Figure 5:

Characterization of CFH proteolysis by FXIa in serum. (A) HUVECs incubated with CFH (200 nM) for 1 hr at 37°C, washed and treated with FXIa (0-30 nM) for 30 min at 37°C. Aprotinin was added to all samples to stop the reaction. Cells lysates were analyzed by Western blotting with an anti-CFH C-terminal domain antibody. (B) Supernatant from activated platelets (2.5 x 10⁸) was incubated with FXIa (30 nM) for selected times (0-180 min). CFH was analyzed by Western blotting with an anti-CFH C-terminal domain antibody. (C) Human plasma or FXI-depleted plasma (FXI−/− plasma) was incubated with aPTT reagent or FXIa (30 nM) for 1 hr at 3 °C. CFH was analyzed by Western blotting with an anti-CFH C-terminal domain antibody. (D) FI-depleted serum was incubated with FXIa (30 nM) for selected times (0-180 min). CFH was analyzed by Western blotting with an anti-CFH C-terminal domain antibody. (E) C3b (300 nM) and FI (5 nM) were incubated in FI-depleted serum (1/5) at 37°C for selected times (10, 20, 40 and 80 min). Samples were separated by SDS-PAGE under reducing conditions and the generation of iC3b was analyzed by Western blotting using an anti-human C3b antibody. In selected experiments FI-depleted serum was incubated with FXIa (30 nM) for 3 hrs. Aprotinin was added to all samples to stop the reaction.