AQ, activation quotient.

FVIII activities (IU/mL) in BHK-M cell supernatants were measured in triplicate using a 1-stage coagulation assay, 1- and 2-stage coagulation assays to calculate AQs, and chromogenic assays for 3 cell lines per FVIII protein. Specific activities (IU/mg) were calculated using the concentrations of FVIII in cell supernatants as measured in an ELISA. The average specific activity for each protein was calculated from the specific activities of the individual cell lines ± standard deviations (SDs) as shown in “1-Stage coagulation assay” and “Chromogenic assay.” Percentage WT (% WT) activity for each BDD-FVIII mutein = (average specific activity or AQ of the cell lines expressing the mutein/average specific activity or AQ of the cell lines expressing WT-BDD-FVIII) × 100. Details regarding individual cell lines are provided in supplemental Figure 4. The WT-BDD-FVIII cell line is HSQ/AvrII/RENEO–expressing BHK-M cells, kindly provided by Pete Lollar’s laboratory (Emory University, Atlanta, GA).

The FVIII activities (IU/mL) of the purified proteins were measured using 1-stage coagulation (n = 4), 1- and 2-stage coagulation to calculate AQs (n = 4), and chromogenic assays (n = 6). Specific activities (IU/mg) were calculated using the concentrations of FVIII determined by measuring A₂₈₀ of the purified protein solutions and using an extinction coefficient of 256 300 M⁻¹cm⁻¹. Research-grade Kogenate, provided by Bayer at a specific activity of 3600 IU/mg, was used as a control for the assay.

AQs are calculated as ratios of 2-stage FVIII activity divided by 1-stage FVIII activity. The expected AQ for human FVIII that has not been significantly preactivated is 15-25.⁴⁷