title: Dynamic antibody-binding properties in the pathogenesis of HIT.
abstract: Rapid laboratory assessment of heparin-induced thrombocytopenia (HIT) is important for disease recognition and management. The utility of contemporary immunoassays to detect antiplatelet factor 4 (PF4)/heparin antibodies is hindered by detection of antibodies unassociated with disease. To begin to distinguish properties of pathogenic anti-PF4/heparin antibodies, we compared isotype-matched monoclonal antibodies that bind to different epitopes: KKO causes thrombocytopenia in an in vivo model of HIT, whereas RTO does not. KKO binding to PF4 and heparin is specifically inhibited by human HIT antibodies that activate platelets, whereas inhibition of RTO binding is not differentially affected. Heparin increased the avidity of KKO binding to PF4 without affecting RTO, but it did not increase total binding or binding to nontetrameric PF4(K50E). Single-molecule forced unbinding demonstrated KKO was 8-fold more reactive toward PF4 tetramers and formed stronger complexes than RTO, but not to PF4(K50E) dimers. KKO, but not RTO, promoted oligomerization of PF4 but not PF4(K50E). This study reveals differences in the properties of anti-PF4 antibodies that cause thrombocytopenia not revealed by ELISA that correlate with oligomerization 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.
authors.affiliation: Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. sachais@mail.med.upenn.edu
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Sachais
authors.forename: Rustem I
authors.initials: RI
authors.lastname: Litvinov
authors.forename: Serge V
authors.initials: SV
authors.lastname: Yarovoi
authors.forename: Lubica
authors.initials: L
authors.lastname: Rauova
authors.forename: Jillian L
authors.initials: JL
authors.lastname: Hinds
authors.forename: Ann H
authors.initials: AH
authors.lastname: Rux
authors.forename: Gowthami M
authors.initials: GM
authors.lastname: Arepally
authors.forename: Mortimer
authors.initials: M
authors.lastname: Poncz
authors.forename: Adam
authors.initials: A
authors.lastname: Cuker
authors.forename: John W
authors.initials: JW
authors.lastname: Weisel
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Cines
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D055910:Antibodies, Immobilized / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000915:Antibody Affinity / Q000235:genetics / Q000502:physiology*; D000937:Antigen-Antibody Reactions / Q000235:genetics / Q000502:physiology; D045744:Cell Line, Tumor; D004330:Drosophila; D004797:Enzyme-Linked Immunosorbent Assay; D006493:Heparin / Q000009:adverse effects; D006801:Humans; D007700:Kinetics; D051379:Mice; D008822:Mice, Transgenic; D010978:Platelet Factor 4 / Q000235:genetics / Q000276:immunology; D011485:Protein Binding / Q000502:physiology; D013921:Thrombocytopenia / Q000209:etiology* / Q000235:genetics / Q000276:immunology* / Q000473:pathology
chemical_list: D000906:Antibodies; D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D010978:Platelet Factor 4; D006493:Heparin