A point mutation Thr(799)Met on the alpha(2) integrin leads to the formation of new human platelet alloantigen Sit(a) and affects collagen-induced aggregation

A new platelet-specific alloantigen, termed Sit(a), was identified in a sev ere case of neonatal alloimmune thrombocytopenia. The Sita alloantigen is o f low frequency (1/400) in the German population. Immunochemical studies de monstrated that the Sita epitopes reside on platelet glycoprotein (GP) la. Nucleotide sequence analysis of GPla cDNA derived from Sit(a)-positive plat elets showed C-2531-->T-2531 point mutation, resulting in Thr(799)Met dimor phism. Analysis of genom ic DNA from 22 Sit(a)-negative normal individuals showed that the Thr(799) is encoded by ACG(2532) (90.9%) Or ACA(2532) (9.1% ) TO establish a DNA typing technique, we elucidated the organization of th e GPla 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. Pol ymerase chain reaction-restriction fragment length polymorphism analysis on DNA derived from 100 donors using the restriction enzyme Mae III showed th at the Met(799) form of GPla is restricted to Sit(a) (+) phenotype. Analysi s of stable Chinese hamster ovary transfectants expressing allele-specific recombinant forms of GPla showed that anti-Sit(a) exclusively reacted with the Glu(505)Met(799), hut not with the Glu(505)Thr(799) and the Lys(505)Thr (799) isoforms, In contrast, anti-Bra (HPA-5b) only recognized the Lys(505) Thr(799) form, whereas anti-Br-b (HPA-5a) reacted with both Glu(505)Thr(799 ) and Glu(505)Met(799) isoforms. These results demonstrated that the Met(79 9) is responsible for formation of the Sit(a) alloantigenic determinants, w hereas amino acid 505 (Lys or Glu) specifically controls the expression of Br-a and Br-b epitopes, respectively. Platelet aggregation responses of Sit (a) (+) individuals were diminished in response to collagen, indicating tha t the Thr(799)Met mutation affects the function of the GPla/IIa complex. (C ) 1999 by The American Society of Hematology.