RESUMO
The tight electrostatic binding of the chemokine platelet factor 4 (PF4) to polyanions induces heparin-induced thrombocytopenia, a prothrombotic adverse drug reaction caused by immunoglobulin G directed against PF4/polyanion complexes. This study demonstrates that nucleic acids, including aptamers, also bind to PF4 and enhance PF4 binding to platelets. Systematic assessment of RNA and DNA constructs, as well as 4 aptamers of different lengths and secondary structures, revealed that increasing length and double-stranded segments of nucleic acids augment complex formation with PF4, while single nucleotides or single-stranded polyA or polyC constructs do not. Aptamers were shown by circular dichroism spectroscopy to induce structural changes in PF4 that resemble those induced by heparin. Moreover, heparin-induced anti-human-PF4/heparin antibodies cross-reacted with human PF4/nucleic acid and PF4/aptamer complexes, as shown by an enzyme immunoassay and a functional platelet activation assay. Finally, administration of PF4/44mer-DNA protein C aptamer complexes in mice induced anti-PF4/aptamer antibodies, which cross-reacted with murine PF4/heparin complexes. These data indicate that the formation of anti-PF4/heparin antibodies in postoperative patients may be augmented by PF4/nucleic acid complexes. Moreover, administration of therapeutic aptamers has the potential to induce anti-PF4/polyanion antibodies and a prothrombotic diathesis.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Ácidos Nucleicos/metabolismo , Fator Plaquetário 4/imunologia , Fator Plaquetário 4/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Aptâmeros de Nucleotídeos/química , Pareamento de Bases , Sequência de Bases , Plaquetas/metabolismo , DNA/química , DNA/metabolismo , Heparina/farmacologia , Humanos , Substâncias Macromoleculares/metabolismo , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ácidos Nucleicos/química , Ativação Plaquetária/imunologia , Polieletrólitos , Polímeros , Ligação Proteica/efeitos dos fármacos , RNA/química , RNA/metabolismoRESUMO
Kinetic analysis of ribozyme reactions is a common method to evaluate and compare activities of catalytic RNAs. The hairpin ribozyme catalyzes the reversible cleavage of a suitable RNA substrate at a specific site. Hairpin ribozyme variants as an allosteric ribozyme responsive to flavine mononucleotide and a hairpin-derived twin ribozyme that catalyzes two cleavage reactions and two ligation events with the result of a fragment exchange have been developed by rational design and were kinetically characterized. Herein, protocols for preparation of ribozymes and dye-labeled substrates as well as for analysis of cleavage, ligation, and fragment exchange reactions are provided.