on July 27, 2006
Published online before print July 27, 2006, doi: 10.1161/01.ATV.0000238350.89477.88
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Submitted on February 27, 2006
Accepted on June 27, 2006
Close Approximation of Two Platelet Factor 4 Tetramers by Charge Neutralization Forms the Antigens Recognized by HIT Antibodies
Andreas Greinacher *;
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.
* To whom correspondence should be addressed. 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>LMWH
fondaparinux) and clinically relevant cross-reactivity (UFH>LMWH>>fondaparinux).
Key words: heparin • heparin-induced thrombocytopenia • platelets • platelet factor 4 • pentasaccharide
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