• β3 fragments;
  • HPA-4;
  • platelets;
  • rare HPA

Summary. Background: Antibodies against human platelet antigens (HPA) are clinically important in fetal–maternal alloimmune thrombocytopenia, refractoriness to platelet transfusions and post-transfusion purpura. Of the 16 HPAs, nine are located on the β3 subunit of the αIIbβ3 integrin. Antibody detection is generally based on platelet-derived αIIbβ3 from HPA-genotyped donors. Recombinant allelic β3 peptides, expressed at high levels would improve consistency in antibody detection, but the expression of soluble and monomeric integrins expressing complex dependent epitopes has previously proved challenging. Objectives: We aimed to generate three recombinant β3 peptides for the detection of antibodies against HPA-4, HPA-8bw and five of the six remaining low frequency β3 alloantigens. Methods: The removal of the specificity-determining loop from the βA domain and fusion of truncated β3 to calmodulin was exploited to obtain expression of monomeric protein. Using site-directed mutagenesis, the mutations for HPA-4b and HPA-8bw were introduced in the ITGB3*001 haplotype. A third peptide for the detection of antibodies against HPA coded by non-synonymous single nucleotide polymorphisms of low frequency was generated by the introduction of five mutations forming the basis of HPA-6bw, -7bw, -10bw, -11bw, and -16bw antigens. Results: Reactivity of the three peptides with β3-specific murine monoclonal antibodies and human HPA-1a phage antibodies confirmed the structural integrity of the recombinant fragments, and reactivity with a unique panel of polyclonal anti-HPA sera confirmed expression of the relevant HPA epitopes. Conclusions: These data demonstrate that β3 integrin domain-deletion fragments are suitable molecular targets for HPA antibody detection.