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

[heparin-induced thrombocytopenia]

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 (PF 4 ) complexes with polyanions . By assessing polyanions of varying length and degree of sulfation using immunoassay and circular dichroism (CD )-spectroscopy , we show that PF 4 structural changes resulting in antiparallel β-sheet content >30 % make PF 4 /polyanion complexes antigenic . Further , we found that polyphosphates (polyP-55 ) induce antigenic changes on PF 4 , whereas fondaparinux does not . We provide a model suggesting that conformational changes exposing antigens on PF 4 /polyanion complexes occur in the hairpin involving AA 32 -38 , which form together with C-terminal AA (66 -70 ) of the adjacent PF 4 monomer a continuous patch on the PF 4 tetramer surface , explaining why only tetrameric PF 4 molecules express "HIT antigens ". The correlation of antibody binding in immunoassays with PF 4 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 PF 4 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 .