Functional characterization of secreted and intracellular wt-fVIII and N1922S-fVIII. BHK-M cells expressing wt-fVIII or N1922S-fVIII were switched to serum-free medium for 24 hours, followed by removal of the medium and extraction of intracellular fVIII from cell lysate, as described in “Heterologous expression of fVIII.” Specific activity of fVIII secreted into the medium or intracellular fVIII was determined as the ratio of fVIII activity measured by one-stage coagulation assay to fVIII antigenic mass measured by ELISA. The inset shows the activation quotients of wt-fVIII and N1922S-fVIII in the medium.