Figure 4. Effect of mevalonate pathway inhibitors on VKD carboxylation.

(A and B) Inhibition of VKD carboxylation by simvastatin (Sim), atorvastatin (Ato), or warfarin (Warf) in FIXgla-PC/HEK293 reporter cells when vitamin K₃ (A) or vitamin K₁ (B) were used as the substrate. Warfarin was used as a control for the inhibition of VKD carboxylation. Reporter cells were incubated with a cell culture medium containing 1 μM vitamin K₃ (A) or 5 μM vitamin K₁ (B) and increasing concentrations of the inhibitors for 24 hours. (C) The dose-dependent response of GGPP to VKD carboxylation when the endogenous GGPP production is blocked by simvastatin (Sim) or atorvastatin (Ato). FIXgla-PC/HEK293 reporter cells were incubated with cell culture medium containing increasing concentrations of GGPP and 10 μM atorvastatin (Ato) or simvastatin (Sim). (D) Conversion of vitamin K₃ to MK-2, MK-3, and MK-4 by UBIAD1 using GPP, FPP, and GGPP as the side chain substrates, respectively. FIXgla-PC/HEK293 cells were incubated with cell culture medium containing 1 μM vitamin K₃, 10 μM atorvastatin or simvastatin, and 20 μM GPP, FPP, or GGPP. Reporter-protein carboxylation was determined as described in the legend of Figure 1B. Carboxylation activity of GGPP was normalized to 100%. Data are presented as mean ± SD of three independent experiments (n=3).