Characterisation of the conformational changes in platelet factor 4 induced by polyanions: towards in vitro prediction of antigenicity

Sven Brandt; Krystin Krauel; Kay E. Gottschalk; Thomas Renné; Christiane A. Helm; Andreas Greinacher; Stephan Block

doi:10.1160/TH13-08-0634

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2014; 112(01): 53-64
DOI: 10.1160/TH13-08-0634

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Characterisation of the conformational changes in platelet factor 4 induced by polyanions: towards in vitro prediction of antigenicity

Sven Brandt

¹ZIK HIKE – Zentrum für Innovationskompetenz “Humorale Immunreaktionen bei kardiovaskulären Erkrankungen”, Greifswald, Germany

,

Krystin Krauel

¹ZIK HIKE – Zentrum für Innovationskompetenz “Humorale Immunreaktionen bei kardiovaskulären Erkrankungen”, Greifswald, Germany

²Institut für Immunologie und Transfusionsmedizin, Greifswald, Germany

,

Kay E. Gottschalk

³Institut für Experimentelle Physik, Universität Ulm, Ulm, Germany

,

Thomas Renné

⁴Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden

⁶Institute for Clinical Chemistry and Laboratory Medicine, University Hospital Hamburg-Eppendorf, Hamburg, Germany

,

Christiane A. Helm

⁵Institut für Physik, Ernst-Moritz-Arndt Universität, Greifswald, Germany

,

Andreas Greinacher^*

²Institut für Immunologie und Transfusionsmedizin, Greifswald, Germany

,

Stephan Block^*

¹ZIK HIKE – Zentrum für Innovationskompetenz “Humorale Immunreaktionen bei kardiovaskulären Erkrankungen”, Greifswald, Germany

› Author Affiliations

Abstract

Summary

Heparin-induced thrombocytopenia (HIT) is the most frequent drug-induced immune reaction affecting blood cells. Its antigen is formed when the chemokine platelet factor 4 (PF4) complexes with polyanions. By assessing polyanions of varying length and degree of sulfation using immunoassay and circular dichroism (CD)-spectroscopy, we show that PF4 structural changes resulting in antiparallel β-sheet content >30% make PF4/polyanion complexes antigenic. Further, we found that polyphosphates (polyP-55) induce antigenic changes on PF4, whereas fondaparinux does not. We provide a model suggesting that conformational changes exposing antigens on PF4/polyanion complexes occur in the hairpin involving AA 32–38, which form together with C-terminal AA (66–70) of the adjacent PF4 monomer a continuous patch on the PF4 tetramer surface, explaining why only tetrameric PF4 molecules express “HIT antigens”. The correlation of antibody binding in immunoassays with PF4 structural changes provides the intriguing possibility that CD-spectroscopy could become the first antibody-independent, in vitro method to predict potential immunogenicity of drugs. CD-spectroscopy could identify compounds during preclinical drug development that induce PF4 structural changes correlated with antigenicity. The clinical relevance can then be specifically addressed during clinical trials. Whether these findings can be transferred to other endogenous proteins requires further studies.

Keywords

Heparin-induced thrombocytopenia - platelet factor 4 - GAGs - CD spectroscopy - antigenicity

Full Text

References

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