Selection of an Aptamer Antidote to the Anticoagulant Drug Bivalirudin
Selection of an Aptamer Antidote to the Anticoagulant Drug Bivalirudin
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DOI：
10.1371/journal.pone.0057341
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发表时间：
2013-03-06
期刊：
影响因子：
3.7
通讯作者：
Tan, Weihong
中科院分区：
文献类型：
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作者：
Martin, Jennifer A.;Parekh, Parag;Tan, Weihong
药物不良反应，包括严重的患者出血，可能发生在抗凝药物的管理。比伐鲁定是一种合成抗凝药物，有时被用作肝素的替代品，肝素是一种常用的抗凝剂，可引起肝素诱导的血小板减少症（HIT）。尽管比伐鲁丁具有不会引起HIT的优点，但一个主要问题是缺乏这种药物的解毒剂。相比之下，医疗专业人员可以使用鱼精蛋白迅速逆转肝素的作用。本报告详细介绍了在缓冲液中作为解毒剂的比伐鲁定适体的选择。这是通过将药物固定在整体柱上，使用低压色谱系统和盐梯度洗脱将结合序列与非结合序列分开来完成的。结合序列的洗脱谱与空白柱（无药物）的洗脱谱进行比较，通过real-time PCR （polymerase chain reaction，聚合酶链反应）分析存在层析差异的部分，用于进一步筛选。序列通过454测序鉴定，仅经过两轮筛选，就通过荧光各向异性显示出低微摩尔解离常数。其中一种适体JPB5在缓冲液中显示出剂量依赖性的凝血时间减少，20 μ M适体达到了几乎完全的解毒剂效果。这项工作预计将导致比伐鲁定具有优越的安全性，从而改善患者护理。
Adverse drug reactions, including severe patient bleeding, may occur following the administration of anticoagulant drugs. Bivalirudin is a synthetic anticoagulant drug sometimes employed as a substitute for heparin, a commonly used anticoagulant that can cause a condition called heparin-induced thrombocytopenia (HIT). Although bivalrudin has the advantage of not causing HIT, a major concern is lack of an antidote for this drug. In contrast, medical professionals can quickly reverse the effects of heparin using protamine. This report details the selection of an aptamer to bivalirudin that functions as an antidote in buffer. This was accomplished by immobilizing the drug on a monolithic column to partition binding sequences from nonbinding sequences using a low-pressure chromatography system and salt gradient elution. The elution profile of binding sequences was compared to that of a blank column (no drug), and fractions with a chromatographic difference were analyzed via real-time PCR (polymerase chain reaction) and used for further selection. Sequences were identified by 454 sequencing and demonstrated low micromolar dissociation constants through fluorescence anisotropy after only two rounds of selection. One aptamer, JPB5, displayed a dose-dependent reduction of the clotting time in buffer, with a 20 mu M aptamer achieving a nearly complete antidote effect. This work is expected to result in a superior safety profile for bivalirudin, resulting in enhanced patient care.