Close approximation of two platelet factor 4 tetramers by charge neutralization forms the antigens recognized by HIT antibodies

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Greinacher, Andreas ; Gopinadhan, Manesh ; Günther, Jens-Uwe ; Omer-Adam, Mahmoud A. ; Strobel, Ulrike ; Warkentin, Theodore E. ; Papastavrou, Georg ; Weitschies, Werner ; Helm, Christiane A.:
Close approximation of two platelet factor 4 tetramers by charge neutralization forms the antigens recognized by HIT antibodies.
In: Arteriosclerosis Thrombosis and Vascular Biology. Bd. 26 (2006) Heft 10 . - S. 2386-2393.
ISSN 1524-4636
DOI: https://doi.org/10.1161/01.ATV.0000238350.89477.88

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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.Methods and 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.Conclusion - 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 approximate to fondaparinux) and clinically relevant cross-reactivity (UFH > LMWH >> fondaparinux).