Institute of Human Genetics, University of Würzburg, Würzburg, Germany.
J Thromb Haemost. 2013 May;11(5):872-80. doi: 10.1111/jth.12185.
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.
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.
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.
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.
华法林通过直接抑制维生素 K 2,3-环氧化物还原酶复合物亚基 1（VKORC1）来发挥抗凝作用。VKORC1 的功能在历史上是通过二硫苏糖醇（DTT）驱动的维生素 K 2,3-环氧化物还原酶（VKOR）测定法在体外进行评估的。华法林通过 DTT-VKOR 测定法抑制野生型 VKORC1 的功能。然而，具有华法林耐药相关错义突变的 VKORC1 变体通常显示出低 VKOR 活性和对华法林的敏感性，而不是耐药性。
开发并表征了一种基于细胞培养的间接 VKOR 测定法，该方法可准确报告野生型和变体 VKORC1 蛋白对华法林的敏感性或耐药性。
在不同华法林浓度下，在标准化条件下共表达人凝血因子（F）IX 和 VKORC1 变体在 HEK 293T 细胞中。分泌的 FIX 活性作为替代标志物，用于报告华法林对共表达的 hVKORC1 野生型、Val29Leu、Val45Ala 和 Leu128Arg 变体的抑制作用。
华法林剂量反应曲线拟合共表达 hVKORC1 野生型、Val29Leu、Val45Ala 和 Leu128Arg 变体的分泌 FIX 活性数据。相应计算出的 IC50 值分别为 24.7、136.4、152.0 和 1226.4nm。所有 VKORC1 变体在不存在华法林的情况下的基础活性与野生型 VKORC1 的活性相似。基于细胞培养的测定法的排序 IC50 值准确反映了 VKORC1 错义突变相关华法林耐药患者需要提高华法林剂量。
