Recombinant Plg activation and effects on BK generation in normal plasma. (A) Coomassie Blue stained SDS–polyacrylamide gels of plasma-derived Plgs (left), recombinant Plgs (center), and recombinant Plms (right). Shown in the center and right panels are 2-μg samples of plasma-derived Glu-Plg/Plm (P), Plg/Plm-Lys³¹¹ (Lys), and Plg/Plm-Glu³¹¹. Positions of molecular mass standards (kDa) are shown at the left of each figure, and positions of standards for zymogen Plg (Z) and the heavy (HC) and light chains (LC) of Plm are shown on the right. (B) Activation of 200 nM of plasma-derived Glu-Plg (green), Lys-Plg (orange), or vehicle (no Plg; purple) by 20 nM of tPA in reaction buffer. (C) Plg-Lys³¹¹ (blue), Plg-Glu³¹¹ (red), Plg-Ala³¹¹ (green), or vehicle (purple), 200 nM each, were incubated in reaction buffer with 20 nM of tPA. In panels B and C, at indicated times, samples were removed for measurement of Plm by chromogenic assay. (D) BK generation in normal plasma supplemented with 600 nM of Plg-Lys³¹¹ (blue), Plg-Ala³¹¹ (green), Plg-Glu³¹¹ (red), or vehicle (purple) after addition of tPA (final, 125 nM). (E) Controls for reactions in panel D. BK generation in normal plasma supplemented with Plg-Glu³¹¹ and tPA (light green), Plg-Glu³¹¹ alone (blue), tPA alone (red), or vehicle (purple). (F) BK generation in normal plasma supplemented with Plg-Glu³¹¹ (600 nM) after adding tPA to 250 (lavender), 125 (steel blue), 62.5 (blue) or 50 nM (magenta). (G) BK generation in normal plasma supplemented with 600 nM of Plg-Lys³¹¹ (blue) or Plg-Glu³¹¹ (red) after addition of tPA (various concentrations) and thrombin (50 nM; to generate fibrin). In panels B to D, error bars indicate standard errors of the mean for duplicate experiments, each with 2 separate measurements. In panels F and G, results are for single representative experiments.