Immunologic and structural analysis of eight novel domain-deletion β₃ integrin peptides designed for detection of HPA-1 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)beta(3) integrin from the platelets of HPA-genotyped donors. Objectives: We set out to define the beta(3) integrin domains required for HPA-1a antibody binding and produce recombinant soluble beta(3) peptides for HPA-1 antibody detection. Methods: We designed two sets (1a and 1b) of four soluble beta(3) domain-deletion peptides (Delta SDL, Delta beta A, 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 beta(3) 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 beta(3) peptides. Testing of FMAIT samples indicated that Delta beta A-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 beta(3)-Leu33 peptides, but there was no correlation between antibody category and clinical severity of FMAIT. Conclusions: Soluble recombinant beta(3) peptides can be used for detection of clinical HPA-1a antibodies.