Abstract Body (Do not enter title and authors here): Background: Unfractionated heparin (UFH), the main anticoagulant used in heart surgeries, is associated with adverse effects such as bleeding and heparin-induced thrombocytopenia. Anticoagulant aptamers targeting coagulation proteases have advantages such as low immunogenicity, low bleeding risk, and reversibility by complementary oligonucleotides. However, the potency of aptamers is often limited as they do not directly inhibit the catalytic sites on their targeted proteases. The natural anticoagulant hirudin achieves potent activity by inhibiting both thrombin’s exosite and active site. We hypothesize that hirudin-like protease inhibitors can be generated de novo by linking EXosite-binding aptamers with small molecule ACTive site inhibitors to create EXACT inhibitors that are much more potent than the small molecule inhibitors or aptamers alone.
Methods: Protease inhibition by EXACT inhibitors were characterized by fluorogenic substrate cleavage assays. The anticoagulation activity of EXACT inhibitors was assessed in normal pooled plasma or whole blood using the standard clinical clotting assays: prothrombin time (PT), activated partial thromboplastin time (aPTT), and active clotting time (ACT). The clinical potential of the EXACT inhibitor was evaluated in an ex vivo Extracorporeal Membrane Oxygenation (ECMO) circuit that mimics the procoagulant environment in many cardiovascular surgical settings.
Results: An EXACT inhibitor termed HD1-12dmA-DAB was synthesized by attaching the DNA aptamer HD1, that binds to thrombin exosite I, through a poly-adenosine linker to the thrombin active site inhibitor dabigatran (DAB). The EXACT inhibitor showed stronger thrombin inhibition (IC₅₀ = 0.2 nM) than DAB (IC₅₀ = 80 nM) or HD1 (IC₅₀ > 1 µM) alone. HD1-12dmA-DAB (2 µM) also achieved equal or higher anticoagulant activity than UFH (5U/mL) in all clotting assays and showed anticoagulation efficacy rivaling UFH in the ex vivo ECMO circuit with minimal clot observed over 120 min. Notably, the EXACT inhibitor’s effect can be rapidly reversed by an antidote oligo. We also used the same strategy to develop a potent EXACT inhibitor against factor Xa.
Conclusions: EXACT inhibitors demonstrate extraordinary anticoagulation activity as potential rapid onset anticoagulants to support cardiovascular procedures. Using this generalizable strategy, selective, potent, and rapidly reversible EXACT inhibitors can be created against many enzymes for numerous therapeutic applications.