Effect of α-FXIIa on fibrin polymerization and fibrin structure. (A-E) Human fibrinogen (1 mg/mL) was incubated with α-FXIIa (0-125nM) in HEPES buffer (25mM HEPES, 150mM NaCl, pH 7.4) for 10 minutes at 37°C before clotting was initiated with CaCl₂ (5mM) and thrombin (0.1-5nM). Turbidity was monitored every 15 seconds at 405 nm at 37°C. (A) Time course of fibrin clot formation with 3 α-FXIIa concentrations (0, 31.25, and 125nM; each in duplicate) initiated with 0.5nM thrombin. (B) Lag time of fibrin formation and (C) maximal turbidity as a function of thrombin concentration. (D-E) To inhibit α-FXIIa, PPACK (1000nM) was incubated with α-FXIIa and removed via dialysis in HEPES buffer (25mM HEPES, 150mM NaCl, 1 mg/mL BSA, pH 7.4). Clotting was initiated with 1nM thrombin. (D) Lag time of fibrin formation and (E) maximal turbidity. (B-E) Data are mean ± range of 2 separate experiments. (F) Representative scanning EM images of clots (n = 6) prepared by incubating fibrinogen (1 mg/mL) with α-FXIIa (0-125nM), thrombin (2.5nM), and CaCl₂ (5mM) in HEPES buffer (20mM HEPES, pH 7.4, 150mM NaCl) for 2 hours at room temperature. To inhibit α-FXIIa, CTI (75 μg/mL) was added to 125nM α-FXIIa. Scale bars represent 1μm.