TY - JOUR
T1 - Competitive tight-binding inhibition of VKORC1 underlies warfarin dosage variation and antidotal efficacy
AU - Li, Shuang
AU - Liu, Shixuan
AU - Liu, Xiaoran Roger
AU - Zhang, Mengru Mira
AU - Li, Weikai
N1 - Funding Information:
The authors thank Evan Sadler for his generous support and invaluable advice to W.L., Charles Eby for critical reading of the manuscript, Guomin Shen for insightful discussion, and Michael Gross for revising the writing. W.L. is supported by the National Institutes of Health, National Heart, Lung, and Blood Institute (R01 HL121718); W. M. Keck Foundation (Basic Science @ the Forefront of Science); Children's Discovery Institute (MC-II-2020-854); National Institutes of Health, National Eye Institute (R21 EY028705); and National Institutes of Health, National Institute of General Medical Sciences (R01 GM131008). LC-MS measurements were supported by National Institutes of Health, National Institute of General Medical Sciences (P41 GM103422).
Funding Information:
W.L. is supported by the National Institutes of Health, National Heart, Lung, and Blood Institute (R01 HL121718); W. M. Keck Foundation (Basic Science @ the Forefront of Science); Children’s Discovery Institute (MC-II-2020-854); National Institutes of Health, National Eye Institute (R21 EY028705); and National Institutes of Health, National Institute of General Medical Sciences (R01 GM131008). LC-MS measurements were supported by National Institutes of Health, National Institute of General Medical Sciences (P41 GM103422).
Publisher Copyright:
© 2020 by The American Society of Hematology
PY - 2020/5/26
Y1 - 2020/5/26
N2 - Dose control of warfarin is a major complication in anticoagulation therapy and overdose is reversed by the vitamin K antidote. Improving the dosage management and antidotal efficacy requires mechanistic understanding. Here we find that effects of the major predictor of warfarin dosage, SNP 21639 G.A, follow a general correlation that warfarin 50% inhibitory concentration decreases with cellular level of vitamin K epoxide reductase (VKORC1), suggesting stoichiometric inhibition. Characterization of the inhibition kinetics required the use of microsomal VKORC1 with a native reductant, glutathione, that enables effective warfarin inhibition in vitro. The kinetics data can be fitted with the Morrison equation, giving a nanomolar inhibition constant and demonstrating that warfarin is a tight-binding inhibitor. However, warfarin is released from purified VKORC1-warfarin complex with increasing amount of vitamin K, indicating competitive inhibition. The competition occurs also in cells, resulting in rescued VKORC1 activity that augments the antidotal effects of vitamin K. Taken together, warfarin is a competitive inhibitor that binds VKORC1 tightly and inhibits at a stoichiometric (1:1) concentration, whereas exceeding the VKORC1 level results in warfarin overdose. Thus, warfarin dosage control should use VKORC1 level as a major indicator, and improved antidotes may be designed based on their competition with warfarin.
AB - Dose control of warfarin is a major complication in anticoagulation therapy and overdose is reversed by the vitamin K antidote. Improving the dosage management and antidotal efficacy requires mechanistic understanding. Here we find that effects of the major predictor of warfarin dosage, SNP 21639 G.A, follow a general correlation that warfarin 50% inhibitory concentration decreases with cellular level of vitamin K epoxide reductase (VKORC1), suggesting stoichiometric inhibition. Characterization of the inhibition kinetics required the use of microsomal VKORC1 with a native reductant, glutathione, that enables effective warfarin inhibition in vitro. The kinetics data can be fitted with the Morrison equation, giving a nanomolar inhibition constant and demonstrating that warfarin is a tight-binding inhibitor. However, warfarin is released from purified VKORC1-warfarin complex with increasing amount of vitamin K, indicating competitive inhibition. The competition occurs also in cells, resulting in rescued VKORC1 activity that augments the antidotal effects of vitamin K. Taken together, warfarin is a competitive inhibitor that binds VKORC1 tightly and inhibits at a stoichiometric (1:1) concentration, whereas exceeding the VKORC1 level results in warfarin overdose. Thus, warfarin dosage control should use VKORC1 level as a major indicator, and improved antidotes may be designed based on their competition with warfarin.
UR - http://www.scopus.com/inward/record.url?scp=85086867817&partnerID=8YFLogxK
U2 - 10.1182/bloodadvances.2020001750
DO - 10.1182/bloodadvances.2020001750
M3 - Article
C2 - 32433747
AN - SCOPUS:85086867817
VL - 4
SP - 2202
EP - 2212
JO - Blood advances
JF - Blood advances
SN - 2473-9529
IS - 10
ER -