An Amino Acid Insertion and Variant Glanzmann Thrombasthenia

The ligand binding site(s) of the a subunit of integrin a IIb b 3 (GPIIb-IIIa), a prototypic non–I domain integrin, remains elusive. In this study, we have characterized a Japanese variant of Glanzmann thrombasthenia, KO, whose platelets express normal amounts of a IIb b 3 . KO platelets failed to bind the activation-independent ligand-mimetic mAb OPG2 and did not bind fibrinogen or the activation-dependent ligand-mimetic mAb PAC-1 following activation of a IIb b 3 under any condition examined. Sequence analysis of PCR fragments derived from KO platelet mRNA revealed a 6-bp insertion leading to a 2-amino-acid insertion (Arg-Thr) between residues 160 and 161 of the a IIb subunit. Introduction of the insertion into wild-type recombinant a IIb b 3 expressed in 293 cells led to the normal expression of a IIb b 3 having the defect in ligand binding function. The insertion is located within the small loop (Cys146-Cys167) in the third NH 2 -terminal repeat of the a IIb subunit. Alanine substitution of each of the oxygenated residues within the loop (Thr150, Ser152, Glu157, Asp159, Ser161, and Asp163) did not significantly affect expression of a IIb b 3 , and only Asp163Ala a IIb b 3 abolished the ligand binding function. In addition, Asp163Ala a IIb b 3 as well as KO mutant a IIb b 3 constitutively expressed the PMI-1 epitope. Our present data suggest that Asp163 of the a IIb subunit is one of the critical residues for ligand binding. ( J. Clin. Invest. 1998. 102:1183–1192.)