Figure 3. Activity in Factor XII Zymogen and ΔFXII.

(A & B) FXII activity. (A) FXII and FXII-T (200 nM) were incubated in the absence of an activator (control, left column), with 70 μM Poly-P (center), or with 50 nM kallikrein without a surface (right). (B) FXII, FXII-T or FXII with the active site serine changed to alanine (FXII-S554A), 200 nM, and PK (200 nM) were incubated at 37°C. For panels A and B, at indicated time points, samples were removed into reducing sample buffer. Samples were size fractionated by SDS-PAGE, followed by western blot using a polyclonal anti-human FXII or anti-PK IgG. Positions of standards for FXII (XII) and PK and the heavy chain (HC) and light chain (LC) of FXIIa and PKa are indicated at the right of each image. Data is from reference 8. (C & D) ΔFXII activation and activity. (C) FXII (○), the full-length FXII precursor used to generate ΔFXII (◻), or ΔFXII (△), 200 nM, were incubated at 37°C with PKa (10 nM). FXIIa activity was measured by chromogenic substrate assay. (D) PK (200 nM) was mixed with 12.5 nM FXII (▽) or FXII-Lys309 (▼). The FXII preparations were preincubated with thrombin, which removes the heavy chain from FXII-Lys309 but not from FXII, prior to addition to PK. PKa generation was determined with a chromogenic substrate assay. Data for panels C and D are from reference 8. (E&F) Effects of ΔFXII on high molecular weight kininogen (HK). (E) Western blots of human FXII-deficient plasma supplemented with FXII or ΔFXII (400 nM). At indicated times, samples were removed into non-reducing sample buffer. Western blots were probed with goat anti-human HK IgG (HK). Positions of standards for HK and cleaved HK (HKa) are shown on the right. (F) Bradykinin generation in normal plasma after addition of ΔFXII (160 nM; black), ΔFXII and an inhibitor of kallikrein (KV999272 10 mM; gray), or vehicle (white). Bradykinin was measured by ELISA (Enzo Life Sciences). Data for panels E and F are from reference 42.