title: [Molecular mechanisms of Glanzmann thrombasthenia caused by alpha II b L721R and Q860X compound heterozygous mutation].
abstract: OBJECTIVE
  To explore the molecular mechanisms of Glanzmann thrombasthenia caused by alpha II b L721R and Q860X compound heterozygous mutation.
  
  
  METHODS
  All exons and exon-intron boundaries of alpha II b and beta3 gene were amplified by PCR and analyzed by direct DNA sequencing. Gene polymorphisms were excluded by direct DNA sequencing. Alpha II b L721R and Q860X mutants expressing vectors were constructed by in vitro site-directed mutagenesis. The expression of alpha II b L721R and Q860X mutants on transfected cell membrane were analyzed by flow cytometry and the whole expression level was confirmed by Western blot. The subcellular localizations of alpha II b L721R and Q860X mutants were determined by immunofluorescent confocal scanning microscopy.
  
  
  RESULTS
  The alpha II b compound heterozygous mutations, T2255G (L721R) and C2671T (Q860X), were identified in the proband, the former being inherited from the maternal side and the latter the paternal side. The 293T cells cotransfected with mutated alpha II b L721R and wild-type beta3 expression plasmids expressed 2.1% of normal amount of alpha II b on the cell surface as shown by FACS, in contrast to 31.9% of normal amount of alpha II b on the cells cotransfected with cDNAs of mutated alpha II b Q860X and wildtype beta3 expression plasmids. Western blot of the cell lysates showed no detectable mature alpha II b in cells lysates with L721R mutant. While, truncated alpha II b protein was detected in cell lystes with Q860X mutant. Immunofluorescence studies demonstrated that both L721R and Q860X mutant pro-alpha II bbeta33 complex colocalized in endoplasmic reticulum, but a little in Golgi.
  
  
  CONCLUSIONS
  The L721R and Q860X mutations of alpha II b prevent transport of the pro-alpha II bbeta3 complex from the endoplasmic reticulum to the Golgi, hindering its maturation and surface expression. The impaired alpha II bbeta3 transport is responsible for the thrombasthenia.
authors.affiliation: Shanghai Institute of Hematology, Affiliated Ruijin Hospital of Shanghai Jiaotong University, Shanghai 200025, China.
authors.forename: Wei-Zhang
authors.initials: WZ
authors.lastname: Shen
authors.forename: Pei-Pei
authors.initials: PP
authors.lastname: Jin
authors.forename: Qiu-Lan
authors.initials: QL
authors.lastname: Ding
authors.forename: Xue-Feng
authors.initials: XF
authors.lastname: Wang
authors.forename: Shu-Mei
authors.initials: SM
authors.lastname: Li
authors.forename: Yu-Zhen
authors.initials: YZ
authors.lastname: Jiang
authors.forename: Hong-Li
authors.initials: HL
authors.lastname: Wang
mesh_terms: D000818:Animals; D016466:CHO Cells; D002675:Child, Preschool; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005822:Genetic Vectors; D006579:Heterozygote; D006801:Humans; D038982:Integrin alpha2beta1 / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013915:Thrombasthenia / Q000235:genetics*; D014162:Transfection
chemical_list: D038982:Integrin alpha2beta1