The α IIbG236E Mutations Causes Glanzmann Thrombasthenia by Impairing Association with β3.
Authors: Artoni Andrea , Gelain Federica , Mannucci Pier Mannuccio 
Published: October 2019

Glanzmann Thrombasthenia (GT) is a recessively inherited bleeding disorder caused by the quantitative or qualitative deficiency of integrin αIIbβ3. The recent solution of the crystal structure of αIIbβ3 revealed that the N-terminal domain of αIIb is folded in a complex structure, a β-propeller formed by seven blades. The role of the β-propeller is to bind fibrinogen and to associate to β3. In the endoplasmic reticulum pro-αIIb associates with β3, it is then transferred to the Golgi, where it is cleaved in two chains disulfide bonded (mature αIIb); mutations that impair the association of pro-αIIb to β3 are able to cause GT. The architecture of the propeller is sustained by several prolines that stabilize the entire structure. From molecular modeling studies it had been postulated that the residue spatially adjacent to proline 258 can only be a glycine because there is no physical space for other residues. Analyzing the GT mutations of Italian patients recently described by D’Andrea et al. (Thromb Haemost., 2002) we found that a patient with type I GT had a αIIb G236E missense substitution, hence we decided to verify if the substitution of glycine 236 was hampering αIIb β3 association. The G236E mutation was introduced by site directed mutagenesis in pCDNA3.1 containing normal human αIIb cDNA. Human embryonic kidney (HEK) cells were transfected either with normal or mutated αIIb in conjunction with pCDNA3.1 containing normal β3. By flow cytometry analysis the percentage of HEK cells transfected with αIIbG236Eβ3 that reacted with 10E5-FITC (anti αIIbβ3) was 7±1% while the percentage of cells transfected with αIIbβ3 that reacted with 10E5-FITC was 37±13%. When the binding of an antibody directed against β3 (VIPL-2-FITC) was evaluated, we observed that cells expressing αIIbG236Eβ3 did bind the antibody, indicating that the normal transfected β3 was associating with the endogenous αV. HEK cells transfected with either αIIbβ3 or αIIbG236Eβ3 were lysed; cells lysates were analysed by SDS-PAGE electrophoresis and immunoblotting. Both lysates were reacting at the same level with antibodies directed against β3. When immunoblotting was done in non-reducing conditions utilizing an antibody reacting with αIIb (mab1990) a band corresponding to 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. Moreover, when tested against lysates of cells transfected with αIIbG236Eβ3, the antibody against αIIb recognized multiple bands of lower molecular weight, presumably degradation products of the mutated protein. Cell lysates were then immunoprecipitated with antibodies against αIIb or αIIbβ3 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 a very weak band at the same level of normal β3 was detected only in immunoprecipitates using 10E5, a complex dependent antibody. In conclusion we demonstrated that αIIbG236E is a mutation that causes GT by impairing the association with β3 in the endoplasmic reticulum; the effect of the mutation is likely to create a severe misfold of the β-propeller and the molecular modeling prediction of the need of a glycine at position 236 is probably confirmed.