HPA-1a phenotype-genotype discrepancy reveals a naturally occurring Arg93Gln substitution in the platelet beta 3 integrin that disrupts the HPA-1a epitope

A single nucleotide polymorphism (SNP) at position 196 in the beta3 integrin causes a Leu33Pro substitution in the mature protein. Alloimmunization against the beta3Leu33 form (human platelet antigen [HPA]-1a, PIA1, Zw(a)) in patients who are beta3Pro33 homozygous (HPA-1b1b, PIA2A2, Zw(bb)) causes neonatal allolmmune thrombocytopenia, posttransfusion purpura, or refractoriness to platelet transfusion. Studies with recombinant proteins have demonstrated that amino acids 1 to 66 and 288 to 490 of the beta3 integrin contribute to HPA-1a epitope formation. In determining the HPA-1a status of more than 6000 donors, we Identified a donor with an HPA-1a(weak) phenotype and an HPA-1a1b genotype. The platelets from this donor had normal levels of surface alphallbbeta3 but reacted only weakly with monoclonal and polyclonal anti-HPA-1a by whole blood enzyme-linked immunosorbent assay (ELISA), flow cytometry, and sandwich ELISA. We reasoned that an alteration in the primary nucleotide sequence of the beta3Leu33 allele of this donor was disrupting the HIPA-la epitope. In agreement with this hypothesis, sequencing platelet RNA-derived alphallb and beta3 cDNA identified a novel G/A SNP at position 376 of the beta3 integrin that encodes for an Arg93Gin replacement in the beta3Leu33 allele. Coexpression of the beta3Leu33Gln93 encoding cDNA in Chinese hamster ovary cells with human allb cDNA showed that the surface-expressed alpha-llbbeta3 reacted normally with beta3 integrin-specific monoclonal antibodies but only weakly with monoclonal anti-HPA-1a. Our results show that an Arg93Gln mutation in the beta3Leu33 encoding allele disrupts the HPA-1a epitope, suggesting that Arg93 contributes to the formation of the HPA-1a beta -cell epitope.