Inhibition of intrinsic factor Xase activity by synthetic peptide 558 to 565. (A) Equal amounts (4 nmol/L) of partially purified FVIII samples were mixed with increasing concentrations of synthetic peptide and assayed for FVIII activity as described in the Materials and Methods. The data represent the average of three independent experiments. FVIII activity for each molecule is expressed as a percent of that obtained in the absence of the peptide. The symbols represent WT FVIII (•), S558F (▴), S558A (▵), I566T (▪) and V634A (⧫). (B) WT and I566T mutant FVIII were treated with N-glycanase for 3 hours at 37°C, then samples were tested in the peptide inhibition assay. The results represent the average of three (filled symbols) and two (open symbols) independent experiments. The symbols represent WT FVIII (•), WT FVIII with N-glycanase (○), I566T (▪) and I566T with N-glycanase (□). (C) Equal amounts (4 nmol/L) of partially purified FVIII samples were mixed with increasing concentrations of scrambled synthetic peptide and assayed for FVIII activity as described in the Materials and Methods. Two independent experiments are shown for WT FVIII (•), S558F (▴), I566T (▪) and V634A (⧫). (D) Four different concentrations of WT FVIII (4 nmol/L [•], 0.8 nmol/L [▪], 0.4 nmol/L [▴], 0.08 nmol/L [⧫]) were tested in the peptide inhibition assay. The results are the average of two independent experiments.