Complex formation with nucleic acids and aptamers alters the antigenic properties of platelet factor 4

Abstract

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...

Antisense Oligonucleotide Therapies

“…While this line of inquiry is promising, questions remain. For instance, it was noted that only about 60% of the anti-PF4/heparin antibodies that induced platelet activation in the presence of heparin also caused platelet activation in the presence of nucleic acids (Jaax et al 2013). There is little or no published data as of yet to support any particular theory, but it is possible that the mechanism may be elucidated in the coming years, given the level of interest in this topic by industry.…”

Spontaneous Heparin-Induced Thrombocytopenia Following Orthopaedic Surgery: Case Report and Review of the Literature

“…Of note, our patient was also ingesting a preoperative supplement containing chondroitin sulfate, serving as another possible inciting factor for the development of spontaneous HIT [9]. Herein, we summarize our patient's presentation and reconcile with a comprehensive review of the literature on spontaneous HIT following orthopaedic surgery in an effort to bring awareness to this rare but potentially fatal complication.…”

“…Interestingly our patient also ingested oral supplements containing chondroitin sulfate pre-operatively. Although there is laboratory evidence that PF4 can form complexes with hypersulfated chondroitin sulfate [9], it is unclear whether oral supplementation with chondroitin sulfate may lead to the formation of systemic platelet-activating PF4 complexes.…”

“…The mechanism of spontaneous HIT following orthopedic surgery is thought to involve the release of glycosaminoglycans such as chondroitin sulfate, a polyanion which structurally resembles heparin and is capable of forming platelet-activating complexes with PF4 [9] [10].…”

A case of heparin-induced thrombocytopenia (HIT) triggered by methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia

“…Especially in gram-negative bacteria, PF4 combines with lipid A and forms a PF4/bacteria complex [9]. Also PF4 binds to nucleic acids generated from breakdown of bacteria and viruses, tissue damage, cell apoptosis, and the release from blood cells such as the formation of neutrophil extracellular traps [10]. IgG against the PF4/bacteria complex has a defensive function such as the promotion of opsonization against bacteria and also combines with the PF4/heparin complex.…”