Key Points• Besides clustering, platelet factor 4/polyanion complexes require input of energy to become immunogenic. • Minute differences in chain length determine the induction of antigenicity of PF4.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 b-sheets exceeding ∼30% (achieved by UFH, LMWH,, (2) formation of multimolecular complexes (UFH,, 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. (Blood. 2014;124(15):2442-2449 IntroductionHeparin, widely used clinically as a parenteral anticoagulant, 1-3 is a polyanion consisting of iduronic acid, glucuronic acid, and glucosamine residues carrying sulfate groups. Pharmaceutical heparin is obtained from porcine gut mucosa and, as biological material, is composed of polysaccharide chains with variable length. Degradation of unfractionated heparin (UFH) results in less polydisperse and smaller low-molecular-weight heparins (LMWHs). The pentasaccharide fondaparinux consists of the shortest sequence able to catalyze the activity of antithrombin. It was the first synthesized heparin approved for clinical use. 4 Currently, several other heparin-based, synthesized polysaccharides are in preclinical development. 5,6 Beside their anticoagulant activity, heparins have other biological effects including potential antitumor activity, 7 which, however, differs depending on the chain length. Clinically, beside bleeding, heparin-induced thrombocytopenia (HIT) is the most important adverse effect of heparin.8 HIT is a lifethreatening immune-driven adverse effect, which occurs in up to 3% of patients receiving UFH after major surgery. 9 HIT is caused by antibodies that recognize platelet factor 4 (PF4), a CXC chemokine family protein, in ultralarge multimolecular complexes with heparin. 10Several of these pathogenic antib...