FXII cleavage by coagulation proteases. (A) Clotting times in an aPTT assay for normal plasma (NPP), FXII-deficient plasma (DP), or FXII-deficient plasma supplemented with recombinant FXII-WT (WT), FXII-Lys³⁰⁹ (309K), FXII-Arg³⁰⁹ (309R), or FXII-3C (3C). Each symbol indicates 1 clotting time. (B) Recombinant FXII (200 nM) incubated in standard buffer with 200 μM S-2302 at 37°C in the presence (+) or absence (−) of 70 μM polyphosphate. Left panel, FXII-Lys³⁰⁹; right panel, FXII-Arg³⁰⁹. Changes in OD 405 nm were continuously monitored. Curves are means of 3 independent runs. (C) Western blot of FXII-deficient plasma supplemented with 400 nM FXII-WT, FXII-Lys³⁰⁹, or FXII-Arg³⁰⁹ induced to clot with TF (2.5 pM) and CaCl₂ (6.25 mM). Fibrin formation was prevented with H-Gly-Pro-Arg-Pro-OH. Western blots of time courses were probed with polyclonal IgG to human FXII. Positions of molecular mass standards in kilodaltons are shown to the left of the images. (D-F) FXII (200 nM) in standard buffer incubated with 25 nM plasmin (D), 10 nM plasmin, thrombin, or FXIa (E), or α-kallikrein (50 nM) (F). Western blots of time courses were probed with a mixture of monoclonal IgGs to the FXII HC (15H8) and LC (1B2). To the right of images in panels C-F are markers for FXII (FXII), the HC and LC of αFXIIa, HC of FXII-Lys³⁰⁹ or FXII-Arg³⁰⁹ cleaved after residue 309 (δHC) and FXII residues Thr³¹⁰ to Ser⁵⁹⁶ (δFXII). (G) FXII (100 nM) incubated in standard buffer with (+) or without (−) 25 nM thrombin for 120 minutes at 37°C. Thrombin was inhibited with argatroban (125 μM) and FXIIa cleavage of S-2302 (500 μM) was determined. The proteins are FXII-WT (WT), FXII-Lys³⁰⁹ (309 Lys), and FXII-Arg³⁰⁹ (309 Arg). Control (XIIa) is 12 nM αFXIIa. Error bars ± 1 standard deviation (SD); n = 6.