The objectives of this research proposal are to define the biochemical role of activated protein C (APC) in regulating the expression of fibrinolytic activity. Our approaches will involve: (a) characterization of the plasminogen activator reported to be responsible for this effect; (b) an examination and characterization of the interaction of APC with cultured cells, and the effect upon expression of plasminogen activator activity; and (c) a characterization of structural and functional aspects of the inhibition of fibrinolysis, and the effect of APC, if any, if any, on these inhibitor molecules. In order to characterize the plasminogen activator responsible for this effect, we will rely on immunological techniques, using specific high-affinity antibodies directed against tissue activator, urokinase and kallikrein. By using the appropriate antibody coupled to Sepharose, we will isolate the plasminogen activator, and determine if a precursor form exists in plasma, capable of undergoing activation in the presence of APC and any necessary cofactor molecules. The binding of 125I-labelled APC to cultured endothelial cells, prostate adenocarcinoma cells, and human monocytes will be thoroughly examined, and the effect on expression of plasminogen activator antigen and activity measured. The binding will be characterized with regard to certain features required of a specific receptor, i.e. high affinity, limited numbers of sites per cell, specificity for APC, and the elicitation of a biological response. Certain structural features of Alpha2-antiplasmin and Alpha2-macroglubulin will also be characterized to further our understanding of the role of these molecules in the inhibition of the fibrinolytic pathway. These studies will include a characterization of the physical properties of these inhibitors and their plasmin complexes by differential scanning calorimetry, examination of the conformational alterations induced in Alpha2M as a consequence of plasmin binding, and a characteization of the structural alteration associated with the inactivation of Alpha2-antiplasmin, and the role of APC, if any, in this process. The long-term objective of this proposal is to explore several areas involved in the regulation of fibrinolysis, with the goal of improving thrombolytic therapy. It is likely that an enzyme with anticoagulant properties, such as APC, that also has been demonstrated to stimulate fibrinolysis, will be an excellent thrombolytic agent.
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