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

Abstract

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 min) and durably (>2 hrs) 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.

Probing the Coagulation Pathway with Aptamers Identifies Combinations that Synergistically Inhibit Blood Clot Formation

“…We have employed this selection method to generate modified RNA aptamers to activated coagulation factor VII (FVIIa) (Layzer and Sullenger, 2007), activated factor IX (FIXa) (Rusconi, et al, 2002), activated factor X (FXa) (Buddai, et al, 2010), and prothrombin (Layzer and Sullenger, 2007). All of the aptamers bind to both the zymogen and enzyme form of their target protein ( e.g., FVII and FVIIa), and mechanistic studies with the FIXa, FXa, and prothrombin aptamers indicate that the aptamers bind a large surface area on the zymogen/enzyme that is critical for procoagulant protein-protein interactions (Bompiani, et al, 2012; Buddai, et al, 2010; Sullenger, et al, 2012). Moreover, we have pioneered the development of two independent types of antidotes that can rapidly modulate aptamer anticoagulant function (Oney, et al, 2009; Rusconi, et al, 2002).…”

Aptamers: Problems, Solutions and Prospects

“…The efficiency of this approach has been confirmed by experiments on animal models. An aptamer was delivered into the bloodstream and exhibited a therapeutic effect, while subsequent injection of an antidote inactivated the aptamer and stopped its action [41, 42]. The high efficiency of aptamer hybridization with an antidote in blood provides a unique opportunity to control the duration of the therapeutic action.…”

Polyvalent nucleic acid aptamers and modulation of their activity: a focus on the thrombin binding aptamer

“…fibrin clot formation, feedback activity and platelet activation). The complementary oligonucleotide antidote can rapidly (b2 min) and durably (>2 h) reverse the aptamer anticoagulation in vitro (Bompiani et al, 2012). Many research groups recently demonstrated that, among the thrombin inhibitors, the aptamers TBA 15 , TBA 29 , and a series of related modified derivatives, were effectively controllable in their anticoagulation activity adopting various methodologies, such as the use of porphyrins, of complementary oligonucleotides or of external stimuli (light or radiofrequency irradiations).…”