A new cell culture‐based assay quantifies vitamin K 2, 3‐epoxide reductase complex subunit 1 function and reveals warfarin resistance phenotypes not shown by the dithiothreitol‐driven VKOR assay. Issue 5 (15th May 2013)

• Record Type: Journal Article
• Authors: Fregin, A.; Czogalla, K. J.; Gansler, J.; Rost, S.; Taverna, M.; Watzka, M.; Bevans, C. G.; MüLler, C. R.; Oldenburg, J.
• Is Part Of: Journal of thrombosis and haemostasis. Volume 11:Issue 5(2013)
• Page Start: 872
• Page End: 880
• Publication Date: 2013-05-15
• Subjects: Thrombosis -- Periodicals; Hemostasis -- Periodicals; Blood coagulation disorders -- Periodicals; 616.1
• DOI: 10.1111/jth.12185
• Languages: English
• ISSNs: 1538-7933

Abstract

<abstract abstract-type="main" id="jth12185-abs-0001"> <title>Summary</title> <sec id="jth12185-sec-0001" sec-type="section"> <title>Background</title> <p>Warfarin directly inhibits the vitamin K 2, 3‐epoxide reductase complex subunit 1 (<italic>VKORC1</italic>) enzyme to effect anticoagulation. <italic>VKORC1</italic> function has historically been assessed <italic>in vitro</italic> 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, <italic>VKORC1</italic> variants with warfarin resistance‐associated missense mutations often show low VKOR activities and warfarin sensitivity instead of resistance.</p> </sec> <sec id="jth12185-sec-0002" sec-type="section"> <title>Objectives</title> <p>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.</p> </sec> <sec id="jth12185-sec-0003" sec-type="section"> <title>Methods</title> <p>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.</p> </sec> <sec id="jth12185-sec-0004" sec-type="section"> <title>Results and conclusions</title> <p>Warfarin dose‐response curves fit to the secreted FIX activity data for coexpressed hVKORC1 wild‐type, Val29Leu, Val45Ala and Leu128Arg variants. The corresponding calculated IC<sub>50</sub> values were 24.7, 136.4, 152.0 and 1226.4 n<sc>m</sc>, respectively. Basal activities in the absence of warfarin for all VKORC1 variants were similar to that of wild‐type VKORC1. Ranked IC<sub>50</sub> values from the cell culture‐based assay accurately reflect elevated warfarin dosages for patients with VKORC1 missense mutation‐associated warfarin resistance.</p> </sec> </abstract>