title: A new cell culture-based assay quantifies vitamin K 2,3-epoxide reductase complex subunit 1 function and reveals warfarin resistance phenotypes not shown by the dithiothreitol-driven VKOR assay.
abstract: BACKGROUND
  Warfarin directly inhibits the vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) enzyme to effect anticoagulation. VKORC1 function has historically been assessed in vitro using a dithiothreitol (DTT)-driven vitamin K 2,3-epoxide reductase (VKOR) assay. Warfarin inhibits wild-type VKORC1 function by the DTT-VKOR assay. However, VKORC1 variants with warfarin resistance-associated missense mutations often show low VKOR activities and warfarin sensitivity instead of resistance.
  
  
  OBJECTIVE
  A cell culture-based, indirect VKOR assay was developed and characterized that accurately reports warfarin sensitivity or resistance for wild-type and variant VKORC1 proteins.
  
  
  METHODS
  Human coagulation factor (F)IX and VKORC1 variants were coexpressed in HEK 293T cells under standardized conditions at various warfarin concentrations. Secreted FIX activity served as surrogate marker to report wild-type and variant VKORC1 inhibition by warfarin.
  
  
  CONCLUSIONS
  Warfarin dose-response curves fit to the secreted FIX activity data for coexpressed hVKORC1 wild-type, Val29Leu, Val45Ala and Leu128Arg variants. The corresponding calculated IC50 values were 24.7, 136.4, 152.0 and 1226.4 nm, respectively. Basal activities in the absence of warfarin for all VKORC1 variants were similar to that of wild-type VKORC1. Ranked IC50 values from the cell culture-based assay accurately reflect elevated warfarin dosages for patients with VKORC1 missense mutation-associated warfarin resistance.
authors: Institute of Human Genetics, University of Würzburg, Würzburg, Germany.
authors: A
authors: A
authors: Fregin
authors: K J
authors: KJ
authors: Czogalla
authors: J
authors: J
authors: Gansler
authors: S
authors: S
authors: Rost
authors: M
authors: M
authors: Taverna
authors: M
authors: M
authors: Watzka
authors: C G
authors: CG
authors: Bevans
authors: C R
authors: CR
authors: Müller
authors: J
authors: J
authors: Oldenburg
mesh_terms: D000925:Anticoagulants / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D008661:Metabolism, Inborn Errors; D020125:Mutation, Missense; D010641:Phenotype; D064417:Vitamin K Epoxide Reductases / Q000235:genetics / Q000378:metabolism*; D014859:Warfarin / Q000494:pharmacology*
chemical_list: D000925:Anticoagulants; D014859:Warfarin; C578523:VKORC1 protein, human; D064417:Vitamin K Epoxide Reductases; D004229:Dithiothreitol