Properties of δFXII-3C and its active form δFXIIa-3C, and their effects on HK cleavage. (A-C) Activation of FXII. FXII-WT (), FXII-3C (□), or δFXII-3C (), 200 nM, were incubated in standard buffer at 37°C with kallikrein (10 nM) (A), plasmin (500 nM) (B), or FXIa (10 nM, subunit concentration) (C). Kallikrein was inhibited with HO3, and FXIa and plasmin were inhibited with aprotinin, and FXIIa generation was determined by chromogenic substrate cleavage. (D) PK activation by FXII-T or δFXII-T. PK (200 nM) was incubated in standard buffer at 37°C with vehicle (), 200 nM FXII-T (□), or δFXII-T (), and cleavage of chromogenic substrate was continuously monitored. (E) PK activation by FXIIa. PK (60 nM) was incubated in standard buffer at 37°C with 50 pM αFXIIa (), βFXIIa (□), or δFXIIa-3C (), or in the absence of FXIIa (). At various times, reactions were stopped with CTI, and kallikrein generation was determined by chromogenic assay. (F) FXI (30 nM) was incubated in standard buffer at 37°C with 10 nM αFXIIa (), βFXII (), or δFXIIa-3C (), or in the absence of FXIIa (). At various times, reactions were stopped with CTI, and FXIa generation was determined by chromogenic assay. For panels A and B, n = 4; C, n = 6; D, n = 3; E, n = 9; and F, n = 6. Error bars ± 1 SD for all panels. (G) HK cleavage in human plasma. Shown are western blots of human FXII-deficient plasma supplemented with FXII-WT, FXII-3C, or δFXII-3C (400 nM) in the absence (−) or presence (+) of PTT-A silica-based reagent. At indicated times, samples were removed into nonreducing sample buffer. Western blots were probed with goat anti-human HK IgG (HK) or goat anti-human C1-INH IgG (C1-INH). Positions of standards for HK, the 2 bands of cleaved HK (HKa), and kallikrein or αFXIIa in complex with C1-INH are shown on the right. Positions of molecular mass standards in kilodaltons are shown to the left of the images.