Characterizing FIX procoagulant activity and FIX antigen of missense variants in FIX’s SP by an in vitro cell system. (A) Sequence of FIX’s SP. The n-region (green), h-region (purple), and c-region (red) are indicated. The residues with reported missense variants were underlined. (B-D) The constructs of the variants were overexpressed in HEK293T cells in the medium with 10 μM vitamin K, and the FIX procoagulant activity (FIX:C), FIX antigen with functional Gla domain, and total FIX antigen were determined by using corresponding methods (see “Methods”). Relative values were normalized by wild-type (WT) FIX, which was defined as 1. The dashed green and red lines indicate the 40% and 5% level of WT FIX, respectively. Error bars are standard deviation calculated from 3 repeats. (B) Normalized FIX:C; (C) Normalized FIX antigen with functional Gla domain; and (D) Normalized total FIX antigen.