Effect of recombinant factor IX mutants on DHG inhibition of plasma thrombin generation. Thrombin generation was initiated with 0.2 pM human TF, 8.3 μM PC:PS vesicles, and 40 μg/mL CTI (plasma concentrations) in factor IX–deficient plasma supplemented with 100% (90 nM) recombinant factor IX WT (A), R170A (B), and R233A (C) in the presence of increasing DHG: 0 μM (●), 0.1 μM (○), 0.25 μM (■), 0.5 μM (□), 1 μM (▴), 2.5 μM (▵), 5 μM (♦), 10 μM (◇). Control reactions are: no TF or factor IX (▾), 0.2 pM TF only (▿), and 90 nM recombinant factor IX only (). The time course of thrombin generation was measured as described in “Fluorogenic assay for detection of plasma thrombin generation.” Thrombin generation curves represent the mean thrombin concentration over the first 30 minutes from replicate determinations (n = 3) and are identified by representative points. The relative velocity index for thrombin generation was plotted versus DHG concentration for each condition, and data were fit as described in “Fluorogenic assay for detection of plasma thrombin generation” to determine the EC₅₀ for inhibition (D). Representative curves are presented.