Department of Molecular Cardiology and.
Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH.
Blood. 2018 Jun 21;131(25):2826-2835. doi: 10.1182/blood-2017-09-804666. Epub 2018 Mar 28.
The anticoagulant warfarin inhibits the vitamin K oxidoreductase (VKORC1), which generates vitamin K hydroquinone (KH) required for the carboxylation and consequent activation of vitamin K-dependent (VKD) proteins. VKORC1 produces KH in 2 reactions: reduction of vitamin K epoxide (KO) to quinone (K), and then KH Our dissection of full reduction vs the individual reactions revealed a surprising mechanism of warfarin inhibition. Warfarin inhibition of KO to K reduction and carboxylation that requires full reduction were compared in wild-type VKORC1 or mutants (Y139H, Y139F) that cause warfarin resistance. Carboxylation was much more strongly inhibited (∼400-fold) than KO reduction (two- to threefold). The K to KH reaction was analyzed using low K concentrations that result from inhibition of KO to K. Carboxylation that required only K to KH reduction was inhibited much less than observed with the KO substrate that requires full VKORC1 reduction (eg, 2.5-fold vs 70-fold, respectively, in cells expressing wild-type VKORC1 and factor IX). The results indicate that warfarin uncouples the 2 reactions that fully reduce KO. Uncoupling was revealed because a second activity, a warfarin-resistant quinone reductase, was not present. In contrast, 293 cells expressing factor IX and this reductase activity showed much less inhibition of carboxylation. This activity therefore appears to cooperate with VKORC1 to accomplish full KO reduction. Cooperation during warfarin therapy would have significant consequences, as VKD proteins function in numerous physiologies in many tissues, but may be poorly carboxylated and dysfunctional if the second activity is not ubiquitously expressed similar to VKORC1.
抗凝剂华法林抑制维生素 K 氧化还原酶（VKORC1），该酶生成维生素 K 氢醌（KH），后者用于维生素 K 依赖性（VKD）蛋白的羧化和随后的激活。VKORC1 通过 2 个反应产生 KH：维生素 K 环氧化物（KO）还原为醌（K），然后是 KH。我们对完全还原与个别反应的剖析揭示了华法林抑制的一个惊人机制。在野生型 VKORC1 或引起华法林耐药的突变体（Y139H、Y139F）中比较了 KO 还原对华法林抑制和羧化的作用。羧化的抑制作用要强得多（约 400 倍），而 KO 还原的抑制作用要强 2-3 倍。使用抑制 KO 到 K 的反应生成的低 K 浓度分析 K 到 KH 的反应。仅需要 K 到 KH 还原的羧化作用的抑制作用比 KO 底物（在表达野生型 VKORC1 和因子 IX 的细胞中分别为 2.5 倍和 70 倍）的抑制作用小得多。结果表明，华法林使完全还原 KO 的 2 个反应解偶联。由于不存在第二种活性，即华法林抗性醌还原酶，因此出现解偶联。相比之下，表达因子 IX 和这种还原酶活性的 293 细胞中羧化作用的抑制作用要小得多。因此，这种活性似乎与 VKORC1 合作完成 KO 的完全还原。如果第二种活性没有像 VKORC1 那样普遍表达，那么在华法林治疗期间的合作将产生重大影响，因为 VKD 蛋白在许多组织中的许多生理过程中发挥作用，但如果不能充分羧化，则可能功能失调。
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