Specific activity of factor VIII mutants relative to WT factor VIII and thrombin generation assays. (A) Specific Activity. Activity values were determined using a 1-stage clotting assay () and 2-stage chromogenic factor Xa generation assay (■). (B,C) Thrombogram of factor VIII proteins. WT (), Glu272Ala (□), Glu272Val (■), Asp519Ala (○), Asp519Val (●), Glu665Ala (△), Glu665Val (▲), Glu1984Ala (◇), and Glu1984Val (♦). (D) Parameter values obtained from thrombin generation assays. Thrombograms show the average values of triplicated samples. The parameter values were expressed as values (%) relative to WT. The actual values for WT were 7.5 plus or minus 0.5 minutes (lag time), 13.7 plus or minus 0.3 minutes (peak time), 157.3 plus or minus 14.7 nM (peak value), and 979.8 plus or minus 37.9 nM/min (ETP). Lag time (□), peak time (), peak value (■), and ETP (▧). Error bars represent SD values averaged from 3 separate determinations.