Characterization of the 3 identified GGCX mutations using the established cell-based assay. (A) Carboxylation of FIXgla-ProT (black bars) and MGP (gray bars) by wild-type GGCX, GGCX_D153G, GGCX_M174R, and GGCX_R325Q in GGCX-deficient cells in the presence of 5 µg/mL vitamin K1. (B) Carboxylation of FIXgla-ProT by wild-type GGCX (circles), GGCX_D153G (solid squares), GGCX_M174R (open squares), and GGCX_R325Q (triangles) with increasing concentrations of vitamin K1; the vertical dashed line indicates the presumed physiological concentration of vitamin K1. (C) Carboxylation of MGP by the wild-type GGCX, GGCX_D153G, GGCX_M174R, and GGCX_R325Q with increasing concentrations of vitamin K1 as in panel B. (D) Carboxylation of FIXgla-ProT by wild-type GGCX with increasing concentrations of vitamin K1 (circles), MK-4 (squares), and MK-7 (triangles). (E) Carboxylation of FIXgla-ProT by GGCX_D153G with increasing concentrations of K vitamins as in panel D. (F) Carboxylation of MGP by GGCX_D153G with increasing concentrations of K vitamins as in panel D. Carboxylation of MGP by the wild-type GGCX with vitamin K1 was used as a control (darker circles).