Cell invasion through connective tissue matrices requires a proteinase cascade, the controlling feature of which is regulated activation of plasminogen by urokinase type plasminogen activator (u-PA). U-PA is synthesized as a minimally active single chain molecule (scu-PA), but can be cleaved to an active two chain form (tcu-PA) by plasmin. This circular, but fundamental process has been puzzling: First, if tcu-PA is required to activate plasminogen, but plasmin is required to activate scu- PA, how does the process start? Second, because this is a """"""""positive feedback loop."""""""" how does the process stop? Furthermore, tcu-PA on the cell u-PA receptor (u-PAR) is efficiently inhibited by plasminogen activator inhibitor (PAI). so how do cells invade through PAI-rich environments? We have used a form of scu-PA, glu158-scu-PA, not cleaved by plasmin to demonstrate that i) scu-PA exhibits enzymatic activity, and that when it and plasminogen are bound to their respective cell surface binding sites, plasmin is generated 100-fold more efficiently than if the reactants are in solution; this activation is carried out by single chain, not two chain u-PA. Hence scu-PA can initiate the process; and ii) scu-PA, in solution binds reversibly to PAI-2, whereas scu-PA on the u-PA receptor is not inhibited. Hence plasminogen activation requires that scu-PA first dissociate from PAI-2, then bind to cell surface receptor to express enzymatic activity even in the presence of excess PAI-2. This probably influences the initiation process, and allows cells to invade through PAI- rich environments. In this application we will focus on testing, each component of the initiating steps of cell surface plasminogen activation. Specifically, we propose to determine. 1) whether the reversible binding of scu-PA to PAI-2 and/or u-PAR has a regulatory role in the initiation of cell surface plasminogen activation (i.e.. is the partitioning of scu-PA between PAI 2 and u-PAR a primary determinant of cell surface plasminogen activation?) 2) The mechanism by which plasminogen is activated by scu-PA 100-fold more efficiently on the monocyte cell surface than in solution. Is it due to an effect on K(m), K(cat), the nature of plasminogen as a substrate, or a combination of these? 3) By analogy to plasminogen activation on fibrin clots whether the enzymatic activity of plasmin plays a role in accelerating plasminogen activation at the cell surface. This work should define regulatory mechanisms for initiating this fundamental property of invading cells.
