Key Points• PF4 binds to nucleic acids and thereby exposes the epitope to which anti-PF4/ heparin antibodies bind.• PF4/aptamer complexes can induce an immune response resembling heparin-induced thrombocytopenia.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. (Blood. 2013;122(2):272-281) IntroductionThe chemokine platelet factor 4 (PF4) is released from platelet a-granules during platelet activation 1 and binds, due to its high positive charge, to many negatively charged polyanions, including heparin. PF4 forms large multimolecular complexes with heparin that are highly immunogenic. 2,3 The resulting immunoglobulin G (IgG) antibodies are the cause of heparin-induced thrombocytopenia (HIT), a prothrombotic adverse drug effect. 4 In HIT, multimolecular complexes composed of PF4, heparin, and anti-PF4/heparin IgG cross-link platelet FcgIIa receptors, 5 triggering platelet activation, microparticle formation, and thrombin generation, with ;50% of affected patients developing thrombosis. 6 Recently, we showed that PF4 binds to polyanions on the surface of bacteria, thereby forming multimolecular complexes that are recognized by human anti-PF4/heparin antibodies. More specifically, we identified the PF4 binding site on gram-negative bacteria as the phosphate groups of lipid A. 7 Based on this observation, we hypothesized that nucleic acids might also form multimolecular complexes with PF4 because they also expose multiple negatively charged phosphate groups. This idea was fostered by previous findings that salmon sperm DNA could substitute heparin in HIT-IgG-induced platelet activation. 8Plasma levels of extracellular nucleic acids are re...

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.