Confirmation of warfarin resistance of naturally occurring VKORC1 variants by coexpression with coagulation factor IX and in silico protein modelling.
BACKGROUND
VKORC1 has been identified some years ago as the gene encoding vitamin K epoxide reductase (VKOR) - the target protein for coumarin derivates like warfarin or phenprocoumon. Resistance against warfarin and other coumarin-type anticoagulants has been frequently reported over the last 50 years in rodents due to problems in pest control as well as in thrombophilic patients showing variable response to anticoagulant treatment. Many different mutations have already been detected in the VKORC1 gene leading to warfarin resistance in rats, mice and in humans. Since the conventional in vitro dithiothreitol (DTT)-driven VKOR enzymatic assay often did not reflect the in vivo status concerning warfarin resistance, we recently developed a cell culture-based method for coexpression of VKORC1 with coagulation factor IX and subsequent measurement of secreted FIX in order to test warfarin inhibition in wild-type and mutated VKORC1.


RESULTS
In the present study, we coexpressed wild-type factor IX with 12 different VKORC1 variants which were previously detected in warfarin resistant rats and mice. The results show that amino acid substitutions in VKORC1 maintain VKOR activity and are associated with warfarin resistance. When we projected in silico the amino acid substitutions onto the published three-dimensional model of the bacterial VKOR enzyme, the predicted effects matched well the catalytic mechanism proposed for the bacterial enzyme.


CONCLUSIONS
The established cell-based system for coexpression of VKORC1 and factor IX uses FIX activity as an indicator of carboxylation efficiency. This system reflects the warfarin resistance status of VKORC1 mutations from anticoagulant resistant rodents more closely than the traditional DTT-driven enzyme assay. All mutations studied were also predicted to be involved in the reaction mechanism.
Elisabeth
E
Müller
Alexander
A
Keller
Andreas
A
Fregin
Clemens R
CR
Müller
Department of Human Genetics, University of Würzburg, Würzburg, Germany. simone.rost@biozentrum.uni-wuerzburg.de.
Simone
S
Rost
D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D005164:Factor IX / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D008661:Metabolism, Inborn Errors / Q000235:genetics*; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D051381:Rats; D046940:Synechococcus / Q000201:enzymology; D064417:Vitamin K Epoxide Reductases / Q000235:genetics*
D001426:Bacterial Proteins; D007527:Isoenzymes; D005164:Factor IX; C578523:VKORC1 protein, human; D064417:Vitamin K Epoxide Reductases