CHARACTERIZATION OF CELLULAR-BINDING SITES AND INTERACTIVE REGIONS WITHIN REACTANTS REQUIRED FOR ENHANCEMENT OF PLASMINOGEN ACTIVATION BY TPA ON THE SURFACE OF LEUKOCYTIC CELLS

Plasminogen and tPA bind to a common set of binding sites on nucleated cells. To assess the functional consequences of cellular binding, we have measured the kinetic changes induced by plasminogen activation by tPA on cell surfaces. These studies were carried out with U937 and TH P-1 monocytoid cells, with Raji, Nalm6 and Molt4 lymphoid cells and wi th peripheral blood monocytes and neutrophils. The interactions of pla sminogen and tPA with cells induced an increase in the rate of plasmin generation which depended upon the cell concentration. With saturatin g amounts of U937 monocytoid cells (1.25 X 10(5)/ml) the rate of plasm in generation was 0.39 nM.s(-1) versus 0.07 and 0.09 nM.s(-1) without cells or without tPA, respectively. The catalytic efficiency of Glu- o r Lys-plasminogen activation by tPA increased by 7.2- and 24.2-fold, r espectively. These changes were induced by a 72-242-fold reduction in the Km of these interactions which was in the range of 0.3-0.9 mu M. T hese values are below the plasminogen concentration in plasma (1-2 mu M). Moreover, we provide new data indicating that 1) only a specific s ubset of plasminogen binding sites, i.e. molecules exposing carboxyl t erminal lysines on the cell surface, promotes plas minogen activation on cells; 2) the first four kringles of plasminogen and the finger of tPA are critical for enhanced plasmin generation on cell surfaces; 3) the simultaneous co-localization of tPA with plasminogen on cell surfa ces is required for enhanced plasminogen activation; 4) modulation of plasminogen/tPA receptor expression induces concomitant modulation of the stimulatory effects of cells on plasminogen activation and 5) in a direct comparison, the mechanism by which cells and fibrin fragments accelerate plasminogen activation are similar but not identical. These data suggest that modulation of plasminogen/tPA binding sites permits local and efficient generation of plasmin on cell surfaces.