In Vitro Evaluation of Aptamer-Based Reversible Inhibition of Anticoagulant Activated Protein C as a Novel Supportive Hemostatic Approach

Hamedani NS, et al., 26(6):355-362, Nucleic Acids Res, 2016

Activated protein C (APC) plays a key role in the regulation of thrombin formation and hence protection against thrombosis. It has been reported that excessive formation of APC is involved in the pathogenesis of trauma-induced coagulopathy (TIC). Therefore, pharmacological inhibition of APC activity could be used for the treatment of acute or chronic bleeding complications. Reported herein is the evaluation of a DNA aptamer, HS02-52G and its ability to restore blood clotting in APC-affected plasma and whole-blood coagulation models. Furthermore, a series of HS02-52G-neutralizing antisense antidote molecules was developed and investigated. The binding kinetics of the aptamer, HS02-52G, to APC and zymogen protein-C (PC) were assessed by using Bio-Layer Interferometry (BLI). A Pall ForteBio BLItz system equipped with High Precision Streptavidin (SAX) Biosensor probes was used to perform BLI experiments. SAX sensor tips were immobilized with 3’-biotinylated HS02-52G. Subsequently, the aptamer loaded SAX sensor tips were immersed in a seral dilution of recombinant (r) APC or zymogen PC (Ceprotin was used as the source of PC). BLI data obtained were analyzed globally using a 1:1 binding model. Association rate constants (kon), dissociation rate constants (koff), and KD values were determined for the binding interactions. The results of this study suggest HS02-52G as highly specific APC inhibitor whose functional activity in plasma and whole blood can be effectively reversed by a short antisense molecule, AD22. The novel aptamer–antidote pair reported herein is a promising treatment option for acute APC-related bleeding events.

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