Novel anticoagulant neutralizing agents for the management of bleeding : studies on the design, mechanism of action and their influence on blood coagulation

Heparins exert anticoagulation by potentiating anti-factor (F)Xa and anti-thrombin activity of antithrombin (AT), whereas oral anticoagulants (DOACs) directly target FXa or thrombin. FXa and thrombin are the key proteases required for blood clotting. Anticoagulants are therefore used for the prophylaxis and treatment of thrombosis and during surgeries. However, anticoagulation associated haemorrhage is a concern. The only approved antidote for unfractionated heparin (UFH), protamine do have limitations including cardiovascular complications. No approved antidotes are available for low molecular weight heparins, fondaparinux and direct FXa inhibitors. Therefore, there is a need for antidotes that are nontoxic and efficient. In this thesis, we reveal the mechanism of action, hemocompatibility and efficiency of three antidote molecules that are under development. UHRA: UHRA is a synthetic antidote developed by the Kizhakkedathu laboratory at the UBC. Thermodynamics based on isothermal titration calorimetry (ITC) and fluorescence studies revealed the molecular design of UHRA, the importance of steric shield produced by PEG brush, the selectivity of UHRA against heparins and its mechanism of action. Clotting studies confirm the antidote activity of UHRA. Studies also show that UHRA even in the absence of heparins, do not interact with fibrinogen, alter fibrin polymerization or abrogate blood clotting. Unlike protamine, UHRA does not incorporate into blood clots, form clots with normal morphology, and lysis profile. Studies in mice reveal that UHRA reverses UFH anticoagulant activity without the lung injury as seen with protamine. Studies confirm the superiority of UHRA compared to protamine. Andexanet Alfa (AnXa) and PER977: AnXa is a truncated FXa recombinant protein developed by Portola Pharmaceuticals and PER977 is a small cationic molecule developed by Perosphere Pharmaceuticals. ITC confirms high-affinity binding of AnXa to heparin/AT complex and to DOACs studied. PER977 shows weak binding to heparins and no binding to DOACs tested. Both antidotes do not influence fibrin polymerization, fibrin and blood clot architecture even in the absence of anticoagulants. Electron micrographs of blood clots containing edoxaban treated with AnXa or PER977 reveal restoration of impaired fibrin formation. However, in clotting assays, PER977 failed to show antidote activity, whereas AnXa neutralized the anticoagulation activity of all tested anticoagulants.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat