The αIIbP258S, a Newly Identified Mutation, and the αIIbG349D Substitutions Cause Glanzmann Thrombasthenia by Impairing αIIb-β3 Association in the Endoplasmic Reticulum.

Glanzmann Thrombasthenia is a recessively inherited hemorrhagic disorder caused by the quantitative or qualitative deficiency of integrin αIIbβ3, platelet fibrinogen receptor. We previously analyzed the phenotypic effects of the αIIbG236E substitution, which is located in a strategic point of the highly organized αIIb propeller. We demonstrated that the effect of αIIbG236E is to hamper the association with β3, impairing the correct assembly of the complex. In the present study we analyze the phenotypic effects of two other mutations in the same domain of αIIb. The first mutation, the αIIbP258S substitution, is a newly found missense substitution that was identified during a genetic screening of a cohort of patients with Glanzmann Thrombasthenia coming from Southern Iran. The presence of the mutation was confirmed in several family members affected by Glanzmann Thrombasthenia; no other additional mutations were identified. The effect of the substitution is to eliminate the key proline from blade 4 of αIIb propeller. The second mutation, the αIIbG349D substitution, has been previously described in an Italian patients with Glanzmann Thrombasthenia. It causes a charge substitution in strand D of blade 5 of the propeller. Both substitutions were inserted by site directed mutagenesis in pCDNA3.1 containing normal αIIb cDNA. Human Embryonic Kidney (HEK) cells were then transfected by electroporation with normal or mutated αIIb in conjunction with pCDNA3.1 containing normal β3. After 48 hours cells were analyzed by flow cytometry: the binding of 10E5-FITC (murine anti-human αIIbβ3) in cells expressing αIIbP258Sβ3 or αIIbG349Dβ3 was approximately at the same level of negative controls (cells transfected with αIIb only) and greatly reduced in comparison to the binding to cells transfected with normal αIIbβ3. HEK cells transfected with either αIIbβ3 or αIIbP258Sβ3 or αIIbG349Dβ3 were lysed; cells lysates were analysed by SDS-PAGE electrophoresis and immunoblotting. All the 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 mutants αIIb. In reducing condition αIIb from cells transfected with αIIbβ3 was nearly all mature, while in cells transfected with αIIbP258Sβ3 or αIIbG349Dβ3 the ratio pro-αIIb: αIIb was 1:1. In conclusion we report the identification of a new mutation responsible for Glanzmann Thrombasthenia located in αIIb propeller. This mutation, in addition with αIIbG349D, causes Glanzmann Thrombasthenia interfering with αIIbβ3 complex formation in the endoplasmic reticulum as shown by the increase amount of pro-αIIb found in cells expressing these mutants.