BK generation with human and mouse HK. For all studies, reactions were run in reaction buffer for 30 minutes at 37°C. BK generation was determined by ELISA. (A) Effect of ε-ACA on Plm-mediated BK generation. Human plasma–derived HK (200 nM) was incubated with PKa (2 nM; orange), Plm-Glu³¹¹ (40 nM; red), Plm-Lys³¹¹ (40 nM; blue), or control vehicle (C; purple) in the absence (left) or presence (right) of 10 mM of ε-ACA. (B) Human plasma–derived HK (left), human recombinant HK (rHK; center), and recombinant full-length mHK1 (right), 200 nM, were incubated with PKa (2 nM; orange), Plm-Glu³¹¹ (40 nM; red), Plm-Lys³¹¹ (40 nM; blue), or C vehicle (purple). (C) Human rHK (left), recombinant full-length mHK1 (center), and mHK3 (right), 200 nM, were incubated with 40 nM of human Plm (H-Plm; mustard), mouse Plm (M-Plm; green), or vehicle C (purple). (D) Recombinant HKs used in panel C were incubated with PKa (2 nM; orange), Plm-Glu³¹¹ (40 nM; red), Plm-Lys³¹¹ (40 nM; blue), or C vehicle (purple). Error bars indicate standard errors of the mean for duplicate experiments, each with 2 separate measurements.