Investigating the structural effects of anti-thrombin anticoagulant aptamers on activation of human prothrombin

Abstract

Disorders of the blood coagulation remain a leading cause of death and disability worldwide raising the search for therapeutic agents able to modulate the coagulation cascade. Different oligonucleotide aptamers have been selected against different coagulation factors and some of them are in preclinical or clinical studies. In particular, anti-thrombin aptamers are promising drugs as they inhibit the activity of the -thrombin and, simultaneously, limit thrombin production via prothrombinase by binding its precursor prothrombin. To investigate the interaction of these aptamers with prothrombin, we performed extensive analyses using calorimetric and spectroscopic techniques, which suggested that they recognize proexosite I of prothrombin and exosite I of thrombin with comparable affinity. SAXS experiments performed on the complex formed by the protein and NU172, the only anti-thrombin aptamer in advanced clinical trials, provided structural insights into aptamer-prothrombin recognition. Interestingly, the aptamer binding to proexosite I shifts the open-closed equilibrium of prothrombin toward the open conformation. A reasonable mechanism underlying the effects of anti-thrombin aptamers towards prothrombin conversion into thrombin has been proposed. Altogether, these results definitively qualify these aptamers as bitargeted drugs, being able to modulate both thrombin function and generation, and supply structural bases to design new anticoagulants, which lack health side effects.