The
chemokine platelet factor 4 (PF4) undergoes conformational changes when complexing with
polyanions. This can induce the antibody-mediated adverse
drug effect of
heparin-induced
thrombocytopenia (HIT). Understanding why the endogenous
protein PF4 becomes immunogenic when complexing with
heparin is important for the development of other negatively charged drugs and may also hint toward more general mechanisms underlying the induction of
autoantibodies to other
proteins. By circular dichroism spectroscopy, atomic force microscopy, and isothermal titration calorimetry we characterized the interaction of PF4 with
unfractionated heparin (UFH), its 16-, 8-, and 6-mer subfractions,
low-molecular-weight heparin (
LMWH), and the pentasaccharide
fondaparinux. To bind anti-PF4/
heparin antibodies, PF4/
heparin complexes require (1) an increase in PF4 antiparallel β-sheets exceeding ∼30% (achieved by UFH,
LMWH, 16-, 8-, 6-mer), (2) formation of multimolecular complexes (UFH, 16-, 8-mer), and (3) energy (needed for a conformational change), which is released by binding of ≥11-mer heparins to PF4, but not by smaller heparins. These findings may help to synthesize safer heparins. Beyond PF4 and HIT, the methods applied in the current study may be relevant to unravel mechanisms making other endogenous
proteins more vulnerable to undergo conformational changes with little energy requirement (eg, point mutations and post-translational modifications) and thereby predisposing them to become immunogenic.