Effect of factor VIII and factor IX activation on inhibition of thrombin generation by DHG. 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 VIII–deficient plasma supplemented with 700 pM recombinant factor VIII (rFVIII) (A) or 700 pM thrombin-activated factor (rFVIIIa) (B); or factor IX–deficient plasma supplemented with 100% (90 nM) plasma-derived factor IX (D) or 100 pM plasma-derived factor IXa in the absence of TF (E) 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 (◇). In panels A and B, control reactions are: no TF or factor VIII(a) (◇), 0.2 pM TF only (▾), and factor VIII(a) only (▿). Control for exogenous thrombin control used in factor VIII activation is not shown. In panels D and E, control reactions are: no TF or factor IX (▾) and 0.2 pM TF 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 in factor VIII–deficient plasma (C) and in factor IX–deficient plasma (F). Representative curves are presented.