FXI activation in normal plasma. (A) Autoactivation of FXII in the presence of 50nM PC/PS vesicles, 50nM CMs, or vehicle control. Reactions were run as described in “Activation of FXII and PK.” (B-D) Thrombin generation in normal plasma depleted of CMs (“Thrombin generation assay”) initiated by 10nM (B) α-IIa, (C) β-IIa, or (D) γ-IIa in the presence or absence of 4μM CTI, or the presence of 300 μg/mL O1A6. (E-F) Western blots of (E) PK or (F) FXII (200nM) incubated with vehicle (C) or 25nM α-IIa (α), β-IIa (β), γ-IIa (γ), FXIa (XIa), FXIIa (XIIa), α-kallikrein (Ka), or 0.01nM trypsin (T). Blots were developed with goat polyclonal antibodies against the relevant protein. α-Kallikrein and FXIIa standards (St) are in the first lanes of each blot, with positions of zymogen PK and FXII (Z) and the heavy chain (HC) and light chain (LC) of α-kallikrein and FXIIa shown to the left of each blot. β-kal indicates bands representing the degradation product β-kallikrein. (G-H) Chromogenic substrate assays for activation of (G) PK or (H) FXII (200nM) by 25nM FXIa (●) or vehicle (□) at 37°C. α-Kallikrein or FXIIa generation was assessed by chromogenic substrate assay as described in “Activation of FXII and PK.”