FXII binding to PK. (A) Standard curve of different concentrations (1-120 μg/mL) of WT FXII or FXIIa binding to plate-immobilized PK. Results are shown as optical density values, measured as described in “Methods.” (B) Binding of WT FXII and FXIIa, and FXII variants (30 μg/mL) to plate-immobilized PK. Values are shown as a percent of the signal for FXII-WT (assigned a value of 100%) ± 1 SD. (C) Plasma FXII or FXIIa (10 μg) was incubated O/N with anti-PK IgG beads alone or with plasma PK (10 μg) at 4°C. (D) Plasma or recombinant FXII (10 μg) were incubated O/N with anti-PK IgG beads and 10 μg PK lacking an active site serine (PK-Ala⁵⁵⁹) at 4°C. For panels C and D. After incubation, the beads were washed, samples were eluted with nonreducing SDS sample buffer, size fractionated by SDS-PAGE, and either (C) stained with Coomassie blue or (D) transferred to nitrocellulose membranes and probed with anti–FXII HRP-conjugated IgG. Positions of molecular mass standards are shown on the left. Positions of standards for PK and FXII/FXIIa are indicated at the right of each image. Shown are representative gels/blots for experiments that were run in duplicate.