Plasminogen activation by tPA mediates bradykinin generation. (A) Representative HMWK western blot (120 KDa) after 3 hours of incubation, showing that plasminogen activation (by tPA+plasminogen) promotes HMWK cleavage and that this effect is a result of plasmin generation, as aprotinin blocked the effect. (B) Time-course of HMWK cleavage by plasmin. This cleavage was prevented by aprotinin. (C) Enzyme-linked immunosorbent assay bradykinin levels showing that either recombinant plasmin or generated plasmin (by tPA-dependent plasminogen activation) generate bradykinin by HMWK cleavage. This effect is prevented by aprotinin. All experiments were performed in triplicate. (D) Schematic representation of the mechanistic link between hydrodynamic transfection of tPA in the liver and subsequent BBB leakage. *P < .05 vs control.