RNA Aptamer Against FXa Synergizes with FXa Catalytic Site Inhibitors to Effectively and Reversibly Anticoagulate Blood in an Ex Vivo Oxygenator Circuit
Authors: Gunaratne Ruwan , Frederiksen James , Thalji Nabil K. , Ho Michelle D , Arepally Gowthami M , Camire Rodney M. , Sullenger Bruce A. 
Published: November 2019

Abstract
Despite notable disadvantages, unfractionated heparin (UFH) remains the standard anticoagulant for clinical procedures requiring potent and reversible anticoagulation such as cardiopulmonary bypass (CPB). Limitations of UFH that contribute to patient morbidity in these settings include the fact that it permits thrombin generation and can cause the antibody-mediated syndrome Heparin-induced Thrombocytopenia (HIT), in addition to independent toxicities associated with its reversal agent, protamine.
11F7t is an anticoagulant RNA aptamer which inhibits FXa but unfortunately achieves less intense anticoagulation than UFH. The latter is also true for clinical FXa catalytic site inhibitors such as rivaroxaban, apixaban, or edoxoban. However, 11F7t does not inhibit FXa's catalytic activity but instead binds a FXa exosite to impede FVa binding and thus prothrombinase assembly. Owing to these different mechanisms, we previously reported that 11F7t can potently synergize with a FXa catalytic site inhibitor to prevent clot formation for >180 minutes (min) in whole blood thromboelastography (TEG) assays, thereby replicating the effect of UFH (5U/mL).
Here we sought to determine whether combinations of 11F7t plus a FXa catalytic site inhibitor can also prevent clotting as effectively as UFH in flowing blood within an ex vivo CPB membrane oxygenator circuit. In addition, we investigated whether efficient and simultaneous reversal of both anticoagulants could also be achieved post-circulation using desGla-Xa-S195A, which is a Gla-domainless catalytically inactive recombinant FXa mutant analogous to Andexanet Alfa, a therapeutic currently in clinical trials as an antidote for several FXa inhibitors. We also quantified levels of thrombin generation during circulation based on Prothrombin fragment 1+2 (F1+2) measurement. Finally, we investigated whether purified IgG obtained from three HIT patients could induce platelet aggregation in the presence of 11F7t, as occurs with UFH.
Human whole blood anticoagulated with either (A) UFH (5U/ml), or a combination of 11F7t (2µM) plus either (B) rivaroxaban (2µM), (C) apixaban (2µM), or (D) edoxaban (2µM) was circulated within a miniature oxygenator circuit at 33°C for 120 min at a 50 mL/min flow rate. While anticoagulation with either 11F7t or each of the FXa catalytic site inhibitors alone failed to maintain circuit blood fluidity, strategies (A) through (D) each prevented visible clot formation for 120 min and achieved therapeutic anticoagulation levels (>400 sec) as measured by the Activated Clotting Time (ACT). In addition, post-circulation scanning electron micrographs of the oxygenator membranes were similar for all four strategies and revealed minimal fibrinous and cellular debris. Successful normalization of the ACT was achieved upon administration of desGla-Xa-S195A (2µM) for strategies (B) through (D), similarly to that observed for UFH reversal by protamine. In addition, elevation of F1+2 levels post-circulation was significantly higher with UFH compared to each of the 11F7t plus FXa catalytic site inhibitor strategies (B-D). Finally, the HIT patient-derived purified IgG only induced platelet aggregation in the presence of UFH but not aptamer 11F7t (strategies B-D).
We have shown that the anticoagulant synergy achieved by combining aptamer 11F7t with a FXa catalytic site inhibitor can prevent blood clotting within an ex vivo oxygenator circuit as effectively as UFH, and may be additionally advantageous in limiting thrombin generation. Moreover, administration of desGla-Xa-S195A or a similar inactive FXa decoy like Andexanet Alfa may enable simultaneous reversal of both anticoagulants. This dual anticoagulant strategy may provide a useful alternative to UFH in clinical settings like CPB that necessitate both potent and reversible anticoagulation, and may be especially valuable for patients with a prior history of HIT.

Disclosures
Arepally: Biokit: Patents & Royalties. Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding; Novo Nordisk: Research Funding; Bayer: Consultancy; sparK: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.