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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:2386.)
© 2006 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Close Approximation of Two Platelet Factor 4 Tetramers by Charge Neutralization Forms the Antigens Recognized by HIT Antibodies

Andreas Greinacher; Manesh Gopinadhan; Jens-Uwe Günther; Mahmoud A. Omer-Adam; Ulrike Strobel; Theodore E. Warkentin; Georg Papastavrou; Werner Weitschies; Christiane A. Helm

From the Institut für Immunologie und Transfusionsmedizin (A.G., U.S.), Ernst-Moritz-Arndt-Universität Greifswald; Institut für angewandte Physik (M.G., J.-U.G., C.A.H.), Ernst-Moritz-Arndt-Universität Greifswald; Institut für Pharmazie (M.A.O.-A., W.W.), Ernst-Moritz-Arndt-Universität Greifswald, Germany; Department of Pathology and Molecular Medicine (T.E.W.), McMaster University, Hamilton, Canada; Department of Inorganic (G.P.), Analytical, and Applied Chemistry, University of Geneva, Switzerland.

Correspondence to Dr Andreas Greinacher, Institute for Immunology and Transfusion Medicine, Ernst-Moritz-Arndt University, Sauerbruchstrasse, 17489 Greifswald, Germany. E-mail greinach{at}uni-greifswald.de

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>LMWHfondaparinux) and clinically relevant cross-reactivity (UFH>LMWH>>fondaparinux).

By atomic force microscopy we show that positively charged platelet factor 4 (PF4) forms clusters with polyanions (UFH>LMWH>>fondaparinux) in which PF4 tetramers lose their intermolecular distance. This generates the binding site for heparin-induced thrombocytopenia antibodies. All 3 polyanions induce the antigen but in very different quantities (UFH>LMWH>>fondaparinux).

Key Words: heparin • heparin-induced thrombocytopenia • platelets • platelet factor 4 • pentasaccharide

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