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Warfarin alters vitamin K metabolism: a surprising mechanism of VKORC1 uncoupling necessitates an additional reductase

Mark A. Rishavy, Kevin W. Hallgren, Lee Wilson, Savita Singh, Kurt W. Runge and Kathleen L. Berkner

Key Points

  • Analysis of full VKORC1 reduction of vitamin K epoxide vs the individual reactions shows that warfarin uncouples the 2 reactions.

  • A mutant becomes more active than wild-type VKORC1 only in the presence of warfarin, revealing a novel mechanism for warfarin resistance.

Publisher's Note: There is a Blood Commentary on this article in this issue.

Abstract

The anticoagulant warfarin inhibits the vitamin K oxidoreductase (VKORC1), which generates vitamin K hydroquinone (KH2) required for the carboxylation and consequent activation of vitamin K–dependent (VKD) proteins. VKORC1 produces KH2 in 2 reactions: reduction of vitamin K epoxide (KO) to quinone (K), and then KH2. Our dissection of full reduction vs the individual reactions revealed a surprising mechanism of warfarin inhibition. Warfarin inhibition of KO to K reduction and carboxylation that requires full reduction were compared in wild-type VKORC1 or mutants (Y139H, Y139F) that cause warfarin resistance. Carboxylation was much more strongly inhibited (∼400-fold) than KO reduction (two- to threefold). The K to KH2 reaction was analyzed using low K concentrations that result from inhibition of KO to K. Carboxylation that required only K to KH2 reduction was inhibited much less than observed with the KO substrate that requires full VKORC1 reduction (eg, 2.5-fold vs 70-fold, respectively, in cells expressing wild-type VKORC1 and factor IX). The results indicate that warfarin uncouples the 2 reactions that fully reduce KO. Uncoupling was revealed because a second activity, a warfarin-resistant quinone reductase, was not present. In contrast, 293 cells expressing factor IX and this reductase activity showed much less inhibition of carboxylation. This activity therefore appears to cooperate with VKORC1 to accomplish full KO reduction. Cooperation during warfarin therapy would have significant consequences, as VKD proteins function in numerous physiologies in many tissues, but may be poorly carboxylated and dysfunctional if the second activity is not ubiquitously expressed similar to VKORC1.

  • Submitted September 1, 2017.
  • Accepted March 7, 2018.
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