To better understand the biosynthesis and release of plasminogen activator, we plan to use tissue culture techniques that permit precise and direct observation of cells and the assay of products which are released into chemically defined media. We will use two different cells, human embryonic kidney cells and a transformed human hepatocyte line to define the biosynthetic pathway from plasminogen proactivator(s) to the different plasminogen activators which will include both urokinase and tissue activators. By the addition of protease inhibitors to the cultures and the use of short culture times, we will maintain a high ratio of proactivator to activator to facilitate the purification of proactivators. In another approach, we will use continuous flow electrophoresis for cell separations to isolate the specific subpopulation of kidney cells responsible for activator biosynthesis. The proactivator(s) and activators will be isolated individually from the culture media by using affinity chromatographic methods with benzamidine-Sepharose and fibrin-celite. We will study the mechanism involved in the activation process and will determine the structural changes that occur. The purified proactivators and activators will be characterized as to molecular weight, chain structure, active site sequence and immunological properties. The enzyme from the culture responsible for the activation will be isolated and its specificity determined. During the biosynthesis, we will test the hypothesis that activator is regulated by neurohumoral control by a plasminogen activator releasing hormone (PARH) by studying the effect of peptides on release of activator from the cell and will establish a structure-function relationship with any positive responses. Our studies will aid in understanding the basic mechanisms involved in the control of the fibrinolytic system and the results could influence the development of the next generation of thrombolytic agents.
