The major objectives of this proposal are to study the basic biological processes involved in the regulation and control of the blood fibrinolytic system in health and disease. They include the biosynthesis of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators in normal mammalian tissues, e.g. monkey and baboon kidney, liver and spleen. These studies include the isolation of specific mRNA, the isolation of translation products by specific immunoprecipitation methods, and the characterization of the activator forms. Abnormal, variant, plasminogens will be isolated form the plasma of patients with thrombotic diseases, and possibly from full-term newborn umbilical cord plasmas, and attempts will be made to determine the molecular defect(s). Plasma u-PA and t-PA forms will be isolated by affinity chromatographic methods, from normal subjects, patients with arterial and venous thrombotic diseases, patients with secondary fibrinolytic states (DIC), and cancer patients in remission treated with stanozolol (an anabolic steroid). The plasma u-PA and t-PA forms (zymogen and enzyme) will be characterized by various chemical, physical and immunologic methods. Their role in the mechanism of physiological and pathological fibrinolysis will be investigated. Activator binding domains, and fibrin-binding domains, on both normal and abnormal plasminogens will be identified using peptide mapping methods by HPLC, and specific antibody probes. Recombinant hybrid activator enzymes will be prepared from both the plasminogen fibrin-binding domains(A chains) and the u-PA/t-PA active center domains (B chains), and their properties will be compared to the parent u-PA and t-PA enzymes.
