They suggest that epitope specificity may be an important parameter in assessing clinical risk, in part because oligomerization of the antigen by heparin or GAGs affects antibody binding and its downstream consequences. by ELISA that correlate with oligomerization 10058-F4 of PF4 and sustained high-avidity interactions that may simulate transient antibody-antigen interactions in vivo. These differences suggest the potential importance of epitope specificity in the pathogenesis of HIT. == Introduction == Heparin-induced thrombocytopenia (HIT) is a thrombotic complication of heparin therapy mediated by antibodies to complexes between platelet factor 4 (PF4) and heparin or glycosaminoglycans (GAGs).13However, antibodies to PF4/heparin are detected by ELISA far more frequently than antibodies that activate platelets or than clinical disease.46For example, anti-PF4/heparin antibodies are detected in 25% to 60% of patients who receive unfractionated heparin after cardiopulmonary bypass surgery and a high proportion of hospitalized patients in other medical settings,4,79an incidence that far exceeds the prevalence of HIT.1,10The reason why only a fraction of patients with anti-PF4 antibodies detected by ELISA develop HIT is unclear and is only partially explained by antibody titer and IgG isotype.5,9,1114 One clue to the differences in the pathogenic potential of anti-PF4/heparin antibodies may begin with the finding that heparin and PF4 form complexes of diverse size that depend on the molar ratio of the reactants.1517HIT antibodies and the HIT-like monoclonal antibody KKO bind and activate platelets and monocytes and promote thrombosis in an animal model over a narrow molar ratio of reactants.18,19At these molar ratios, ultralarge complexes (ULCs) form in solution between heparin and multiple PF4 tetramers capable of binding multiple antibody molecules16that in 10058-F4 the case of platelets may lead to sustained engagement of FcRIIA, which initiates aggregation.2022 Molecular replacement 10058-F4 studies reveal a track of amino acids on the surface of the PF4 tetramer required for binding of a HIT-like pathogenic monoclonal antibody KKO.23,24Heparin approximates PF4 tetramers as assessed by atomic force microscopy25and in doing so may expose this region or other neoepitopes recognized by pathogenic, but not by nonpathogenic antibodies, or reorganization may promote antibody avidity. To begin to understand the structural basis for the binding of pathogenic antibodies, we compared the effect of heparin on the binding of KKO and platelet-activating anti-PF4/heparin antibodies and anti-PF4/heparin antibodies not associated with platelet activation from patients suspected of HIT. The data presented here suggest there is a NFKB1 fundamental difference between the binding properties of pathogenic and nonpathogenic anti-PF4 antibodies that is not evident by ELISA, and they suggest potential new approaches toward identifying clinically relevant HIT antibodies in the future. == Methods == == Generation of human PF4 in Schnieder 2 insect cells == cDNAs encoding human wild-type (WT) PF4 and PF4K50Ewere cloned into the plasmid pMT/BiP/V5-His (Invitrogen) for expression in Drosophila Expression System (Invitrogen). Cloning was performed using BglII and AgeI cloning sites. A hexanucleotide encoding the BglII site was then eliminated by site-directed mutagenesis so that the expressed protein contained full-length WT PF4 or PF4K50Ewith an identical sequence as their counterparts expressed inEscherichia coli.26PF4 expression was induced by adding copper sulfate (0.5mM), and the protein was collected in serum-free medium Insect-Xpress (Lonza Walkersville) for 3 to 5 5 days; sodium azide (0.02% final concentration) and EDTA (2.5mM final concentration) were added, and the medium was filtered through Express Plus 0.22-mm filter (Millipore). PF4 was purified from the media on a heparin HiTrap column (GE Healthcare) on an ATKA Prime FPLC (GE Healthcare) at 4C using a 10mM Tris and 1mM EDTA, pH 8, buffer system. Medium was loaded in buffer containing 0.5M NaCl and PF4 eluted at 1.8M NaCl using a linear gradient. Fractions containing purified PF4 detected by silver staining of 12% polyacrylamide gels (SDS-PAGE) were pooled and then concentrated, and buffer was exchanged into 50mM HEPES and 0.5M NaCl, pH 7.2, using an Amicon Ultra filter (3000 molecular weight cut-off; Millipore). Protein was quantified using a bicinchoninic acid assay (Thermo Fisher Scientific). PF4K50Ewas purified in the same way as WT PF4, with several modifications: the buffer system used was 50mM MES, 1mM EDTA, pH 6.5; medium was loaded in buffer containing 0.3M NaCl, and the protein was eluted at 1.3M NaCl using a linear gradient. == Antihuman PF4 monoclonal antibodies 10058-F4 to PF4 == KKO and RTO hybridoma cells were generated and characterized as described previously.27In brief, KKO and RTO are IgG2b monoclonal antihuman PF4 antibodies generated concurrently in mice injected with complexes of human PF4 and unfractionated heparin at an equimolar ratio. The IgG fractions were purified from conditioned PFHM-II media (Invitrogen) using protein A agarose (Invitrogen) as recommended by the manufacturer..