Effect of N-glycanase for I566T mutant FVIII. (A) Immunoprecipitated WT and I566T mutant from radiolabeled CM were treated with nothing (IIa⁻, N-Gly⁻) or thrombin (IIa⁺, N-Gly⁻) or N-glycanase after thrombin (IIa⁺, N-Gly⁺) before SDS-PAGE as described in the Materials and Methods. Molecular weight size markers are shown on the left. A3-C1-C2, A1, A2, and mutant A2 fragments are indicated at the right. (B) FVIII in conditioned medium as well as in patient's plasma were incubated with 10 U/mL of N-glycanase at 37°C. FVIII activity after increasing times was determined by one stage clotting assay. Data are plotted as the percent activity of the mutant compared with the WT-recombinant (○) or plasma-derived (•) FVIII, respectively. Data represent the average of two independent experiments.