Induced forms of α₂-macroglobulin neutralize heparin and direct oral anticoagulant effects

Alpha₂-macroglobulin (α₂M) is a physiological macromolecule that facilitates the clearance of many proteinases, cytokines and growth factors in human. Here, we explored the effect of induced forms of α₂M on anticoagulant drugs. Gla-domainless factor Xa (GDFXa) and methylamine (MA)-induced α₂M were prepared and characterized by electrophoresis, immunonephelometry, chromogenic, clot waveform and rotational thromboelastometry assays. Samples from healthy volunteers and anticoagulated patients were included. In vivo neutralization of anticoagulants was evaluated in C57Bl/6JRj mouse bleeding-model. Anticoagulant binding sites on induced α₂M were depicted by computer-aided energy minimization modeling. GDFXa-induced α₂M neutralized dabigatran and heparins in plasma and whole blood. In mice, a single IV dose of GDFXa-induced α₂M following anticoagulant administration significantly reduced blood loss and bleeding time. Being far easier to prepare, we investigated the efficacy of MA-induced α₂M. It neutralized rivaroxaban, apixaban, dabigatran and heparins in spiked samples in a concentration-dependent manner and in samples from treated patients. Molecular docking analysis evidenced the ability of MA-induced α₂M to bind non-covalently these compounds via some deeply buried binding sites. Induced forms of α₂M have the potential to neutralize direct oral anticoagulants and heparins, and might be developed as a universal antidote in case of major bleeding or urgent surgery.