HK cleavage in human plasma and in mice. (A) Human FXII-deficient plasma was supplemented with FXII-WT, ΔFXII, FXII-Ala³⁶, FXII-Lys²⁵³, and FXII-Ala³⁴⁶ (140 nM) and incubated at 37°C. At indicated times, samples were removed into non-reducing sample buffer, size fractionated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with goat anti-human HK IgG. Positions of markers for uncleaved HK (white arrow) and cleaved HKa forms (black arrows) are indicated to the right of each image. Western blots underwent densitometry scanning to generate the curves shown below each western blot. Curves show the disappearance of HK (blue), appearance of HKa intermediate (single cleavage, gray), and appearance of the final form of HKa (2 cleavages, red). Percentile values were assigned to each band based on comparison to the density of the HK band at time 0 (assigned a value of 100%). Data are averages of 2 experiments. (B) FXII-deficient mice received intravenous infusions of FXII-WT, ΔFXII, FXII-Ala36, FXII-Lys²⁵³, or FXII-Ala³⁴⁶ to achieve estimated plasma concentrations of 40 nM. Shown are nonreducing western blots of plasma collected 0, 15, or 30 minutes, or ∼16 hours (O/N) after infusion. Blots were developed with anti-murine HK IgG. Positions of markers for HK (white arrows) and cleaved HKa forms (black arrows) are indicated to the right of each image. Positions of molecular mass markers are shown on the left. Shown are representative blots for experiments that were run in duplicate.