title: A Conjugate of Two tPA-Binding RNA Aptamers Efficiently Inhibits Fibrinolysis.
abstract: Uncontrolled bleeding is a major cause of mortality. Lysine analogues are routinely used in the management of bleeding, but several studies indicate a risk of serious detrimental effects upon their administration. In this study, we report a bivalent conjugate "3218" of two RNA aptamers selected for binding to the serine protease tissue-type plasminogen activator (tPA), the principal initiator of fibrinolysis in mammals. The constituent monomeric aptamers, K32v2 and K18v2, were previously demonstrated to weakly inhibit fibrinolysis. We now show that K32v2 and K18v2 recognize distinct binding sites, presumably in the A- and B-chain of tPA, respectively. Both aptamers bind tPA with low nanomolar affinity and inhibit tPA-mediated activities in a way that is consistent with the proposed localization of their binding sites. The 3218 conjugate possesses the inhibitory activities of both K32v2 and K18v2 and additionally exhibits increased inhibitory efficiency relative to the monomeric aptamers. The 3218 conjugate proved an efficient inhibitor of fibrinolysis and may find application in the management of bleeding as a substitute for, or in combination with, currently used lysine analogues.
authors.affiliation: 1 Department of Molecular Biology and Genetics, Aarhus University , Aarhus, Denmark .
authors.forename: Nils
authors.initials: N
authors.lastname: Bjerregaard
authors.affiliation: 1 Department of Molecular Biology and Genetics, Aarhus University , Aarhus, Denmark .|2 Interdisciplinary Nanoscience Center (iNANO), Aarhus University , Aarhus, Denmark .
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Dupont
authors.affiliation: 1 Department of Molecular Biology and Genetics, Aarhus University , Aarhus, Denmark .
authors.forename: Peter A
authors.initials: PA
authors.lastname: Andreasen
mesh_terms: D052157:Aptamers, Nucleotide / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D020029:Base Pairing; D001665:Binding Sites; D020134:Catalytic Domain; D005342:Fibrinolysis / Q000187:drug effects*; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013936:Thymidine / Q000031:analogs & derivatives / Q000737:chemistry; D014158:Transcription, Genetic
chemical_list: D052157:Aptamers, Nucleotide; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; C068636:2'-fluorothymidine; D013936:Thymidine
keywords: 2′-fluoropyrimidine modified RNA; bivalent conjugate; fibrinolysis; nucleic acid aptamer; serine protease; tissue-type plasminogen activator