The long term objective of this project is to understand the biological significance in health and disease of plasminogen activator receptors. Localized expression of plasminogen activator activity on cell surfaces has been implicated in a wide variety of biological processes including fibrinolysis and migration of normal and malignant cells. Endothelial cells, which are key cells in the maintenance of blood fluidity and in the processes of angiogenesis and vascular repair, also express binding sites for plasminogen activators. We hope to isolate and purify the urokinase-type plasminogen activator (u-PA) receptor from human endothelial cells in sufficient quantity and characterize it in sufficient detail to enable its molecular cloning and the production of monoclonal antibodies to it. We will utilize affinity chromatography with u-PA-Sepharose to purify u-PA receptor from cultured human umbilical vein endothelial cells. We will perform N-terminal amino acid sequence analysis as well as study internal sequences after limited protease digestion. We will utilize this purified protein to prepare specific monoclonal antibodies. We will screen these antibodies for their ability to compete for the binding of u-PA to endothelial cells. We will utilize ligand blotting, antibodies and/or oligonucleotides to screen a lambda gt11 endothelial cell cDNA expression library to enable cloning of the u-PA receptor. We will demonstrate that the cDNA clones isolated code for receptor by transfecting them into cells that do not express u-PA receptor, and by demonstrating the acquisition of the ability to bind labeled u-PA. Obtaining the antibodies to and the cDNA for the u-PA receptor will enable us to study the expression and regulation of this receptor in endothelial cells. We will study the effects of cytokines, growth factors and plasminogen activators on receptor expression. We will also study the effects of receptor occupancy on endothelial cell growth and migration. Down regulation of receptors may be pathophysiologic in thrombotic disorders whereas up regulation may be important in promoting wound healing, vascular repair, and protection from thrombosis. Up regulation could be harmful as well in such conditions as pathologic neovascularization (e.g. diabetic retinopathy) and in tumor metastasis. Understanding the control and functional significance of u-PA binding to cells in general will hopefully enable the design of therapies to optimize the beneficial aspects and minimize any harmful effects of this interaction.
