Functional study of VKOR and its mutants. (A) Cell-based activity assay of VKOR and its mutants. Data are presented as mean ± SD (n = 3). Warfarin-resistance study of the Y139A mutant (B), the Y25A mutant (C), and the A26T mutant (D) were performed by culturing the VKOR/VKORC1L1-knockout FIXgla-PC/HEK293 reporter cells expressing the individual mutant with 5 µM KO containing increasing concentrations of warfarin. Warfarin resistance, as determined by IC₅₀, is available in supplemental Table 2. (E) The response of apparent Vmax to VKOR concentrations in the presence and absence of warfarin. Apparent Vmax was determined under different enzyme concentrations, with 0 nM, 2 nM, and 10 nM warfarin. VKOR concentrations were controlled by seeding different numbers of the reporter cells into the multiwell cell culture plate. Because the reporter cell line was derived from a single cell colony, we assume that each individual cell has a similar expression level of the endogenous VKOR, and VKOR concentration is proportional to the cell numbers. (F) Removal of warfarin from the VKOR-warfarin complex. VKOR-containing microsomes were incubated with 30 µM warfarin on ice for 30 minutes; warfarin was then removed by dilution and ultracentrifugation for activity assay. VKOR, VKOR-containing microsomes; VKOR+warfarin, VKOR-containing microsomes incubated with 30 µM warfarin; Wash, warfarin-treated VKOR-containing microsomes after wash.