Abstract Number: PB0038
Meeting: ISTH 2021 Congress
Background: Coagulation cascade cofactors are essential proteins that control homeostasis and are appealing targets for anticoagulants. However, targeting such proteins has been challenging due to their lack of an active site.
Aims: Development of FV/FVa and FVIII-inhibiting aptamers as potent and reversible anticoagulants that can be used to aid (or replace) heparin for therapeutic anticoagulation. Reveal new mechanisms of cofactor inhibition.
Methods: 2’F modified RNA Aptamers were identified via in vitro systematic evolution of ligand by exponential enrichment (SELEX) and post-SELEX truncation. Aptamer affinity was characterized by nitrocellulose filter binding assays and surface plasmon resonance. Aptamer bioactivity was analyzed using clotting assays and biochemical assays. Aptamers’ binding mechanism was determined using fluorescent anisotropy, fluorescence resonance energy transfer, dynamic light scattering, and molecular modeling.
Results: An aptamer termed T18.3 binds to both human FV/FVa with KD ~10 nM and prolongs aPTT in normal human plasma by 3.5-fold at a concentration of 500 nM. The aptamer inhibits membrane docking of FVa by binding to FV/FVa light chain, thereby reduces prothrombinase assembly and thrombin generation. Notably, the aptamer showed similar anticoagulant potency in normal, FV Leiden, and COVID-19 patient plasma. The aptamer also synergistically inhibits clotting with enoxaparin and can be rapidly reversed by protamine in in vitro assays. Another aptamer termed F8-3.1 binds to human FVIII with a KD of 0.67 nM and prolongs aPTT in normal human plasma by 1.9-fold at a concentration of 500 nM. F8-3.1 also has full cross-reactivity to canine FVIII (KD = 0.68 nM) and significantly prolongs the aPTT in canine and porcine plasma.
Conclusions: The work describes the generation of aptamers targeting FV/FVa and FVIII that can achieve clinically relevant anticoagulant activity. These results not only demonstrate the feasibility of using cofactor-binding aptamers as therapeutic anticoagulants but also reveal a novel mechanism of aptamer-mediated protein inhibition by interrupting protein-membrane interactions.
To cite this abstract in AMA style:Yu H, Soule E, Olson L, Naqvi I, Kumar S, Krishnaswamy S, Sullenger B. Generation of Anticoagulant RNA Aptamers Targeting Cofactors that Inhibit Clotting in Normal and COVID-19 Patient Samples [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/generation-of-anticoagulant-rna-aptamers-targeting-cofactors-that-inhibit-clotting-in-normal-and-covid-19-patient-samples/. Accessed January 23, 2022.
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