Regular Article THROMBOSIS AND HEMOSTASIS A cellular system for quantitation of vitamin K cycle activity: structure-activity effects on vitamin K antagonism by warfarin metabolites

• Factor IX glutamyl carboxylation in engineered HEK 293 cells recapitulates in vivo anticoagulant inhibition of vitamin K cycle activity. • Warfarin metabolite structureactivity analysis on vitamin K cycle antagonism determines their contributions to in vivo anticoagulation. Warfarin and other 4-hydroxycoumarins inhibit vitamin K epoxide reductase (VKOR) by depleting reduced vitamin K that is required for posttranslational modification of vitamin K–dependent clotting factors. In vitro prediction of the in vivo potency of vitamin K antagonists is complicated by the complex multicomponent nature of the vitamin K cycle. Herewe describe a sensitive assay that enables quantitative analysis of g-glutamyl carboxylation and its antagonism in live cells.We engineered a human embryonic kidney (HEK) 293–derived cell line (HEK 293-C3) to express a chimeric protein (F9CH) comprising the Gla domain of factor IX fused to the transmembrane and cytoplasmic regions of proline-rich Gla protein 2. Maximal g-glutamyl carboxylation of F9CH required vitamin K supplementation, and was dose-dependently inhibited by racemic warfarin at a physiologically relevant concentration. Cellular g-glutamyl carboxylation also exhibited differential VKOR inhibition by warfarin enantiomers (S > R ) consistent with their in vivo potencies. We further analyzed the structure-activity relationship for inhibition of g-glutamyl carboxylation by warfarin metabolites, observing tolerance to phenolic substitution at the C-5 and especially C-6, but not C-7 or C-8, positions on the 4-hydroxycoumarin nucleus. After correction for in vivo concentration and protein binding, 10-hydroxywarfarin and warfarin alcohols were predicted to be the most potent inhibitory metabolites in vivo. (Blood. 2014;123(4):582-589)