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A mutation in the β 3 cytoplasmic tail causes variant Glanzmann thrombasthenia by abrogating transition of α IIb β 3 to an active state
Author(s) -
HAUSCHNER H.,
MORCOHEN R.,
SELIGSOHN U.,
ROSENBERG N.
Publication year - 2012
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2011.04577.x
Subject(s) - mutant , mutation , thrombasthenia , transition (genetics) , microbiology and biotechnology , frameshift mutation , baby hamster kidney cell , chemistry , glanzmann's thrombasthenia , biology , biochemistry , gene , cell , platelet , immunology , platelet aggregation
Summary. Background: The cytoplasmic tails of α IIb and β 3 regulate essential α IIb β 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 IIb β 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 IIb β 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 IIb β 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 IIb β 3 in a resting conformation.