A spontaneous mutation of integrin α_IIbβ₃ (platelet glycoprotein IIb-IIIa) helps define a ligand binding site

This work characterizes a mutant integrin α_IIbβ₃ (glycoprotein (GP) IIb-IIIa) from a thrombasthenic patient, ET, whose platelets fail to aggregate in response to stimuli. The nature of the defect was defined by the reduced ability of synthetic peptide ligands, corresponding to the carboxyl terminus of the fibrinogen γ chain (γ402-411) and Arg-Gly-Asp (RGD), to increase the binding of the occupancy-dependent anti-LIBS1 antibody to mutant α_IIbβ₃ and the reduced binding of mutant α_IIbβ₃ to an immobilized RGD peptide. In addition, ET's platelets failed to bind the ligand-mimetic monoclonal anti-α_IIbβ₃, PAC1. DNA sequence analysis of amplified ET genomic DNA revealed a single G → A base change which encoded substitution of R214 by Q in mature β₃. Introduction of this point mutation into recombinant wild type α_IIbβ₃ expressed in Chinese hamster ovary cells reproduced the ET platelet α_IIbβ₃ deficits in binding of fibrinogen, mAb PAC1, and synthetic peptide ligands. Furthermore, substitution of R214 by Q in the synthetic peptide containing the sequence of β₃(211-222) resulted in decreased ability of this peptide to block fibrinogen binding to purified α_IIbβ₃. These findings suggest that substitution of β₃ R214 by Q is responsible for the functional defect in α_IIbβ₃ and that R214 is proximal to or part of a ligand binding domain in α_IIbβ₃.