title: Defining a second epitope for heparin-induced thrombocytopenia/thrombosis antibodies using KKO, a murine HIT-like monoclonal antibody.
abstract: Heparin-induced thrombocytopenia/thrombosis (HIT/T) is a common complication of heparin therapy that is caused by antibodies to platelet factor 4 (PF4) complexed with heparin. The immune response is polyclonal and polyspecific, ie, more than one neoepitope on PF4 is recognized by HIT/T antibodies. One such epitope has been previously identified; it involves the domain between the third and fourth cysteine residues in PF4 (site 1). However, the binding sites for other HIT/T antibodies remain to be defined. To explore this issue, the binding site of KKO, an HIT/T-like murine monoclonal antibody, was defined. KKO shares a binding site with many HIT/T antibodies on PF4/heparin, but does not bind to site 1 or recognize mouse PF4/heparin. Therefore, the binding of KKO to a series of mouse/human PF4 chimeras complexed with heparin was examined. KKO recognizes a site that requires both the N terminus of PF4 and Pro34, which immediately precedes the third cysteine. Both regions lie on the surface of the PF4 tetramer in sufficient proximity (within 0.74 nm) to form a contiguous antigenic determinant. The 10 of 14 HIT/T sera that require the N terminus of PF4 for antigen recognition also require Pro34 to bind. This epitope, termed site 2, lies adjacent to site 1 in the crystal structure of the PF4 tetramer. Yet sites 1 and 2 can be recognized by distinct populations of antibodies. These studies further help to define a portion of the PF4 tetramer to which self-reactive antibodies develop in patients exposed to heparin.
authors.affiliation: Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia, USA.
authors.forename: Zhong Q
authors.initials: ZQ
authors.lastname: Li
authors.forename: Weiyi
authors.initials: W
authors.lastname: Liu
authors.forename: Kwang S
authors.initials: KS
authors.lastname: Park
authors.forename: Brue S
authors.initials: BS
authors.lastname: Sachais
authors.forename: Gowthani M
authors.initials: GM
authors.lastname: Arepally
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Cines
authors.forename: Mortimer
authors.initials: M
authors.lastname: Poncz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D006493:Heparin / Q000009:adverse effects / Q000276:immunology*; D006801:Humans; D051379:Mice; D010978:Platelet Factor 4 / Q000737:chemistry / Q000276:immunology; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D013921:Thrombocytopenia / Q000139:chemically induced / Q000276:immunology*; D013927:Thrombosis / Q000139:chemically induced / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D010978:Platelet Factor 4; D006493:Heparin