Cell-based activity assay and analysis of warfarin responses

Assays of the epoxide reductase activity were performed as previously described [5] using a cell line established with a chimeric FIXgla-Protein C gene and with endogenous VKORC1 and VKORL1 genes knocked out. Wild-type and mutant VKORL1 clones, along with a luciferase gene, were transfected into this double-knockout cell line. The carboxylation level of secreted FIXgla-PC was measured by a sandwich ELISA by using the cell-culture medium, with luciferase activity serving as the control for transfection efficiency. The chimeric FIXgla-Protein C was used because antibody that specifically recognizes fully carboxylated FIX-gla is commercially available (FIXgla mAb from Green Mountain Antibodies) [27]. In addition, over 90% of the uncarboxylated protein C is degraded and not secreted into the culture medium, significantly reducing the possible background. The ELISA assay was conducted following a protocol reported by the Stafford group [27]. Briefly, ELISA plates were coated with FIXgla mAb overnight at 4°C, and subsequently blocked by bovine serum albumin. Samples of secreted FIXgla-PC and protein standards with 5 mM CaCl₂ were added and incubated for 2 hours at room temperature. After washing with TBST buffer (20mM Tris-HCl, pH 7.6, 150mM NaCl, and 0.1% Tween 20) containing CaCl₂, the HRP conjugated anti–human protein C IgG (Affinity Biologicals) was added to each well and incubated for 45 minutes at room temperature. The HRP activity was analyzed by incubating with 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and the absorbance was measured at 405 nm using a ELISA plate reader (Molecular Devices).

To measure IC50s of warfarin (and 4-HC) in the VKORL1 constructs, the transfected cells were treated with 11 different concentrations of warfarin, with the concentration range optimized according to the warfarin response of each construct. The IC50 of each construct was analyzed using GraphPad Prism. The normalized warfarin resistance (nR_war) was determined by dividing IC50s of the mutants to the IC50 of wild type VKORL1, measured in the same set of experiments. Error propagations were calculated accordingly. In our experience with this assay, nR_war > 3 is generally a reliable result and nR_war > 5 indicates substantial resistance (Fig. 1). The relative normalized resistance of VKORL1 and VKORC1 constructs to warfarin (Table S1) was given by (nR_war of VKORL1 / nR_war of VKORC1), with error propagations calculated accordingly.