A mutation in the β ₃ cytoplasmic tail causes variant Glanzmann thrombasthenia by abrogating transition of α _IIbβ ₃ to an active state

Background:The cytoplasmic tails of α _IIb and β ₃ regulate essential α _IIbβ ₃ functions. We previously described a variant Glanzmann thrombasthenia mutation in the β ₃ cytoplasmic tail, IVS14: -3C>G, which causes a frameshift with an extension of β ₃ by 40 residues. Objectives:The aim of this study was to characterize the mechanism by which the mutation abrogates transition of α _IIbβ ₃ from a resting state to an active state. Methods:We expressed the natural mutation, termed 742ins, and three artificial mutations in baby hamster kidney (BHK) cells along with wild-type (WT) α _IIb as follows: β ₃-742stop, a truncated mutant to evaluate the effect of deleted residues; β ₃-749stop, a truncated mutant that preserves the NPLY conserved sequence; and β ₃-749ins, in which the aberrant tail begins after the conserved sequence. Flow cytometry was used to determine ligand binding to BHK cells. Results and conclusions:Surface expression of α _IIbβ ₃ of all four mutants was at least 60% of WT expression, but there was almost no binding of soluble fibrinogen following activation with activating antibodies (anti-ligand-induced-binding-site 6 [antiLIBS6] or PT25-2). Activation of the α _IIbβ ₃ mutants was only achieved when both PT25-2 and antiLIBS6 were used together or following treatment with dithiothreitol. These data suggest that the ectodomain of the four mutants is tightly locked in a resting conformation but can be forced to become active by strong stimuli. These data and those of others indicate that the middle part of the β ₃ tail is important for maintaining α _IIbβ ₃ in a resting conformation.