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Keywords:

  • Glanzmann thrombasthenia;
  • integrins;
  • variant Glanzmann thrombasthenia;
  • αIIb;
  • β3

Summary.  Background: The cytoplasmic tails of αIIb and β3 regulate essential αIIbβ3 functions. We previously described a variant Glanzmann thrombasthenia mutation in the β3 cytoplasmic tail, IVS14: −3C>G, which causes a frameshift with an extension of β3 by 40 residues. Objectives: The aim of this study was to characterize the mechanism by which the mutation abrogates transition of αIIbβ3 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) αIIb as follows: β3-742stop, a truncated mutant to evaluate the effect of deleted residues; β3-749stop, a truncated mutant that preserves the NPLY conserved sequence; and β3-749ins, 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 αIIbβ3 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 αIIbβ3 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 β3 tail is important for maintaining αIIbβ3 in a resting conformation.