Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213 (USA).
Chembiochem. 2014 Jan 24;15(2):205-8. doi: 10.1002/cbic.201300693. Epub 2013 Dec 12.
Thrombin is the typical target in anticlotting therapy for many serious diseases such as heart attack and stroke. DNA aptamers are well-known thrombin inhibitors that prevent fibrinogen hydrolysis. We have discovered that exosite-targeting antithrombin aptamers enhance the activity of thrombin toward a small peptide substrate, Sar(N-methylglycine)-Pro-Arg-paranitroanilide, and that the activation of the enzyme by these aptamers is strongly inhibited by their complementary DNAs. Our study reveals that treatment with mixed aptamers or with a dual-aptamer construct led to an 8.6- or 7.8-fold enhancement in peptide hydrolysis relative to thrombin alone, a synergistic effect much higher than the activation observed with a monofunctional aptamer (1.5-fold for Apt27 or 2.7-fold for Apt15). In addition, we discovered that Apt27 is a biofunctional molecule for thrombin because of its activation effect. An enzyme kinetic study indicates that the binding of aptamers to exosites I and II significantly activates thrombin towards the peptide substrate, thus illustrating that binding of aptamers to exosites can allosterically regulate the active site of thrombin. Our study suggests the necessity of considering possible side effects when DNA aptamers are used for clinical applications involving the inhibition of thrombin-mediated clotting.
凝血酶是许多严重疾病（如心脏病发作和中风）抗凝血治疗的典型靶点。DNA 适体是众所周知的凝血酶抑制剂，可阻止纤维蛋白原水解。我们发现，外位靶抗凝血酶适体增强了凝血酶对小分子肽底物 Sar（N-甲基甘氨酸）-Pro-Arg-对硝基苯胺的活性，并且这些适体对酶的激活被其互补 DNA 强烈抑制。我们的研究表明，用混合适体或双适体构建体进行治疗可使肽水解相对于单独的凝血酶增强 8.6 或 7.8 倍，协同效应远高于用单功能适体观察到的激活（Apt27 为 1.5 倍或 Apt15 为 2.7 倍）。此外，我们发现 Apt27 是凝血酶的生物功能分子，因为它具有激活作用。酶动力学研究表明，适体与外位 I 和 II 的结合显着激活了凝血酶对肽底物的作用，从而表明适体与外位的结合可以变构调节凝血酶的活性位点。我们的研究表明，在涉及抑制凝血酶介导的凝血的临床应用中使用 DNA 适体时，有必要考虑可能的副作用。
