A high affinity,antidote‐controllable prothrombin and thrombin‐binding RNA aptamer inhibits thrombin generation and thrombin activity

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 (RNA_R9D‐14T), we also explored whether complementary antidote oligonucleotides can rapidly modulate the optimized aptamer’s anticoagulant activity. Methods and Results:  RNA_R9D‐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). RNA_R9D‐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 min) and durably (>2 h) reverse RNA_R9D‐14T anticoagulation in vitro. Conclusions:  Powerful anticoagulation, in conjunction with antidote reversibility, suggests that RNA_R9D‐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.