Effect of recombinant factor IXa mutants on DHG inhibition of plasma thrombin generation. Thrombin generation was initiated with 100 pM recombinant factor IXa WT (A), R170A (B), or R233A (C), 8.3 μM PC:PS vesicles, and 40 μg/mL CTI (plasma concentrations) in factor IX–deficient plasma 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 without recombinant factor IXa are presented (▾). 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 “Methods” to determine the EC50 for inhibition (D). Representative curves are presented.
