title: Binding of anti-platelet factor 4/heparin antibodies depends on the thermodynamics of conformational changes in platelet factor 4.
abstract: The chemokine platelet factor 4 (PF4) undergoes conformational changes when complexing with polyanions. This can induce the antibody-mediated adverse drug effect of heparin-induced thrombocytopenia (HIT). Understanding why the endogenous protein PF4 becomes immunogenic when complexing with heparin is important for the development of other negatively charged drugs and may also hint toward more general mechanisms underlying the induction of autoantibodies to other proteins. By circular dichroism spectroscopy, atomic force microscopy, and isothermal titration calorimetry we characterized the interaction of PF4 with unfractionated heparin (UFH), its 16-, 8-, and 6-mer subfractions, low-molecular-weight heparin (LMWH), and the pentasaccharide fondaparinux. To bind anti-PF4/heparin antibodies, PF4/heparin complexes require (1) an increase in PF4 antiparallel β-sheets exceeding ∼30% (achieved by UFH, LMWH, 16-, 8-, 6-mer), (2) formation of multimolecular complexes (UFH, 16-, 8-mer), and (3) energy (needed for a conformational change), which is released by binding of ≥11-mer heparins to PF4, but not by smaller heparins. These findings may help to synthesize safer heparins. Beyond PF4 and HIT, the methods applied in the current study may be relevant to unravel mechanisms making other endogenous proteins more vulnerable to undergo conformational changes with little energy requirement (eg, point mutations and post-translational modifications) and thereby predisposing them to become immunogenic.
authors.affiliation: ZIK HIKE-Zentrum für Innovationskompetenz, Humorale Immunreaktionen bei Kardiovaskulären Erkrankungen, Greifswald, Germany;
authors.forename: Martin
authors.initials: M
authors.lastname: Kreimann
authors.affiliation: ZIK HIKE-Zentrum für Innovationskompetenz, Humorale Immunreaktionen bei Kardiovaskulären Erkrankungen, Greifswald, Germany;
authors.forename: Sven
authors.initials: S
authors.lastname: Brandt
authors.affiliation: Institut für Immunologie und Transfusionsmedizin, Greifswald, Germany;
authors.forename: Krystin
authors.initials: K
authors.lastname: Krauel
authors.affiliation: ZIK HIKE-Zentrum für Innovationskompetenz, Humorale Immunreaktionen bei Kardiovaskulären Erkrankungen, Greifswald, Germany;
authors.forename: Stephan
authors.initials: S
authors.lastname: Block
authors.affiliation: Institut für Physik, Greifswald, Germany; and.
authors.forename: Christiane A
authors.initials: CA
authors.lastname: Helm
authors.affiliation: Institut für Pharmazie, Greifswald, Germany.
authors.forename: Werner
authors.initials: W
authors.lastname: Weitschies
authors.affiliation: Institut für Immunologie und Transfusionsmedizin, Greifswald, Germany;
authors.forename: Andreas
authors.initials: A
authors.lastname: Greinacher
authors.affiliation: ZIK HIKE-Zentrum für Innovationskompetenz, Humorale Immunreaktionen bei Kardiovaskulären Erkrankungen, Greifswald, Germany;
authors.forename: Mihaela
authors.initials: M
authors.lastname: Delcea
mesh_terms: D000906:Antibodies / Q000378:metabolism*; D002151:Calorimetry; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D000077425:Fondaparinux; D006495:Heparin, Low-Molecular-Weight / Q000737:chemistry; D006801:Humans; D018625:Microscopy, Atomic Force; D010978:Platelet Factor 4 / Q000737:chemistry* / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013816:Thermodynamics
chemical_list: D000906:Antibodies; D006495:Heparin, Low-Molecular-Weight; D011134:Polysaccharides; D010978:Platelet Factor 4; D000077425:Fondaparinux