title: Close approximation of two platelet factor 4 tetramers by charge neutralization forms the antigens recognized by HIT antibodies.
abstract: OBJECTIVE
  Heparin-induced thrombocytopenia (HIT) is a prothrombotic drug reaction caused by antibodies that recognize positively charged platelet factor 4 (PF4), bound to the polyanion, heparin. The resulting immune complexes activate platelets. Unfractionated heparin (UFH) causes HIT more frequently than low-molecular-weight heparin (LMWH), whereas the smallest heparin-like molecule (the pentasaccharide, fondaparinux), induces anti-PF4/heparin antibodies as frequently as LMWH, but without exhibiting cross-reactivity with these antibodies. To better understand these findings, we analyzed the molecular structure of the complexes formed between PF4 and UFH, LMWH, or fondaparinux.
  
  
  RESULTS
  By atomic force microscopy and photon correlation spectroscopy, we show that with any of the 3 polyanions, but in the order, UFH>LMWH>>fondaparinux--PF4 forms clusters in which PF4 tetramers become closely apposed, and to which anti-PF4/heparin antibodies bind. By immunoassay, HIT antibodies bind strongly to PF4/H/PF4 complexes, but only weakly to single PF4/heparin molecules.
  
  
  CONCLUSIONS
  HIT antigens are formed when charge neutralization by polyanion allows positively charged PF4 tetramers to undergo close approximation. Whereas such a model could explain why all 3 polyanions form antibodies with similar specificities, the striking differences in the relative size and amount of complexes formed likely correspond to the observed differences in immunogenicity (UFH>LMWH approximately fondaparinux) and clinically relevant cross-reactivity (UFH>LMWH>>fondaparinux).
authors.affiliation: Institute for Immunology and Transfusion Medicine, Ernst-Moritz-Arndt University, Sauerbruchstrasse, 17489 Greifswald, Germany. greinach@uni-greifswald.de
authors.forename: Andreas
authors.initials: A
authors.lastname: Greinacher
authors.forename: Manesh
authors.initials: M
authors.lastname: Gopinadhan
authors.forename: Jens-Uwe
authors.initials: JU
authors.lastname: Günther
authors.forename: Mahmoud A
authors.initials: MA
authors.lastname: Omer-Adam
authors.forename: Ulrike
authors.initials: U
authors.lastname: Strobel
authors.forename: Theodore E
authors.initials: TE
authors.lastname: Warkentin
authors.forename: Georg
authors.initials: G
authors.lastname: Papastavrou
authors.forename: Werner
authors.initials: W
authors.lastname: Weitschies
authors.forename: Christiane A
authors.initials: CA
authors.lastname: Helm
mesh_terms: D000327:Adsorption; D000906:Antibodies / Q000276:immunology*; D000917:Antibody Formation; D004797:Enzyme-Linked Immunosorbent Assay; D000077425:Fondaparinux; D006493:Heparin / Q000009:adverse effects* / Q000276:immunology; D006495:Heparin, Low-Molecular-Weight / Q000276:immunology; D006801:Humans; D018625:Microscopy, Atomic Force; D017785:Photons; D010978:Platelet Factor 4 / Q000737:chemistry* / Q000276:immunology*; D011134:Polysaccharides / Q000276:immunology; D013057:Spectrum Analysis / Q000379:methods; D013921:Thrombocytopenia / Q000139:chemically induced* / Q000276:immunology*
chemical_list: D000906:Antibodies; D006495:Heparin, Low-Molecular-Weight; D011134:Polysaccharides; D010978:Platelet Factor 4; D006493:Heparin; D000077425:Fondaparinux