title: Selection of an aptamer antidote to the anticoagulant drug bivalirudin.
abstract: Adverse drug reactions, including severe patient bleeding, may occur following the administration of anticoagulant drugs. Bivalirudin is a synthetic anticoagulant drug sometimes employed as a substitute for heparin, a commonly used anticoagulant that can cause a condition called heparin-induced thrombocytopenia (HIT). Although bivalrudin has the advantage of not causing HIT, a major concern is lack of an antidote for this drug. In contrast, medical professionals can quickly reverse the effects of heparin using protamine. This report details the selection of an aptamer to bivalirudin that functions as an antidote in buffer. This was accomplished by immobilizing the drug on a monolithic column to partition binding sequences from nonbinding sequences using a low-pressure chromatography system and salt gradient elution. The elution profile of binding sequences was compared to that of a blank column (no drug), and fractions with a chromatographic difference were analyzed via real-time PCR (polymerase chain reaction) and used for further selection. Sequences were identified by 454 sequencing and demonstrated low micromolar dissociation constants through fluorescence anisotropy after only two rounds of selection. One aptamer, JPB5, displayed a dose-dependent reduction of the clotting time in buffer, with a 20 µM aptamer achieving a nearly complete antidote effect. This work is expected to result in a superior safety profile for bivalirudin, resulting in enhanced patient care.
authors.affiliation: Department of Chemistry and Department of Physiology and Functional Genomics, Shands Cancer Center and Center for Research at the Bio/nano Interface, University of Florida, Gainesville, FL, USA.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Martin
authors.forename: Parag
authors.initials: P
authors.lastname: Parekh
authors.forename: Youngmi
authors.initials: Y
authors.lastname: Kim
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Morey
authors.forename: Kwame
authors.initials: K
authors.lastname: Sefah
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Gravenstein
authors.forename: Donn M
authors.initials: DM
authors.lastname: Dennis
authors.forename: Weihong
authors.initials: W
authors.lastname: Tan
mesh_terms: D000925:Anticoagulants / Q000009:adverse effects*; D000931:Antidotes / Q000494:pharmacology*; D052157:Aptamers, Nucleotide / Q000494:pharmacology*; D001777:Blood Coagulation / Q000187:drug effects; D002021:Buffers; D005434:Flow Cytometry; D005454:Fluorescence Polarization; D006629:Hirudins / Q000009:adverse effects*; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000009:adverse effects*; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000009:adverse effects; D052156:SELEX Aptamer Technique / Q000379:methods*; D017422:Sequence Analysis, DNA
chemical_list: D000925:Anticoagulants; D000931:Antidotes; D052157:Aptamers, Nucleotide; D002021:Buffers; D006629:Hirudins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C074619:bivalirudin