Endothelial cell strains of venous and arterial origin obtained from fetal calves and calves of different ages will be established and cloned using a fibrin-dish method developed in our laboratory. The influence of age of the animal, site of origin and passage number of the endothelial cells on the production and secretion of both plasminogen activator and protease inhibitors respectively will be determined using human plasminogen and culture dishes covered with 125I-fibrin. The cell cycle dependency of synthesis and secretion of this protease will be determined using synchronized cell cultures. Screening for compounds that either enhance (e.g., cyclic AMP-like agents, diguanides, stanozolol, nicotinic acid, etc.) or inhibit (e.g., dexamethasone and related compounds) the production and secretion of plasminogen activator will be done on endothelial cells colonies using a fibrin-agarose overlay method, and active agents so detected will be further examined in mass culture on 125 I-fibrin. The mechanisms regulating these induction and inhibition phenomena will also be studied. The serine protease(s) secreted by endothelial cells of arterial and venous origin will be purified by means of salt precipitation and chromatography and compared to other known plasminogen activators such as urokinase using SDS gel-electrophoresis and antisera obtained from rabbits immunized with these proteases. We shall also attempt to culture human endothelial cells obtained from different veins and arteries and, if successful, perform similar studies as outlined for endothelial cells of bovine origin. These studies should elucidate some aspects of the activation of fibrinolysis in blood and eventually compounds and enhancing or inhibiting effect on the plasminogen activator production will be detected that could later be tested for prevention and treatment of thrombosis in vivo.
