title: Construction of a human platelet alloantigen-1a epitope(s) within murine glycoprotein IIIa: identification of residues critical to the conformation of the antibody binding site(s).
abstract: The human platelet alloantigen 1 system (HPA-1) is determined by a polymorphism at position 33 in the N-terminus of human glycoprotein IIIa (GPIIIa). This naturally occurring substitution creates a conformation in the HPA-1a allelic form that can be antigenic when presented to an individual expressing the HPA-1b form. Anti-HPA-1a antibodies generated by this immune response can lead to the destruction of platelets, as seen in the clinical disorders, neonatal alloimmune thrombocytopenia (NAIT) and posttransfusion purpura (PTP). To understand better the structural requirements for recognition by these pathogenic antibodies, we investigated the N-terminal 66 amino acids from the HPA-1a form of human GPIIIa and the analogous amino acids from the nonimmunogenic murine homolog. Our objectives were to define further the boundaries of the HPA-1a epitope(s) in the N-terminus of human GPIIIa, to isolate the murine 5' nucleotide sequence and compare the deduced murine N-terminal sequence to that of human, and to mutate the murine sequence systematically to include an HPA-1a epitope(s). Murine amino acids that differed from human were changed by site-directed mutagenesis to the analogous residues in the HPA-1a form of human GPIIIa, starting and radiating from murine position 33 (site of human polymorphism). This systematic approach allowed us to pinpoint amino acids critical to a conformation recognized by anti-HPA-1a antibodies. Our results show that an HPA-1a epitope can be created within the N-terminus of murine GPIIIa and raise the possibility that murine models of HPA-1a sensitization can be developed.
authors.affiliation: Departments of Pediatrics and Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: E A
authors.initials: EA
authors.lastname: Barron-Casella
authors.forename: G
authors.initials: G
authors.lastname: Nebbia
authors.forename: O C
authors.initials: OC
authors.lastname: Rogers
authors.forename: K E
authors.initials: KE
authors.lastname: King
authors.forename: T S
authors.initials: TS
authors.lastname: Kickler
authors.forename: J F
authors.initials: JF
authors.lastname: Casella
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal; D016824:Antigens, Human Platelet / Q000737:chemistry* / Q000276:immunology*; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D000939:Epitopes / Q000737:chemistry*; D006801:Humans; D039661:Integrin beta3; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
chemical_list: D000911:Antibodies, Monoclonal; D016824:Antigens, Human Platelet; D000939:Epitopes; C061248:ITGB3 protein, human; D039661:Integrin beta3; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins