In vitro evaluation of aptamer-based reversible inhibition of anticoagulant activated protein C as a novel supportive hemostatic approach

N. Shahidi Hamedani1, H. Rühl1, S. Gasper1, J. J. Zimmermann1, T. Heiseler2, J. Oldenburg1, G. Mayer1, B. Pötzsch1, J. Müller1 (1Bonn, Germany, 2Dreieich, Germany)

Innovation and Novelty
Date: 16.02.2017,
Time: 14:00 - 15:15

Objective: It has been shown that overwhelming formation of activated protein C (APC) may play an important role in the pathogenesis of trauma induced coagulopathy (TIC). In addition, APC has been recognized as a candidate target for the adjuvant treatment of hemophilia. Thus, drugs that inhibit the anticoagulant activity of APC might be useful for the treatment of acute or chronic bleeding complications. The objective of the present study was the in vitro evaluation of HS02-52G, an APC-binding DNA-aptamer, as a reversible inhibitor of APC.

Methods: The binding kinetics of HS02-52G were determined by Biolayer Interferometry (BLI). A series of single-stranded (ss) DNA molecules (AD series), complementary to different parts of HS02-52G, were designed in order to identify an effective HS02-52G-neutralizing antidote molecule. The specificity as well as the APC-inhibitory potential of HS02-52G and the performance of the AD-molecules were assessed by various in vitro coagulation models, based on, inter alia, plasma or whole blood aPTT assays and the thrombin generation assay (calibrated automated thrombogram [CAT]).

Results: Regarding the binding of HS02-52G to APC, BLI analysis revealed on- and off-rates of 1.118 ± 0.013 × 10^5 M^−1 s^−1 and 1.234 ± 0.033 x 10^-3 s^−1, respectively, corresponding to a KD of 11 nM. In contrast, only negligible binding to the zymogen protein C was observed. Even when applied at concentrations of 1 µM, no aptamer-specific effects on global coagulation assays could be observed, proving the high target-specificity of HS02-52G. Accordingly, functional analysis revealed HS02-52G as a potent inhibitor of APC in plasma and whole blood with IC50 values <= 30 nM. Regarding the neutralization of the APC-inhibitory activity of HS02-52G, the complementary ssDNA-molecule AD22 was found to be an effective HS02-52G-antidote in plasma and whole blood (found IC50 values <= 200 nM).

Conclusion: HS02-52G has been introduced as a highly specific APC inhibitor. It could be demonstrated that the aptamer retains its high inhibitory activity in plasma and whole blood and that its functional activity within these matrices can be effectively reversed by the short antisense molecule AD22. In the first place, these features qualify the novel native aptamer-antidote pair as a save candidate treatment option for acute APC-related bleeding complications as, for instance, potentially observed in TIC.