A two-amino acid insertion in the Cys146-Cys167 loop of the α(IIb) subunit is associated with a variant of Glanzmann thrombasthenia: Critical role of Asp163 in ligand binding

Abstract

The ligand binding site(s) of the α subunit of integrin α(IIb)β3 (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 α(IIb)β3. KO platelets failed to bind the activation-independent ligand-mimetic mAb OP-G2 and did not bind fibrinogen or the activation-dependent ligand-mimetic mAb PAC-1 following activation of α(IIb)β3 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 α(IIb) subunit. Introduction of the insertion into wild-type recombinant α(IIb)β3 expressed in 293 cells led to the normal expression of α(IIb)β3 having the defect in ligand binding function. The insertion is located within the small loop (Cys146-Cys167) in the third NH2-terminal repeat of the α(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 α(IIb)β3, and only Asp163Alaα(IIb)β3 abolished the ligand binding function. In addition, Asp163-Alaα(IIb)β3 as well as KO mutant α(IIb)β3 constitutively expressed the PMI-1 epitope. Our present data suggest that Asp163 of the α(IIb) subunit is one of the critical residues for ligand binding.