The objective of this proposal is a study of the regulation of fibrinolysis in human endothelial cells. The vascular endothelium plays a major role in the regulation of both hemostasis and fibrinolysis. The latter is regulated, at least in part, by the balance between plasmiogen activators (PAs), which initiate the fibrinolytic process, and specific plasminogen activator-inhibitors (PAIs).
The aim of this proposal is to study the mechanisms of regulation of tissue-type PA (tPA) and PAI in cultured human endothelial cells by a variety of mediators including thrombin, bacterial endotoxin, the monokines interleukin-1 and tumor necrosis factor, glucocorticoids, and by tPA and PAI themselves. The unifying factor among these apparently disparate molecules is that they all may be produced in response to injury to the vascular system whether traumatic, infectious or inflammatory. We will first establish the fibrinolytic parameters of early passage human umbilical vein endothelial cells, measuring both the functional activity and amount of tPA and PAI by biochemical and immunological assays. We will then examine the effects of selected mediators on the rates of synthesis and degradation of tPA and PAI, on levels of specific mRNA, and on rates of tanscription and mRNA stability in order to define the mechanisms of action of these agents. We will carry out similar studies on early passage cultures of adult human vascular endothelium to establish the physiologic relevance of these studies. Finally, we will study the regulation of fibrinolysis in established cell lines of endothelial origin to determine their suitability as experimental model systems, and will attempt to isolate variant cell lines altered in the regulation of fibrinolysis. Biochemical and genetic analysis of such variants should enhance our ability to define the mechanisms of regulation of fibrinolysis. The role of fibrinolytic abnormalities in thromboembolic disease is now well established. The necessary biochemical, immunological and molecular tools are available for a comprehensive analysis of the mechanisms of regulation of fibrinolysis. A better understanding of these mechanisms should have considerable clinical relevance.
