FXIa activation of FIX. (A) FIX activation by FXIa with FXI-A3 domains. Human FIX (200 nM) was incubated at 37°C with vehicle (no FXIa), 2 nM human FXIa-WT (human FXI-A3), or 2 nM human FXIa with a platypus FXI-A3 domain replacing the human A3 domain (FXIa/PlatXIA3, platypus FXI-A3). At various times, samples were removed into reducing sample buffer, size-fractionated by SDS-PAGE and stained with Coomassie blue (top row). Positions of standards for FIX, the heavy chain of the intermediate α-FIX (α), the heavy chain of the final product FIXaβ (HC), and light chain of α-FIX and FIXaβ (LC) are shown on the right of each image; positions of molecular mass markers in kilodaltons are shown to the left of the images. Stained gels underwent densitometry scanning to generate the curves in the bottom row. Values for each band were compared with those for FIX at 0 minutes, which was assigned a value of 100%. Curves show the disappearance of FIX (Δ), and the appearance of the heavy chain of the intermediate α-FIX (□), the heavy chain of FIXaβ (∇), and the light chain of α-FIX and FIXaβ (○). (B) FIX activation by FXIa with PK-A3 domains. Human FIX was incubated as in panel A with 2 nM human FXI with a human PK-A3 domain replacing the FXI A3 domain (FXIa-PKA3, human PK-A3) or human FXI with a platypus PK-A3 domain replacing the FXI A3 domain (FXIa/PlatPKA3, platypus PK-A3). Time course experiments were run and analyzed as in panel A.