BACKGROUND: The single-nucleotide polymorphism (SNP) rs5918 in the ITGB3 gene defines the human platelet antigen-1 (HPA-1) system encoding a Leu (HPA-1a) or Pro (HPA-1b) at position 33. HPA-1 antibodies are clinically the most relevant in the Caucasoid population, but detection currently requires alpha(IIb)beta3 integrin from the platelets of HPA-genotyped donors. OBJECTIVES: We set out to define the beta3 integrin domains required for HPA-1a antibody binding and produce recombinant soluble beta3 peptides for HPA-1 antibody detection. METHODS: We designed two sets (1a and 1b) of four soluble beta3 domain-deletion peptides (deltaSDL, deltabetaA, PSIHybrid, PSI), informed by crystallography studies and computer modeling. The footprints of three human HPA-1a-specific phage antibodies were defined by analyzing binding patterns to the beta3 peptides and canine platelets, and models of antibody-antigen interfaces were derived. Specificity and sensitivity for HPA-1a detection were assessed using sera from 140 cases of fetomaternal alloimmune thrombocytopenia (FMAIT). RESULTS: Fusion of recombinant proteins to calmodulin resulted in high-level expression in Drosophila S2 cells of all eight beta3 peptides. Testing of FMAIT samples indicated that deltabetaA-Leu33 is the superior peptide for HPA-1a antibody detection, with 96% sensitivity and 95% specificity. The existence of type I and II categories of HPA-1a antibodies was confirmed by the study of HPA-1a phage antibody footprints and the reactivity pattern of clinical samples with the four beta3-Leu33 peptides, but there was no correlation between antibody category and clinical severity of FMAIT. CONCLUSIONS: Soluble recombinant beta3 peptides can be used for detection of clinical HPA-1a antibodies.
BACKGROUND: The single-nucleotide polymorphism (SNP) rs5918 in the ITGB3 gene defines the human platelet antigen-1 (HPA-1) system encoding a Leu (HPA-1a) or Pro (HPA-1b) at position 33. HPA-1 antibodies are clinically the most relevant in the Caucasoid population, but detection currently requires alpha(IIb)beta3 integrin from the platelets of HPA-genotyped donors. OBJECTIVES: We set out to define the beta3 integrin domains required for HPA-1a antibody binding and produce recombinant soluble beta3 peptides for HPA-1 antibody detection. METHODS: We designed two sets (1a and 1b) of four soluble beta3 domain-deletion peptides (deltaSDL, deltabetaA, PSIHybrid, PSI), informed by crystallography studies and computer modeling. The footprints of three human HPA-1a-specific phage antibodies were defined by analyzing binding patterns to the beta3 peptides and canine platelets, and models of antibody-antigen interfaces were derived. Specificity and sensitivity for HPA-1a detection were assessed using sera from 140 cases of fetomaternal alloimmune thrombocytopenia (FMAIT). RESULTS: Fusion of recombinant proteins to calmodulin resulted in high-level expression in Drosophila S2 cells of all eight beta3 peptides. Testing of FMAIT samples indicated that deltabetaA-Leu33 is the superior peptide for HPA-1a antibody detection, with 96% sensitivity and 95% specificity. The existence of type I and II categories of HPA-1a antibodies was confirmed by the study of HPA-1a phage antibody footprints and the reactivity pattern of clinical samples with the four beta3-Leu33 peptides, but there was no correlation between antibody category and clinical severity of FMAIT. CONCLUSIONS: Soluble recombinant beta3 peptides can be used for detection of clinical HPA-1a antibodies.
Authors: Aye Myat Myat Thinn; Zhengli Wang; Dongwen Zhou; Yan Zhao; Brian R Curtis; Jieqing Zhu Journal: Proc Natl Acad Sci U S A Date: 2018-09-12 Impact factor: 11.205
Authors: Cedric Ghevaert; David A Wilcox; Juan Fang; Kathryn L Armour; Mike R Clark; Willem H Ouwehand; Lorna M Williamson Journal: J Clin Invest Date: 2008-08 Impact factor: 14.808
Authors: Vincent Jallu; Pierre Poulain; Patrick F J Fuchs; Cecile Kaplan; Alexandre G de Brevern Journal: PLoS One Date: 2012-11-14 Impact factor: 3.240