Functional characterization of FIXa with substitutions in exosite II. (A) Binding of FIXa variants to the A2 subunit of FVIII was measured by SPR as described in “Materials and methods.” The graph shows association and dissociation curves for wt-FIXa (blue), FIXaK293A{126_CT} (red), FIXaR333A{165_CT} (pink), FIXaR338A{170_CT} (orange), FIXaK341A{173_CT} (purple), FIXaK400A{230_CT} (green), FIXaR403A{233_CT} (yellow), and the double mutant FIXaR333A+R403A{165+233_CT} (gray) at a concentration of 400 nM. (B) FX activation in the presence of FVIIIa was performed with various concentrations of FVIII. FVIII was incubated with phospholipids (100 µM), FX (200 nM), and 0.1 nM of wt-FIXa (blue), FIXaK293A{126_CT} (red), FIXaR333A{165_CT} (pink), FIXaR338A{170_CT} (orange), FIXaK341A{173_CT} (purple), FIXaK400A{230_CT} (green), FIXaR403A{233_CT} (yellow), or FIXaR333A+R403A{165+233_CT} (gray). Data represent the mean of 2 independent experiments. (C) Activation of FX (0-50 nM) by 0.3 nM of FIXaR333A{165_CT} (pink) or FIXaR333A+R403A{165+233_CT} (gray). The inset shows the same data with wt-FIXa included. FX activation was assessed in the presence of 100 μM phospholipids and 0.3 nM FVIIIa. Data represent the mean of 2 to 3 independent experiments. (D) Basic residues of the α-helix 126-132_CT, 162-helix_CT, and α-helix 236-240_CT were mutated into alanine residues. Chymotrypsin numbering is indicated (FIXa PDB 2wpm).