Amino acid screen at FIX position 338. Specific activity of hFIX variants (A) and cFIX-variants (B) relative to hFIX-WT (R338) and cFIX-WT (R338), respectively. FIX-WT (R338) is highlighted in in blue, and R338L (Padua) is highlighted in red. Variants expressed in HEK-293 cells. cFIX-R338I is highlighted in black in 2B. *Statistically significant difference in specific activity from FIX-WT at the .05 level. (C) Scatter plot of specific activities of hFIX and cFIX R338 substituted variants (green letters). Line is linear correlation (y = (0.9 ± 0.2)x – (0.5 ± 0.2); Pearson’s r = .62). Total activity and total antigen hFIX variants (D) and cFIX variants (E) relative to hFIX-WT (R338) and cFIX-WT (R338), respectively. Bars or square points represent the mean specific activity of 3 to 8 independent transient transfections, and errors bars represent ± standard error of the mean.