Characterization of plasminogen activation by glycosylphosphatidylinositol-anchored urokinase

Abstract

The characteristics of plasminogen activation by glycosylphosphatidylinositol (GPI)-anchored urokinase were evaluated and compared with those reported previously for receptor-bound urokinase. When expressed in cultured bovine aortic endothelial cells, GPI anchoring of single-chain urokinase plasminogen activator (scu-PA) potentiated plasmin generation as compared with GPI-anchored scu-PA that had been released into solution from the cell surface by enzymatic cleavage of the GPI anchor ("released" scu-PA). The potentiation of plasmin generation by GPI-anchored scu-PA was inhibited in a dose-dependent manner by 6-aminohexanoic acid, a lysine analog, suggesting that the augmentation of plasmin generation by GPI-anchored scu-PA was dependent on simultaneous binding of plasminogen to the cell surface. GPI-anchored two-chain urokinase (tcu)-PA cleaved a peptide substrate at a rate equivalent to that of released urokinase. However, at a plasminogen concentration of 0.5 microM, GPI-anchored tcu-PA activated plasminogen less rapidly than did released urokinase. Modeling of kinetics of individual reactions revealed that cell-associated plasminogen activation by GPI-anchored tcu-PA was characterized by a Km of approximately 0.15 microM. This value of Km was 70-fold below that for activation of solution plasminogen by GPI-anchored urokinase. There was a concomitant decrease in Vmax for plasminogen activation by anchored tcu-PA. These alterations in kinetic parameters are similar to those reported previously for the activation of plasminogen by receptor-bound tcu-PA. In addition, GPI-anchored tcu-PA exhibited a modest resistance to plasminogen activator inhibitor 1 inactivation. The enzymatic characteristics of GPI-anchored urokinase reported here resemble closely those reported previously for receptor-bound urokinase. These data suggest that the urokinase receptor may regulate plasmin generation through a relatively nonspecific localization of urokinase to the cell surface rather than through any intrinsic property of the urokinase receptor.