Characterization of Antithrombin-Specific RNA Aptamers for Use in Anticoagulant Therapy

Historically, unfractionated heparin (a complex polysaccharide classified as a glycosaminoglycan) has been used in the treatment and prevention of thromboembolic disorders. However, due to complications associated with heparin (such as bleeding and heparin-induced thrombocytopenia), low molecular weight heparins (LMWHs) have been developed and are being used in place of unfractionated heparin. LMWHs have proven effective as antithrombotic agents; however, the effects of LMWHs cannot be readily reversed, while unfractionated heparin can be reversed by protamine sulfate. The overall goal of this project was to characterize an antithrombin (AT)-specific RNA aptamer that will combine the safety and efficacy of LMWH with the reversibility of unfractionated heparin. I have developed an RNA molecule (aptamer) that binds to AT. Using Systematic Evolution of Ligands by EXponential enrichment (SELEX) we created a combinatorial library consisting of single stranded RNA molecules with 20-40 randomized positions resulting in approximately 10¹⁴ different sequences. The RNA libraries after round 12 consisted of several different aptamers, and there was a progressive decrease in library complexity. We isolated four individual RNA molecules that bind in the nanomolar range to AT. Each displayed the same general properties, they accelerated factor Xa inhibition by AT in a dose-dependent manner. It should be noted that as anticipated, the aptamers did not promote the enhanced inhibition of thrombin by AT. Overall, our results show that these molecules are able to enhance the inhibition of factor Xa by AT. We suspect that these aptamers mimic the action of LMWH by binding to the D-helix of AT. Our previous AT specific RNA aptamer, Aptamer 7-4.16 was shown to prolonged bleeding in vivo in a vascular injury model. These data showed that Aptamer 7-4.16 is as effective as heparin in preventing clotting after vascular injury. We suspect that these new aptamers will have a similar effect in vivo. Proof of the concept was established by the work of Rusconi et al. (Nature, 2002), who developed an RNA aptamer and RNA aptamer antidote directed against coagulation factor IXa. The factor IXa aptamer completely inhibits the activation of factor X. Its anticoagulant action is controlled by the antidote, which is able to reverse the effects of the factor IXa aptamer within seconds. This aptamer/antidote pair is currently being tested clinically. Our data suggesting that antidotes to our AT aptamers have the ability to reverse its activity. If successful, this aptamer/antidote pair will have advantages over LMWHs because not only will it be able to control thrombosis in a fashion similar to LMWHs, but the availability of an antidote will allow for better therapeutic regulation and intervention.