FXII activation. (A-C) Activation of FXII by (A-B) PKa or (C) FXIa. One hundred nanomolar FXII-WT, FXII lacking most of its heavy chain region (ΔFXII), or FXII variants with single–amino acid substitutions were incubated with PKa (12.5 nM) or FXIa (2.5 nM) at 37°C. For panels A-C, at indicated times, aliquots were removed and tested for FXIIa generation by chromogenic assay. Results represent averages ± 1 standard deviation (SD) for at least 3 experiments. (D) FXII cleavage by PKa. One micromolar FXII-WT (left) or the variant FXII-Ala³⁴⁶ (right), was incubated with 125 nM PKa. At indicated time, samples were removed into reducing sample buffer and size fractionated by SDS-PAGE. Positions of molecular mass standards are shown on the left. Shown on the right are positions of standards for the zymogen FXII band (Z) and the heavy chain (HC) and light chain (LC) of FXIIa. (E) Densitometric quantification of FXII Z band disappearance in reactions with PKa identical to those shown in panel D. The curves show changes in Z band intensity as a percent of the intensity at 0 minutes. Each point represents the average for 2 experiments. (F) FXII-Ala⁵⁴⁴ (left), FXII-Ala²⁶⁸, Ala⁵⁴⁴ (middle), and FXII-Ala⁵⁰², Ala⁵⁴⁴ (right), 100 nM each, was incubated with 12.5 nM PKa at 37°C. At indicated times, samples were removed into reducing SDS sample buffer, size fractionated by SDS-PAGE, and transferred to nitrocellulose membranes. Immunoblotting was with an HRP-conjugated anti-FXII IgG. Positions of molecular mass standards are shown on the left. Positions of standards for the Z band, and the HC, and LC of FXIIa are indicated at the right of each image. Shown are representative blots for experiments that were run in duplicate.