title: Aptamers act as activators for the thrombin mediated-hydrolysis of peptide substrates.
abstract: 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.
authors: Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213 (USA). tanxiao@andrew.cmu.edu.
authors: Xiaohong
authors: X
authors: Tan
authors: Sourav Kumar
authors: SK
authors: Dey
authors: Cheryl
authors: C
authors: Telmer
authors: Xiaoliang
authors: X
authors: Zhang
authors: Bruce A
authors: BA
authors: Armitage
authors: Marcel P
authors: MP
authors: Bruchez
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics / Q000494:pharmacology*; D001483:Base Sequence; D055162:Biocatalysis; D020134:Catalytic Domain; D004789:Enzyme Activation / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D009842:Oligopeptides / Q000378:metabolism*; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D052157:Aptamers, Nucleotide; D009842:Oligopeptides; D013917:Thrombin
keywords: activators; allosterism; aptamers; thrombin