A High Affinity, Antidote-Controllable Prothrombin and Thrombin-Binding RNA Aptamer Inhibits Thrombin Generation and Thrombin Activity

Summary. Background:  The conversion of prothrombin to thrombin is one of two non-duplicated enzymatic reactions during coagulation. Thrombin has long been considered an optimal anticoagulant target because it plays a crucial role in fibrin clot formation by catalyzing the cleavage of fibrinogen, upstream coagulation cofactors and platelet receptors. Although a number of anti-thrombin therapeutics exist, it is challenging to use them clinically due to their propensity to induce bleeding. Previously, we isolated a modified RNA aptamer (R9D-14) that binds prothrombin with high affinity and is a potent anticoagulant in vitro. Objectives:  We sought to explore the structure of R9D-14 and elucidate its anticoagulant mechanism(s). In addition to designing an optimized aptamer (RNAR9D-14T), we also explored whether complementary antidote oligonucleotides can rapidly modulate the optimized aptamer’s anticoagulant activity. Methods and Results:  RNAR9D-14T binds prothrombin and thrombin pro/exosite I with high affinity and inhibits both thrombin generation and thrombin exosite I-mediated activity (i.e. fibrin clot formation, feedback activity and platelet activation). RNAR9D-14T significantly prolongs the aPTT, PT and TCT clotting assays, and is a more potent inhibitor than the thrombin exosite I DNA aptamer ARC-183. Moreover, a complementary oligonucleotide antidote can rapidly ( 2 h) reverse RNAR9D-14T anticoagulation in vitro. Conclusions:  Powerful anticoagulation, in conjunction with antidote reversibility, suggests that RNAR9D-14T may be ideal for clinical anticoagulation in settings that require rapid and robust anticoagulation, such as cardiopulmonary bypass, deep vein thrombosis, stroke or percutaneous coronary intervention.