CHARACTERIZATION OF PLASMINOGEN ACTIVATION BY GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED UROKINASE

The characteristics of plasminogen activation by glycosylphosphatidyli nositol (GPI)-anchored urokinase were evaluated and compared with thos e reported previously for receptor-bound urokinase. When expressed in cultured bovine aortic endothelial cells, GPI anchoring of single-chai n urokinase plasminogen activator (scu-PA) potentiated plasmin generat ion as compared with GPI-anchored scu-PA that had been released into s olution from the cell surface by enzymatic cleavage of the GPI anchor (''released'' scu-PA). The potentiation of plasmin generation by GPI-a nchored scu-PA was inhibited in a dose-dependent manner by 6-aminohexa noic acid, a lysine analog, suggesting that the augmentation of plasmi n generation by GPI-anchored scu-PA was dependent on simultaneous bind ing of plasminogen to the cell surface. GPI-anchored two-chain urokina se (tcu)-PA cleaved a peptide substrate at a rate equivalent to that o f released urokinase. However, at a plasminogen concentration of 0.5 m uM, GPI-anchored tcu-PA activated plasminogen less rapidly than did re leased urokinase. Modeling of kinetics of individual reactions reveale d that cell-associated plasminogen activation by GPI-anchored tcu-PA w as characterized by a K(m) of approximately 0.15 muM. This value of K( m) was 70-fold below that for activation of solution plasminogen by GP I-anchored urokinase. There was a concomitant decrease in V(max) for p lasminogen activation by anchored tcu-PA. These alterations in kinetic parameters are similar to those reported previously for the activatio n of plasminogen by receptor-bound tcu-PA. In addition, GPI-anchored t cu-PA exhibited a modest resistance to plasminogen activator inhibitor 1 inactivation. The enzymatic characteristics of GPI-anchored urokina se reported here resemble closely those reported previously for recept or-bound urokinase. These data suggest that the urokinase receptor may regulate plasmin generation through a relatively nonspecific localiza tion of urokinase to the cell Surface rather than through any intrinsi c property of the urokinase receptor.