华法林耐药突变体减弱人维生素K环氧化物还原酶与华法林的结合能力  

Warfarin-resistant mutations weaken the binding of human vitamin K epoxide reductase to warfarin

作　　者：王雯琪 沈滟[1,2] 李晓明 沈国民[1,2] Wang Wenqi;Shen Yan;Li Xiaoming;Shen Guomin(College of Basic Medicine and Forensic Medicine,Henan University of Science and Technology,Luoyang 471023;Henan Province International Joint Laboratory of Thrombosis and Hemostasis,Luoyang 471023)

机构地区：[1]河南科技大学基础医学与法医学院医学遗传学教研室,洛阳471023 [2]河南省血栓与止血国际联合实验室,洛阳471023

出　　处：《安徽医科大学学报》2024年第9期1629-1635,共7页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81770140、82170133);河南省科技攻关计划(编号:212102310629、212102310877)。

摘　　要：目的探讨人维生素K环氧化物还原酶(VKORC1)耐药突变体对华法林耐药的机制。方法利用与华法林结合导致VKORC1电泳迁移率变化的原理进行电泳迁移实验,并通过酶联免疫吸附实验检测VKORC1的活性和华法林抑制VKORC1的半抑制浓度(IC_(50)),评价VKORC1耐药突变体与华法林的结合能力。结果华法林与VKORC1结合后,保护其柔性跨膜螺旋1(TM1)中的一个半胱氨酸免受N-乙基马来酰亚胺(NEM)的修饰,从而增加VKORC1的电泳迁移率。增加华法林浓度可以增加具有快速迁移效应的VKORC1。与野生型VKORC1相比,华法林耐药突变体的快速迁移效应减弱或消失,表明华法林与耐药突变体的结合能力减弱;且突变体快速迁移效应越弱,其对华法林的耐药性越强。结论VKORC1耐药突变体与华法林的结合能力减弱是华法林耐药的主要原因。Objective To understand how warfarin inhibition is hindered by the vitamin K epoxide reductase(VKORC1)resistant mutations.Methods An electrophoretic mobility assay was conducted based on the principle that binding with warfarin resulted in changes in VKORC1 electrophoretic mobility.The activity of VKORC1 and the half maximal inhibitory concentration(IC_(50))of warfarin inhibiting VKORC1 were detected by ELISA.These experiments evaluated the binding ability of VKORC1 resistant mutants to warfarin.Results With warfarin bound,VKORC1 was protected from N-ethylmaleimide(NEM)modification and showed increased electrophoretic mobility,which was dependent on an unmodified cysteine on the flexible transmembrane helix 1(TM1)of VKORC1.Increasing the warfarin concentration could shift more VKORC1 towards the species with the fast mobility.Compared to the wildtype VKORC1,the fast mobility fraction became less or disappeared in warfarin-resistant mutants,indicating weakened binding of warfarin.In addition,VKORC1 mutants with the weaker electrophoretic mobility shift indicated the stronger warfarin resistant.Conclusion Weakened warfarin binding is the primary cause of warfarin resistance.

关 键 词：凝血 耐药 电泳迁移实验 维生素K 维生素K环氧化还原酶 华法林耐药突变体 华法林 抗凝药 

分 类 号：R596[医药卫生—内科学]