Tissue plasminogen activator (TPA) is the key enzyme in physiologic fibrinolysis but the factors controlling its synthesis and release are not well understood. Utilizing a novel enzyme linked immunosorbent assay (ELISA) for alpha2-plasmin inhibitor - plasmin complexes, we have demonstrated in vivo that aspirin (ASA) inhibits the rise of TPA induced by venous occlusion in human volunteers. Since both PA and PGI2 are produced by the endothelium and ASA inhibits endothelial cell PGI2 production, we will test the hypothesis that PGI2 or other eicosanoids stimulate the production or release of TPA. To determine the time and dose dependence of the ASA effect on TPA and whether transacetylation or inhibition of cyclooxygenase is necessary for the inhibition of TPA by ASA, TPA release will be induced in vivo by forearm venous occlusion after subjects have ingested no drug, various doses of ASA, indomethacin, and salicylate. TPA activity and antigen in plasma will be measured by ELISA while platelet function will be measured by aggregation and TXB2 production by RIA. For in vitro studies, human saphenous vein segments and human and bovin endothelial and vascular smooth muscle cells will be grown in tissue culture and stimulated to produce TPA with hypoxia, elevated pressure, DDAVP, phorbol esters, and vasoactive amines alone and in combination. The role of monocytes polymorphonuclear leukocytes, platelets and red cells isolated by centrifugal elutriation, neurotransmitters and Protein C in mediating TPA production by vascular cells will be explored. TPA production will be measured by ELISA and the 125I-fibrin plate assay and characterized by SDS-PAGE and fibrin gel autography. Inhibition of PA production will be attempted with cyclooxygenase and lipoxygenase inhibitors. Incubation of inhibited cells with PGI2, TXA2, HETE and other lipoxygenase products will then demonstrate whether any of these metabolites can directly induce TPA production. If novel eicosanoids are demonstrated, these will be characterized by HPLC and GC-MS. Because both arachidonate metabolites and fibrinolysis may play major roles in atherogenesis and the pathophysiology of myocardial ischemia, this research, characterizing an interaction between the two systems, should be of major significance and may result in an improved approach to the secondary prevention of myocardial infarction and stroke.
