Collagen enhances conversion of FXII into active FXIIa. (A) Collagen-induced FXII activation in plasma-free system. Assay samples contained 95 nM FXII, 30 nM prekallikrein (PK), 30 nM high-molecular-weight kininogen (HK), 5 μg/mL collagen type I/H, and 4 μM CTI, as indicated. Chromogenic FXIIa activity was determined at 37°C from the cleavage of Pefachrome FXIIa substrate. (B) Collagen-induced activation of FXII in coagulating plasma. PRP (10⁸ platelets/mL) was activated with 16.6 mM CaCl₂ in the presence of 40 μg/mL collagen type I/H or vehicle solvent (top panel). Shown is representative Western blot of plasma immunoprecipitates stained for FXII(a); bars give increased ratio of cleaved FXIIa light chain (28-30 kDa) to uncleaved FXII (78-80 kDa) normalized to the condition without collagen (n = 2). In addition, PRP was activated similarly but also with kaolin (50 μg/mL) present (bottom panel). Given is representative Western blot of diluted plasma stained for FXII(a). Vertical line has been inserted to indicate a repositioned gel lane. (C,D) Binding of FXII to collagen. Coverslips with immobilized type I collagen were incubated for 10 minutes with FXII plus or minus PK/HK (concentrations as in panel A), then rinsed and stained with biotin-labeled anti-FXII(a) mAb and FITC-avidin. (C) Two-photon laser scanning images (180 × 180 μm) of fibers of FXII-stained collagen type I/H. Negative control represents condition where primary anti-FXII(a) mAb was omitted. (D) Integrated fluorescence intensity (arbitrary units) of immunostaining for FXII with various type I collagens. Controls were incubations with heat-treated FXII (5 minutes at 56°C) or biotin-labeled IgG. Mean ± SEM; n = 3-4; *P < .05 vs condition without collagen.