Warfarin uncouples the 2 VKORC1 reactions that fully reduce KO to KH₂. (A) r-wt VKORC1/r-fIX BHK cells were incubated without vitamin K (−K) or with KO or K (both at 2 µM) in the absence (−) or presence (+) of warfarin (2 µM). Secreted fIX was analyzed in westerns for carboxylation (anti-Gla²¹ ) or total fIX (anti-fIX). The differences in fIX migration are a result of glycosylation.³⁴  (B) r-fIX BHK and r-wt VKORC1/r-fIX BHK cells incubated with KO (2 µM) were monitored for KO reduction by isolation of intracellular vitamin K, followed by high-performance liquid chromatography analysis to separate and quantitate individual forms. fIX carboxylation was monitored by western analysis with anti-Gla antibody. (C) r-wt VKORC1/r-fIX BHK cells were incubated with KO (2 µM) and varying amounts of warfarin. KO reduction and fIX carboxylation were monitored as in (B). The KO to K curve is only slightly affected by subsequent K reduction, as only ∼15% of the K resulted in Gla product. (D) The K to KH₂ reaction and consequent carboxylation was analyzed using low K levels that result from inhibition of the KO to K reaction. Cells were incubated with warfarin (50 nM) and KO (2 µM) or K (0.4-2 µM). A range of K concentrations was used to identify cells with the appropriate intracellular K concentration for comparison with cells incubated in KO. Warfarin decreased KO to K reduction 2.5-fold, giving an intracellular K level of 1 nmol for 10⁷ cells. Left: warfarin (50 nM) inhibition of carboxylation in cells containing this intracellular K level. Right: warfarin inhibition of cells incubated with KO (2 µM).