Heterologous expression of N1922S fVIII and wt-fVIII by BHK-M cells. (A) Domain structure of fVIII. X marks the site of the N1922S substitution. (B) Substitution site in N1922S-fVIII. The NH₂-terminal and COOH-terminal subdomains are shown in red and blue, respectively. The N1922 backbone and side chain are shown at the interface of the 2 domains using CPK coloring. (C) BHK-M cells were stably transfected with a plasmid encoding a geneticin-resistance gene and either the N1922S or wt-fVIII cDNA, as described in “Methods.” The top N1922S-fVIII clone and a stock wt-fVIII clone were grown to greater than 90% confluence. FVIII activity was measured by one-stage coagulation assay 24 hours after switching to serum-free medium. FVIII antigen also was measured at 24 hours by indirect ELISA using an anti-A2 domain MAb, 4A4, and a biotinylated anti-C2 MAb, 2-117, as capture and detection antibodies, respectively. Results are reported as mean and sample standard deviation of 6 samples per construct.