Single amino acid substitution in human platelet glycoprotein Ibβ is responsible for the formation of the platelet-specific alloantigen Iya

Abstract

We recently described a new low-frequency platelet alloantigen on the human platelet glycoprotein (GP) Ib-IX complex, termed Iya, which was implicated in a severe case of neonatal alloimmune thrombocytopenia. Immunoprecipitation studies with trypsin-treated platelets indicated that the Iyaalloantigenic determinants are formed by the membrane-associated remnant moiety of GP Ib (GP Ibr) together with GP Ibβ and GP IX. To elucidate the molecular basis underlying the Iya alloantigen, we amplifiedGPIbr, GPIbβ, andGPIX genes by polymerase chain reaction (PCR). Nucleotide-sequence analysis of these 3 genes showed a G to A transition at position 141 on GPIbβ gene in a subject positive for Iya. This transition resulted in a Gly15Glu dimorphism on the N-terminal domain ofGPIbβ. This finding was confirmed by genotyping analysis of 6 Iya-positive subjects by restriction fragment length polymorphism (RFLP) studies using NarI endonuclease. In 300 randomly selected healthy blood donors, one Iya-positive individual was found. Phenotypes determined by monoclonal antibody-specific immobilization of platelet antigens assay and genotypes determined by RFLP were identical in this population. Analysis of Iya-positive platelets showed that the point mutation affected neither the degree of surface expression nor the function of the GP Ib-GP Ibβ-IX complex on the platelet surface. Transient expression of the GP Ib-IX complex in CHO cells using wild-type GP Ibβ (Gly15) or mutant GP Ibβ (Glu15) allowed us to demonstrate that this single amino acid substitution is sufficient to induce Iya epitope(s).