Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1), expressed in HEK 293T cells and localized exclusively to membranes of the endoplasmic reticulum, was found to support both vitamin K 2,3-epoxide reductase (VKOR) and vitamin K reductase enzymatic activities. Michaelis-Menten kinetic parameters for dithiothreitol-driven VKOR activity were:

Key Points• In vitro analysis of VKORC1 mutations perfectly reflects patients' warfarin resistance phenotypes.• In silico docking of warfarin on a VKORC1 model reveals a putative docking site in agreement with the locations of OACR-associated mutations.Since the discovery of warfarin-sensitive vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1), 26 human VKORC1 (hVKORC1) missense mutations have been associated with oral anticoagulant resistance (OACR). Assessment of warfarin resistance using the "classical" dithiothreitol-driven vitamin K 2,3-epoxide reductase (VKOR) assay has not reflected clinical resistance phenotypes for most mutations. Here, we present half maximal inhibitory concentrations (IC 50 ) results for 21 further hVKORC1 mutations obtained using a recently validated cell-based assay (J Thromb Haemost 11(5):872). In contrast to results from the dithiothreitol-driven VKOR assay, all mutations exhibited basal VKOR activity and warfarin IC 50 values that correspond well to patient OACR phenotypes. Thus, the present assay is useful for functional investigations of VKORC1 and oral anticoagulant inhibition of the vitamin K cycle. Additionally, we modeled hVKORC1 on the previously solved structure of a homologous bacterial enzyme and performed in silico docking of warfarin on this model. We identified one binding site delineated by 3 putative binding interfaces. These interfaces comprise linear sequences of the endoplasmic reticulum-lumenal loop (Ser52-Phe55) and the first (Leu22-Lys30) and fourth (Phe131-Thr137) transmembrane helices. All known OACR-associated hVKORC1 mutations are located in or around these putative interfaces, supporting our model. (Blood. 2013;122(15):2743-2750

BackgroundThe phenotypic diagnosis of von Willebrand disease (VWD) is a multistep process with classification dependent on the quantification of von Willebrand factor (VWF) multimeric structure. VWF multimer analysis is a technically challenging, lengthy and non‐standardised assay, usually performed in specialist laboratories. Recently, a new semi‐automated multimer assay, the Hydragel 5 von Willebrand multimers (H5VWM) has become available.ObjectivesThis study, performed in two European centres, compared existing in‐house multimer assays to the H5VWM in individuals with and without VWD.ResultsOverall agreement of 91.1% was observed in 74 individuals with normal VWF levels, 57 patients grouped as type 1 VWD, 33 type 2A, 16 type 2B, 28 type 2M, 11 type 2N. Patients tested following Desmopressin or VWF concentrate, with thrombotic thrombocytopenic purpura and acquired von Willebrand syndrome were also evaluated. Many of the discrepancies between methods were in patients with genetic mutations linked to more than one type of VWD including p.R1374C/H and p.R1315C. Quantifiable multimer results were available within one working day. Densitometry improved the interpretation of the multimers with slight structural variations that were not apparent by visual inspection of the in‐house method.Conclusions5VWM was a rapid, sensitive, standardised assay which used existing technology and could be included as an initial screen of VWF multimers in a VWD diagnostic algorithm in conjunction with traditional multimer analysis.

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VKORC1 and VKORC1L1 are enzymes that both catalyze the reduction of vitamin K2,3-epoxide via vitamin K quinone to vitamin K hydroquinone. VKORC1 is the key enzyme of the classical vitamin K cycle by which vitamin K-dependent (VKD) proteins are γ-carboxylated by the hepatic γ-glutamyl carboxylase (GGCX). In contrast, the VKORC1 paralog enzyme, VKORC1L1, is chiefly responsible for antioxidative function by reduction of vitamin K to prevent damage by intracellular reactive oxygen species. To investigate tissue-specific vitamin K 2,3-epoxide reductase (VKOR) function of both enzymes, we quantified mRNA levels for VKORC1, VKORC1L1, GGCX, and NQO1 and measured VKOR enzymatic activities in 29 different mouse tissues. VKORC1 and GGCX are highly expressed in liver, lung and exocrine tissues including mammary gland, salivary gland and prostate suggesting important extrahepatic roles for the vitamin K cycle. Interestingly, VKORC1L1 showed highest transcription levels in brain. Due to the absence of detectable NQO1 transcription in liver, we assume this enzyme has no bypass function with respect to activation of VKD coagulation proteins. Our data strongly suggest diverse functions for the vitamin K cycle in extrahepatic biological pathways.