FXI activation by thrombin. (A) Western blot of FXI-Ala557 (100nM) incubated with 25nM α-IIa, β-IIa, or γ-IIa, or 0.1nM bovine trypsin. FXI was detected with a polyclonal anti–human FXI antibody. Incubation times in hours are indicated below the blot. The position of the zymogen FXI band (Z) and the heavy chain (HC) and light chain (LC) of FXIa are shown to the left of the gel. (B) FXI-WT (100nM) was incubated with 25nM α-IIa (■), β-IIa (□), γ-IIa (●), or vehicle (○) at 37°C. FXIa generation was determine with a chromogenic substrate assay on aliquots at various time points as described in “Activation of FXI by thrombin.” (C-F) Thrombin generation assays were run as described in “Thrombin generation assay.” (C) Thrombin generation initiated by 10nM α-IIa, β-IIa, or γ-IIa in FXII-deficient plasma #1. (D) Same as in panel C, but with FXII-deficient plasma #2. (E) Same as in panel C with the addition of 300nM IgG O1A6 to block FIX activation by FXIa. (F) FXII-deficient plasma #1 using 10nM trypsin as the initiator. The signal here is probably background from trypsin cleavage of the fluorogenic substrate.
