Journal
BLOOD
Volume 123, Issue 4, Pages 582-589Publisher
AMER SOC HEMATOLOGY
DOI: 10.1182/blood-2013-05-505123
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Funding
- National Institutes of Health, National Institutes of General Medical Sciences [P01GM32165, R01GM109743, U01GM92676]
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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. Here we describe a sensitive assay that enables quantitative analysis of gamma-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 gamma-glutamyl carboxylation of F9CH required vitamin K supplementation, and was dose-dependently inhibited by racemic warfarin at a physiologically relevant concentration. Cellular gamma-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 gamma-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.
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