A Point Mutation Thr799Met on the α2 Integrin Leads to the Formation of New Human Platelet Alloantigen Sita and Affects Collagen-Induced Aggregation

Abstract

AbstractA new platelet-specific alloantigen, termed Sita, was identified in a severe case of neonatal alloimmune thrombocytopenia. The Sita alloantigen is of low frequency (1/400) in the German population. Immunochemical studies demonstrated that the Sita epitopes reside on platelet glycoprotein (GP) Ia. Nucleotide sequence analysis of GPIa cDNA derived from Sita-positive platelets showed C2531→T2531 point mutation, resulting in Thr799Met dimorphism. Analysis of genomic DNA from 22 Sita-negative normal individuals showed that the Thr799 is encoded by ACG2532 (90.9%) or ACA2532 (9.1%). To establish a DNA typing technique, we elucidated the organization of the GPIa gene adjacent to the polymorphic bases. The introns (421 bp and 1.2 kb) encompass a 142-bp exon with the 2 polymorphic bases 2531 and 2532. Polymerase chain reaction-restriction fragment length polymorphism analysis on DNA derived from 100 donors using the restriction enzyme MaeIII showed that the Met799 form of GPIa is restricted to Sita (+) phenotype. Analysis of stable Chinese hamster ovary transfectants expressing allele-specific recombinant forms of GPIa showed that anti-Sita exclusively reacted with the Glu505Met799, but not with the Glu505Thr799 and the Lys505Thr799 isoforms. In contrast, anti-Bra (HPA-5b) only recognized the Lys505Thr799 form, whereas anti-Brb(HPA-5a) reacted with both Glu505Thr799 and Glu505Met799 isoforms. These results demonstrated that the Met799 is responsible for formation of the Sita alloantigenic determinants, whereas amino acid 505 (Lys or Glu) specifically controls the expression of Bra and Brb epitopes, respectively. Platelet aggregation responses of Sita (+) individuals were diminished in response to collagen, indicating that the Thr799Met mutation affects the function of the GPIa/IIa complex.