title: A mutation in the β3 cytoplasmic tail causes variant Glanzmann thrombasthenia by abrogating transition of αIIb β3 to an active state.
abstract: 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.
  
  
  OBJECTIVE
  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.
  
  
  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.
authors.affiliation: Amalia Biron Research Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel Hashomer and Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel.
authors.forename: H
authors.initials: H
authors.lastname: Hauschner
authors.forename: R
authors.initials: R
authors.lastname: Mor-Cohen
authors.forename: U
authors.initials: U
authors.lastname: Seligsohn
authors.forename: N
authors.initials: N
authors.lastname: Rosenberg
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D005340:Fibrinogen / Q000378:metabolism; D005434:Flow Cytometry; D039661:Integrin beta3 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013915:Thrombasthenia / Q000097:blood / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: C061248:ITGB3 protein, human; D039661:Integrin beta3; D008024:Ligands; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D005340:Fibrinogen