Glanzmann thrombasthenia (GT) is a recessively inherited bleeding disorder caused by the quantitative or qualitative deficiency of the platelet fibrinogen receptor, integrin αIIbβ3. The N-terminal domain of the αIIb subunit is folded in a β-propeller that plays the role of binding fibrinogen and associating with the ligand-binding region of β3. Analysing the mutations of Italian GT patients we found that a patient had a αIIb G236E missense substitution that substitutes a glycine from the highly conserved ΦΦGΦ motif of blade 4 of the β-propeller. To verify experimentally the effect of the substitution of glycine 236 human embryonic kidney (HEK) cells were transfected with normal or mutated αIIb in conjunction with normal β3. Using flow cytometry analysis we found the percentage of HEK cells transfected with αIIbG236Eβ3 that reacted with anti αIIbβ3 was very low. In HEK cells transfected with either αIIbβ3 or αIIbG236Eβ3 and lysed, when immunoblotting was done in non-reducing conditions a band reacting with an antibody against αIIb was present in both lysates, although less intense in cells transfected with αIIbG236Eβ3. In reducing condition αIIb from cells transfected with αIIbβ3 was nearly all mature, while in cells transfected with αIIbG236Eβ3 the ratio pro-αIIb: αIIb was 1 : 1, with signs of degradation of the mutated protein. Cell lysates were then immunoprecipitated with antibodies against αIIb and immunoblotted with an antibody reacting with β3. While in immunoblots from cells transfected with αIIbβ3 a band corresponding to β3 was strongly detectable, in immunoblots originating from cells transfected with αIIbG236Eβ3 no band at the same level of normal β3 was detected. Immunofluorescence studies showed accumulation of αIIbG236Eβ3 in the endoplasmic reticulum and minimal transport to the Golgi. In conclusion we demonstrated that the αIIbG236E mutation causes GT by impairing the association with β3 during biogenesis of the receptor.
|Titolo:||AlphaIIbG236E causes Glanzmann thrombasthenia by impairing association with beta3|
MANNUCCI, PIER MANNUCCIO (Ultimo)
|Parole Chiave:||6IIb73; Glanzmann thrombasthenia|
|Settore Scientifico Disciplinare:||Settore MED/09 - Medicina Interna|
|Data di pubblicazione:||ago-2008|
|Digital Object Identifier (DOI):||10.1080/09537100802123365|
|Appare nelle tipologie:||01 - Articolo su periodico|