PK activation by FXII and δFXII. (A) Western blot of FXII/PK reciprocal activation. Plasma-derived FXII (200 nM) and PK (200 nM) were incubated in standard buffer at 37°C. At indicated times, samples were removed into reducing sample buffer. Western blots were probed with goat anti-human FXII IgG or sheep anti-human PK IgG. Positions of markers for FXII, PK, and the HC and LC of αFXIIa and kallikrein are indicated to the right of each image. (B) Kallikrein generation in reciprocal reactions with FXII. FXII-WT (, ), FXII-Lys³⁰⁹ (, ), or FXII-Arg³⁰⁹ (, ), 100 nM in standard buffer was incubated with (, , ) or without (, , ) thrombin (25 nM) for 2 hours at 37°C. Reactions were stopped with argatroban (125 μM). PK (60 nM) was mixed with 12.5 nM of the preincubated FXII. At various times, aliquots were removed, FXIIa was inhibited with CTI, and kallikrein concentration was determined by chromogenic assay. (C) FXII amino acid sequence within the proline-rich region showing the amino acids inserted to create a cleavage site for 3CP in the protein FXII-3C. The image to the right of the arrow shows the N and C termini after cleavage of FXII-3C with 3CP. (D) Schematic diagrams of FXII with the sequence Leu-Glu-Val-Leu-Phe-Gln-Gly inserted between FXII residues Gln³⁰⁷ and Pro³⁰⁸ to create a cleavage site for 3C protease in FXII-3C. Cleavage of FXII-3C with 3C creates a mixture of δHC (with a slightly different C terminus than in panel B), and δFXII-3C. The latter differs from δFXII in having an additional Gly-Pro at the N terminus. Cleavage of δFXII after Arg³⁵³ creates the fully active species δFXIIa-3C. (E) Nonreducing 20% Tris–2-(N-morpholino)ethanesulfonic acid (MES) SDS-PAGE of FXII-3C cleaved by 3C protease and the purified FXII-3C δHC and δFXII-3C. Positions of molecular mass standards in kilodaltons are shown to the left of the image. (F) Kallikrein generation in reciprocal reactions with FXII-3C and δFXII-3C. PK (60 nM) was incubated in standard buffer at 37°C with 12.5 nM FXII-WT (), FXII-3C (□), FXII-3C cleaved with 3C protease () or purified δFXII-3C (), or in the absence of FXII (). Reactions were run in the absence (left panel) or presence (right panel) of 70 μM Poly-P. At the indicated times, aliquots were removed and tested for kallikrein generation by chromogenic assay. For panels B and F, error bars ± 1 SD; n = 5 or 6.