UROKINASE HAS DIRECT CATALYTIC ACTIVITY AGAINST FIBRINOGEN AND RENDERS IT LESS CLOTTABLE BY THROMBIN

Recently, we demonstrated that tissue plasminogen activator directly releases fibrinopeptides A and B (FPA and FPB) from fibrinogen. The purpose of this study was to determine whether urokinase has similar activity. Incubation of urokinase with fibrinogen or heparinized plasma results in concentration-dependent FPB release unaccompanied by FPA cleavage. For equivalent amidolytic activity, high molecular weight urokinase releases twofold more FPB than the low molecular weight species. In contrast, prourokinase does not release FPB until activated to urokinase. Contaminating thrombin or plasmin is not responsible for urokinase-mediated FPB release because this activity is unaccompanied by FPA or Bβ1-42 cleavage, and is unaffected by heparin, hirudin, a monospecific antibody against thrombin, aprotinin, or α2-antiplasmin. FPB release reflects a direct action of urokinase on fibrinogen because release is completely inhibited by a monospecific antibody against the enzyme. Further, urokinase releases FPB from the FPB-containing substrate Bβ1-42, thus confirming its specificity for the Bβ14(Arg)-Bβ15 (Gly) bond. In addition to FPB release, SDS-PAGE analysis of the time course of urokinase-mediated fibrinogenolysis indicates progressive proteolysis of both the Aα- and Bβ-chains of fibrinogen that occurs after FPB release is completed. As a consequence of urokinase-mediated fibrinogenolysis, there is progressive prolongation of the thrombin clotting time. These studies indicate that urokinase has direct catalytic activity against fibrinogen. By releasing FPB, a potent chemoattractant, and by rendering fibrinogen less clottable by thrombin, urokinase may participate in processes extending beyond fibrinolysis.