The circulating plasminogen activators, tissue-type (tPA) and urokinase- type (uPA) plasminogen activators, which are essential for the normal activation of the fibrinolytic system in blood, are thought to originate mainly from the vascular endothelium. Decreased fibrinolytic potential of blood has been found to be associated with an increased risk of thromboembolic events in both the venous and arterial systems. Recent studies in our laboratory have shown that the synthesis and release of tPA and uPA from vascular endothelial cells can be enhanced by specific pharmacological agents, including sulfonylureas, nicotine and cotinine, polyamines, and retinoids. A pharmacological mechanism involving stimulation of tPA/uPA synthesis and release, or inhibition of plasminogen activator inhibitor (PAI) synthesis and release, may represent a promising method for the long-term prevention of thrombotic processes. The overall goals of this research proposal are to elucidate the biology and pharmacology of plasminogen activator synthesis and release in endothelial cells. Such information will be of significant value for any further evaluation of the feasibility of such an antithrombotic mechanism, in addition to providing fundamental information regarding the regulation of tPA/uPA synthesis.
The specific aims of this research proposal are as follows: 1) To characterize interrelationships between cellular transport and secretion of tPA, uPA and PAI in endothelial cells. This will include experiments in control cells and cells treated with sulfonylureas and nicotine and related agents on cellular transport of tPA, uPA and PAI, localization of PA-PAI complex formation, potential desensitization, and relationships between tPA/uPA and PAI synthesis. 2) To elucidate the cellular mechanism(s) underlying pharmacological modulation of tPA and uPA expression by sulfonylureas and nicotine and related agents. This will involve assays of tPA, uPA, and PAI mRNA levels after treatment with these pharmacological agents, using cDNA-mRNA hybridization techniques and nuclear runoff transcription assays. 3) To determine the mechanisms involved in the regulation of coexpression of tPA and uPA in endothelial cells after pharmacological stimulation with sulfonylureas. This will involve a series of structural and functional experiments on the regulatory components of the tPA and uPA genes, including promoter deletion mapping coupled with the use of expression vectors and transfection in endothelial cells, gel mobility shift assays, and DNA-protein cross-linking. 4) To evaluate the potential role of two specific endothelial cell receptors involved in the pharmacological stimulation of tPA and uPA synthesis and secretion. This will involve experiments focusing on sulfonylurea binding sites and associated ATP-dependent K+ channels and on specific muscarinic cholinergic receptors. Overall, the proposed experiments are aimed at enhancing our understanding of the biological and pharmacological mechanisms involved in the regulation of tPA/uPA synthesis and release, and will thus enable further evaluation of the feasibility of this promising pharmacological mechanism.
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