Activation by thrombin and PAI-1 resistance of the scFv/uPA-T. (A) Specific activation of scFv/uPA-T by thrombin. scFv/uPA-T was incubated with various serine proteases for 1 hour. Thrombin (5 nM) generated amidolytic activity from scFv/uPA-T, whereas the other serine proteases or heat-inactivated thrombin did not, even when 40 nM enzyme was added. APC indicates activated protein C. Error bars represent SEM. (B) Activity of scFv/uPA-T measured by zymography before or after incubation with thrombin under reduced or nonreduced conditions. (C) Fibrinolytic activity. The indicated amounts of scFv/uPA-T, thrombin-treated scFv/uPA-T, lmw-tcuPA, lmw-scuPA, and thrombin-treated lmw-scuPA were incubated on a fibrin-coated plate at 37°C. Lytic zones were counterstained using impregnation of fibrin by trypan blue. (D) Susceptibility of scFv/uPA-T to PAI-1. Native scFv/uPA-T does not bind PAI-1. After addition of thrombin, a dose-dependent increase in lmw-tcuPA-PAI-1 complexes is evident.