Human VKORC1 mutations cause variable degrees of 4-hydroxycoumarin resistance and affect putative warfarin binding...

Area of Science:

• Biochemistry
• Pharmacology
• Genetics

Background:

• 26 human VKORC1 mutations are linked to oral anticoagulant resistance (OACR).
• The classical dithiothreitol-driven VKOR assay poorly reflects clinical warfarin resistance phenotypes for most mutations.

Purpose of the Study:

• To evaluate 21 additional hVKORC1 mutations using a validated cell-based assay.
• To compare cell-based assay results with the classical VKOR assay and clinical OACR phenotypes.
• To model hVKORC1 and identify warfarin binding sites.

Main Methods:

• Utilized a recently validated cell-based assay to determine IC50 values for 21 hVKORC1 mutations.
• Performed in silico modeling of hVKORC1 based on a homologous bacterial enzyme structure.
• Conducted in silico docking of warfarin onto the hVKORC1 model.

Main Results:

• The cell-based assay yielded IC50 values that correlate well with patient OACR phenotypes, unlike the dithiothreitol-driven VKOR assay.
• All tested mutations showed basal VKOR activity and warfarin IC50 values consistent with clinical resistance.
• A single warfarin binding site was identified, involving specific loops and transmembrane helices, with all known OACR mutations located nearby.

Conclusions:

• The cell-based assay is a reliable tool for functional investigation of VKORC1 mutations and their impact on oral anticoagulant inhibition.
• The identified warfarin binding site and mutation locations provide structural insights into OACR mechanisms.
• This assay improves the understanding of vitamin K cycle inhibition and warfarin resistance.