title: Characterisation of the conformational changes in platelet factor 4 induced by polyanions: towards in vitro prediction of antigenicity.
abstract: 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.
authors.forename: S
authors.initials: S
authors.lastname: Brandt
authors.forename: K
authors.initials: K
authors.lastname: Krauel
authors.forename: K E
authors.initials: KE
authors.lastname: Gottschalk
authors.forename: T
authors.initials: T
authors.lastname: Renné
authors.forename: C A
authors.initials: CA
authors.lastname: Helm
authors.affiliation: Prof. Dr. med. Andreas Greinacher, Institut für Immunologie und Transfusionsmedizin, Sauerbruchstrasse, 17475 Greifswald, Germany, Tel.: +49 3834 865482, Fax: +49 3834 865489, E-mail: greinach@uni-greifswald.de.
authors.forename: A
authors.initials: A
authors.lastname: Greinacher
authors.affiliation: Stephan Block, PhD, Applied Physics, Chalmers University of Technology, Fysikgränd 3, S-412 96 Gothenburg, Sweden, E-mail: block@physik.uni-greifswald.de, stephan.block@chalmers.se.
authors.forename: S
authors.initials: S
authors.lastname: Block
mesh_terms: D000906:Antibodies / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000378:metabolism; D000941:Antigens / Q000276:immunology*; D002942:Circular Dichroism; D000077425:Fondaparinux; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D010978:Platelet Factor 4 / Q000276:immunology / Q000378:metabolism*; D000071228:Polyelectrolytes; D011108:Polymers / Q000378:metabolism*; D011122:Polyphosphates / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D013463:Sulfuric Acid Esters / Q000378:metabolism; D013921:Thrombocytopenia / Q000139:chemically induced / Q000276:immunology / Q000378:metabolism*
chemical_list: D000906:Antibodies; D000936:Antigen-Antibody Complex; D000941:Antigens; D000077425:Fondaparinux; D006493:Heparin; D010978:Platelet Factor 4; D000071228:Polyelectrolytes; D011108:Polymers; D011122:Polyphosphates; D011134:Polysaccharides; D013463:Sulfuric Acid Esters
keywords: CD spectroscopy; GAGs; Heparin-induced thrombocytopenia; antigenicity; platelet factor 4