• antibodies;
  • β3 integrin deletion peptides;
  • HPA-1;
  • platelets

Summary. 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 αIIbβ3 integrin from the platelets of HPA-genotyped donors.Objectives: We set out to define the β3 integrin domains required for HPA-1a antibody binding and produce recombinant soluble β3 peptides for HPA-1 antibody detection.Methods: We designed two sets (1a and 1b) of four soluble β3 domain-deletion peptides (ΔSDL, ΔβA, 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 β3 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 β3 peptides. Testing of FMAIT samples indicated that ΔβA-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 β3-Leu33 peptides, but there was no correlation between antibody category and clinical severity of FMAIT. Conclusions: Soluble recombinant β3 peptides can be used for detection of clinical HPA-1a antibodies.