Abnormal thrombus formation and dissolution are associated with several cardiovascular diseases including atherosclerosis and both thromboembolic and hemorrhogic conditions. The fibrinolytic system thus assumes pivotal importance in maintaining the hemostatic balance. It is clear that many of the fibrinolytic components of plasma either originate from endothelial cells (ECs) (e.g., tissue-type plasminogen activator) or bind to ECs (e.g., urokinase-like plasminogen activator), and that EC- mediated fibrinolysis must therefore be precisely regulated. The unexpected finding that cultured ECs also produce two immunological distinct plasminogen activator inhibitors, including both type 1 (PAI-1) and type 2 (PAL-2), introduces a previously unsuspected level of complexity to our understanding of this system, and raises the possibility that the altered fibrinolytic activity of cells following various treatments, or of blood in certain human diseases, reflect changes in either one of these inhibitors. Although PAI-1 has been studied extensively recently, little information is available concerning the production of PAI-2 by ECs. The long term objective of this application is to develop an accurate understanding of the role of PAI-2 in human disease.
The specific aim of this proposal is to define the role of this inhibitor in regulating EC-mediated fibrinolysis. PAI-2 will be purified in sufficient quantities to permit the development of specific antibodies and immunologic assays for both PAI-2 activity and antigen. These techniques will by employed, in conjunction with immunofluorescence studies, to define its cellular location (e.g., presence in cytosol, membrane fraction, or extracellular matrix) in confluent umbilical vein ECs, as well as in other types of ECs. Several structural features (i.e., molecular weight, isoelectric point, presence of carbohydrate) of PAI-2 produced by confluent human ECs will be established and compared to PAI-2 purified from the histocytic lymphoma cell line, U-937. These studies will provide a framework to determine the basal rate of PAI-2 synthesis and secretion using immunologic assays complemented by immunoprecipitation experiments employing biosynthetically labeled PAI-2. The effect of several factors (e.g., cell growth, cell passage, serum, endotoxin, monokines, etc.) on the production of PAI-2 will then be investigated. These studies are essential to delineate the role of PAI-2 in a variety of processes that require EC-mediated proteolysis, such as clot dissolution, tissue repair, angiogenesis, etc., and to accurately define the contribution of PAI-2 to altered fibrinolytic states.
