Modulation of Heparin Therapy by RNA Aptamers and Andexanet Alfa

Unfractionated heparin (UFH) is the primary anticoagulant for highly procoagulant indications including cardiopulmonary bypass (CPB) and more recently, severe COVID-19. UFH, however, is unable to fully inhibit thrombin generation leading to continuous activation of both coagulation and inflammatory cascades, increasing the risk for thrombo-inflammatory adverse events. The widespread use of UFH makes it important to improve on its action by addressing its incomplete inhibition of thrombin generation. Additionally, there is a need to better understand how to effectively administer UFH in relation to other therapeutics to avoid counter interactions that leave patients vulnerable to life threatening complications. Known limitations of unfractionated heparin (UFH) have encouraged the evaluation of anticoagulant aptamers as alternatives to UFH in highly procoagulant settings such as cardiopulmonary bypass (CPB). Despite progress, these efforts have not been totally successful. In the first part of this work, we take a different approach and explore whether properties of an anticoagulant aptamer can complement UFH, rather than replace it, to address shortcomings with UFH use. Combining RNA aptamer 11F7t, which targets Factor X/Xa, with UFH (or Low Molecular Weight Heparin) yields a significantly enhanced anticoagulant cocktail effective in normal and COVID-19 patient blood. In these studies we determined that this aptamer-UFH combination a.) supports continuous circulation of human blood through an ex vivo membrane oxygenation circuit, as is required for patients undergoing CPB and COVID-19 patients requiring extracorporeal membrane oxygenation, b.) allows for a reduced level of UFH to be employed c.) more effectively limits thrombin generation compared to UFH alone and d.) is rapidly reversed by the administration of protamine sulfate, the standard treatment for reversing UFH clinically following CPB. Thus, the combination of Factor X/Xa aptamer and UFH has significantly improved anticoagulant properties compared to UFH alone and underscores the potential of RNA aptamers to improve medical management of acute care patients requiring potent yet rapidly reversible anticoagulation. In the second part of this work, we focused on understanding the interactions between Andexanet alfa (AnXa), a reversal agent for FXa targeted direct oral anticoagulants (DOACs), and UFH. While AnXa was designed to control bleeding associated with the use of DOACs, no studies on its effect on subsequent UFH anticoagulation prior to surgery were carried out. Following FDA approval of AnXa in 2018, several cases have been reported where prior AnXa administration cause heparin resistance during CPB. In our studies, we established that the concomitant presence of AnXa and UFH in whole human blood diminishes the anticoagulant effect of heparin. Furthermore, AnXa addition to ex vivo membrane oxygenation circuit, where UFH anticoagulated blood was being circulated led to the formation of macroscopic and microscopic clots within the circuit. Further studies established potential countermeasures to alleviate heparin resistance in this context through administration of antithrombin (AT) and excess UFH. These results give insight into the interaction of AnXa and UFH during extracorporeal oxygenation and how to circumvent the thrombotic sequalae associated with this interaction. We believe this work will greatly inform and improve patient outcomes. We hope it will also encourage issuance of a warning against concurrent or sequential administration of indirect inhibitors of FXa (UFH, LMWH and Fondaparinux) and AnXa.