title: 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.
  
  
  OBJECTIVE
  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.
  
  
  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.
authors.affiliation: Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Bompiani
authors.forename: D M
authors.initials: DM
authors.lastname: Monroe
authors.forename: F C
authors.initials: FC
authors.lastname: Church
authors.forename: B A
authors.initials: BA
authors.lastname: Sullenger
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D000931:Antidotes / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D001667:Binding, Competitive; D001777:Blood Coagulation / Q000187:drug effects*; D020134:Catalytic Domain; D002417:Cattle; D004285:Dogs; D004355:Drug Stability; D004789:Enzyme Activation; D015943:Factor Va / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010314:Partial Thromboplastin Time; D015539:Platelet Activation / Q000187:drug effects; D011485:Protein Binding; D011516:Prothrombin / Q000378:metabolism*; D011517:Prothrombin Time; D011817:Rabbits; D051381:Rats; D012260:Ribonucleases / Q000378:metabolism; D052156:SELEX Aptamer Technique; D012756:Sheep; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013552:Swine; D013917:Thrombin / Q000378:metabolism*; D013918:Thrombin Time
chemical_list: D000925:Anticoagulants; D000931:Antidotes; D052157:Aptamers, Nucleotide; D015943:Factor Va; D011516:Prothrombin; D012260:Ribonucleases; D013917:Thrombin