG acquired a 12.7-fold increase in catalytic efficiency of activation by c-PA. Similarly, the ability of t-PA to activate PLG was preserved when t-PA was associated with the cell surface. The current studies will examine the molecular mechanisms by which PLG and t-PA interact with the endothelial cell surface. The applicant will (1) study the mechanism by which surface associated PLG acquires enhanced """"""""activatability,"""""""" by examining the possible role of modified forms of PLG in this interaction; (2) identify potential PLG and t-PA binding sites using detergent extracts of isolated HUVEC plasma membranes in ligand blotting, affinity chromatography, bifunctional cross- linking, and monoclonal antibody immunodetection procedures; (3) identify the cell surface binding domain of t-PA in experiments employing specific deletion mutants of t-PA, chemically modified forms of t-PA, and anti-t-PA monoclonal antibodies with defined epitope specificity; regulation of PLG or t-PA binding by cytokines known to influence HUVEC study the synthesis and secretion of t-PA and its major inhibitor, PAI; and (5) examine the relationship of PLG binding sites on HUVEC to apolipoprotein a """"""""kringle-""""""""containing protein which is correlated with atherosclerosis, and highly homologous to PLG.
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