RESUMEN
Unfractionated heparin (UFH), the standard anticoagulant for cardiopulmonary bypass (CPB) surgery, carries a risk of post-operative bleeding and is potentially harmful in patients with heparin-induced thrombocytopenia-associated antibodies. To improve the activity of an alternative anticoagulant, the RNA aptamer 11F7t, we solved X-ray crystal structures of the aptamer bound to factor Xa (FXa). The finding that 11F7t did not bind the catalytic site suggested that it could complement small-molecule FXa inhibitors. We demonstrate that combinations of 11F7t and catalytic-site FXa inhibitors enhance anticoagulation in purified reaction mixtures and plasma. Aptamer-drug combinations prevented clot formation as effectively as UFH in human blood circulated in an extracorporeal oxygenator circuit that mimicked CPB, while avoiding side effects of UFH. An antidote could promptly neutralize the anticoagulant effects of both FXa inhibitors. Our results suggest that drugs and aptamers with shared targets can be combined to exert more specific and potent effects than either agent alone.
Asunto(s)
Anticoagulantes/administración & dosificación , Inhibidores del Factor Xa/administración & dosificación , Factor Xa/química , Hemorragia Posoperatoria/tratamiento farmacológico , Anticoagulantes/química , Aptámeros de Nucleótidos/administración & dosificación , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/genética , Puente Cardiopulmonar/efectos adversos , Cristalografía por Rayos X , Combinación de Medicamentos , Factor Xa/genética , Inhibidores del Factor Xa/química , Heparina/efectos adversos , Humanos , Hemorragia Posoperatoria/genética , Hemorragia Posoperatoria/patología , Conformación Proteica/efectos de los fármacosRESUMEN
Coordinated enzymatic reactions regulate blood clot generation. To explore the contributions of various coagulation enzymes in this process, we utilized a panel of aptamers against factors VIIa, IXa, Xa, and prothrombin. Each aptamer dose-dependently inhibited clot formation, yet none was able to completely impede this process in highly procoagulant settings. However, several combinations of two aptamers synergistically impaired clot formation. One extremely potent aptamer combination was able to maintain human blood fluidity even during extracorporeal circulation, a highly procoagulant setting encountered during cardiopulmonary bypass surgery. Moreover, this aptamer cocktail could be rapidly reversed with antidotes to restore normal hemostasis, indicating that even highly potent aptamer combinations can be rapidly controlled. These studies highlight the potential utility of using sets of aptamers to probe the functions of proteins in molecular pathways for research and therapeutic ends.