title: Determination of the warfarin inhibition constant Ki for vitamin K 2,3-epoxide reductase complex subunit-1 (VKORC1) using an in vitro DTT-driven assay.
abstract: BACKGROUND
  Warfarin directly inhibits vitamin K 2,3-epoxide reductase (VKOR) enzymes. Since the early 1970s, warfarin inhibition of vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1), an essential enzyme for proper function of blood coagulation in higher vertebrates, has been studied using an in vitro dithiothreitol (DTT) driven enzymatic assay. However, various studies based on this assay have reported warfarin dose-response data, usually summarized as half-maximal inhibitory concentration (IC50), that vary over orders of magnitude and reflect the broad range of conditions used to obtain VKOR assay data.
  
  
  METHODS
  We standardized the implementation of the DTT-driven VKOR activity assay to measure enzymatic Michaelis constants (Km) and warfarin IC50 for human VKORC1. A data transformation is defined, based on the previously confirmed bi bi ping-pong mechanism for VKORC1, that relates assay condition-dependent IC50 to condition-independent Ki.
  
  
  RESULTS
  Determination of the warfarin Ki specifically depends on measuring both substrate concentrations, both Michaelis constants for the VKORC1 enzyme, and pH in the assay.
  
  
  CONCLUSIONS
  The Ki is not equal to the IC50 value directly measured using the DTT-driven VKOR assay.
  
  
  CONCLUSIONS
  In contrast to warfarin IC50 values determined in previous studies, warfarin inhibition expressed as Ki can now be compared between studies, even when the specific DTT-driven VKOR assay conditions differ. This implies that warfarin inhibition reported for wild-type and variant VKORC1 enzymes from previous reports should be reassessed and new determinations of Ki are required to accurately report and compare in vitro warfarin inhibition results.
authors.affiliation: Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt-am-Main, Germany.
authors.forename: Carville G
authors.initials: CG
authors.lastname: Bevans
authors.forename: Christoph
authors.initials: C
authors.lastname: Krettler
authors.forename: Christoph
authors.initials: C
authors.lastname: Reinhart
authors.forename: Hélène
authors.initials: H
authors.lastname: Tran
authors.forename: Katja
authors.initials: K
authors.lastname: Koßmann
authors.forename: Matthias
authors.initials: M
authors.lastname: Watzka
authors.forename: Johannes
authors.initials: J
authors.lastname: Oldenburg
mesh_terms: D004229:Dithiothreitol / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors*; D064417:Vitamin K Epoxide Reductases; D014859:Warfarin / Q000494:pharmacology*
chemical_list: D014859:Warfarin; D006899:Mixed Function Oxygenases; C578523:VKORC1 protein, human; D064417:Vitamin K Epoxide Reductases; D004229:Dithiothreitol