Human endothelial cells in culture continue to produce an secrete tissue plasminogen activator (tPA) and plasminogen activator (PAI-1 and respond to specific agonists by altering the production of these fibrinolytic proteins. Activation of protein kinase C by phorbol esters is associated with the stimulation tPA antigen secretion and an increase in the level of tPA mRNA. Elevation of cAMP potentiates this response 5 to 10 fold while having no effect on tPA secretion by itself. cAMP has second important effect on endothelial cell fibrinolysis elevated levels suppress the basal rate of PAI-1 secretion and attenuate phorbol ester-induced secretion of PAI-1. Thus cAMP appears to play a pivotol role in establishing a profibrinolytic state in the endothelial cell environment. Evidence also suggests that these two pathways may be involved with stimulation of tPA secretion by more physiological agonists such as histamine and thrombin. Employing human endothelial cells in culture it is the objective of this study to identify and characterize those intracellular pathways that are responsible for responsible-signal coupling in the regulation tPA production and secretion. Emphasis will be placed on the role of the protein kinase C and cAMP pathways and whether they, as suggested by the data, mediate agonist dependent stimulation of tPA secretion. The mechanisms by which tPA mRNA levels are modified, the protein intermediates that are involved following agonist treatment, and the interrelationship between the protein kinase C and cAMP pathways that may explain potentiation or depression of phorbol ester-mediated effects will be examined. The possibility that physiologic agonist control tPA secretion through both protein kinase C cAMP pathways will agonists control tPA secretion through both protein kinase C and cAMP pathways will also be considered. Such studies will also be applied to the depression of PAI-1 secretion by cAMP and the attenuation of phorbol ester induced PAI-1 secretion. These studies will address important and how specific issues dealing with tPA regulation in endothelial cells and how changes in the components of the extracellular environment impact on the level of fibrinolysis in the vascular system.
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