[Molecular mechanisms of Glanzmann thrombasthenia caused by alphaII b P126H mutation: an in vitro experiment].
OBJECTIVE
To explore the molecular mechanisms of Glanzmann thrombasthenia caused by alpha IIb P126H mutation.


METHODS
Eukaryotic vector of alpha IIb P126H was constructed by PCR site-directed mutagenesis and then co-transfected with eukaryotic vector PCDM8 II a expressing the subunit beta3 into human renal epithelial cells of the line 293& and Chinese hamster ovarian cancer cells of the line CHO after sequencing. The membrane expression of alpha IIb P126H mutant was analyzed by flow cytometry and the whole expression was confirmed by Western blotting. The alpha IIb P126H mutant subcellular localization was determined by laser confocal scanning microscopy.


RESULTS
The 293T cells cotransfected with cDNAs of mutated alpha IIb and wild-type beta3 failed to express alpha II bbeta3 on the cell surface as shown by FACS. Western blotting of the cell lysate showed no detectable mature alpha IIb. Immunofluorescence studies demonstrated proa II bbeta3 complex colocalized with an endoplasmic reticulum (ER) marker, but showed minimal colocalization with an Golgi marker.


CONCLUSIONS
The P126H mutation in alpha IIb prevents transport of the pro-alpha II bbeta3 complex from ER to the Golgi body, thus hindering its maturation and surface expression. The impaired alpha II bp33 transport is responsible for the thrombasthenia.
Shen
Wei-zhang
WZ
Department of Hematology, Second Affiliated Hospital of Jilin University, Changchun 130041, China.
Li
Shu-mei
SM
Jin
Pei-pei
PP
Ding
Qiu-lan
QL
Wang
Xue-feng
XF
Jiang
Yu-zhen
YZ
Wang
Hong-li
HL
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D018613:Microscopy, Confocal; D009154:Mutation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics* / Q000378:metabolism; D021381:Protein Transport; D013915:Thrombasthenia / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D019039:Platelet Glycoprotein GPIIb-IIIa Complex