RESUMO
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.
Assuntos
Anticoagulantes/farmacologia , Antídotos/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Protrombina/metabolismo , Trombina/metabolismo , Animais , Anticoagulantes/química , Anticoagulantes/metabolismo , Antídotos/química , Antídotos/metabolismo , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Ligação Competitiva , Domínio Catalítico , Bovinos , Cães , Estabilidade de Medicamentos , Ativação Enzimática , Fator Va/metabolismo , Meia-Vida , Humanos , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Tempo de Tromboplastina Parcial , Ativação Plaquetária/efeitos dos fármacos , Ligação Proteica , Tempo de Protrombina , Coelhos , Ratos , Ribonucleases/metabolismo , Técnica de Seleção de Aptâmeros , Ovinos , Especificidade da Espécie , Relação Estrutura-Atividade , Suínos , Tempo de TrombinaRESUMO
Aptamers, or nucleic acid ligands, have gained clinical interest over the past 20 years due to their unique characteristics, which are a combination of the best facets of small molecules and antibodies. The high binding affinity and specificity of aptamers allows for isolation of an artificial ligand for theoretically any therapeutic target of interest. Chemical manipulations of aptamers also allow for fine-tuning of their bioavailability, and antidote control greatly expands their clinical use. Here we review the various methods of antidote control of aptamer therapeutics--matched oligonucleotide antidotes and universal antidotes. We also describe the development, recent progress, and potential future therapeutic applications of these types of aptamer-antidote pairs.
