Three Separate Glanzmann Thrombasthenia Mutants Affecting the αIIb β-Propeller Result in Production of Normally Stable Pro-αIIb, but Impaired Progression from Endoplasmic Reticulum to Golgi.

The platelet αIIbβ3 (GPIIb/IIIa) integrin plays an important role in platelet function. Mutations in either αIIb or β3 can cause Glanzmann thrombasthenia by interfering with its biogenesis and/or function. We have previously reported three missense mutations in the β-propeller domain of αIIb, namely G128S, S287L and G357S, among three unrelated patients with Glanzmann thrombasthenia in southern India. Immunoblot analysis of platelet lysates showed no detectable αIIb in any of the patients and trace amounts of β3 in the latter two patients. Here, we describe further characterization of these mutations carried out by expression studies. Transfections were performed using HEK 293 cell lines with each of the following constructs: mock (vector only), αIIb only, normal αIIbβ3, and mutant αIIbβ3 (mutant αIIb G128S, S287L, or G357S co-transfected with normal β3). Stable cell lines were generated by G418 selection. While 52% of the normal αIIbβ3 cell lines bound antibody 10E5 (background = 5% positive), and the mean fluorescence intensity (MFI) of this subpopulation was 43 units, only 0, 2 and 8% of the mutant cell lines bound 10E5 above background, and the extent of binding among the positive cells was low (MFIs 14, 14 and 35).

Immunoprecipitation of the cell lysates and immunoblotting showed no detectable mature αIIb in the G128S mutant. In contrast, when compared with results of normal αIIbβ3, the S287L mutant showed 7% of the normal value of mature αIIb, and the G357S mutant showed 24% of the normal value. Pulse-chase analysis showed maturation of pro-αIIb to mature, cleaved αIIb in the normal αIIbβ3 cell line over 0–24 h. All three mutants demonstrated pro-αIIb bands comparable to the normal pro-αIIb band, but there was no conversion to mature αIIb in the G128S mutant, and trace conversion to mature αIIb in the S287L and G357S mutants. The disappearance of pro-αIIb in the three mutants was very similar to that of the normal αIIbβ3 and the αIIb only cell lines, with about 50% lost after 4 h. Thus, the failure of the mutant pro-αIIb to progress to mature αIIb was not a result of rapid degradation of pro-αIIb. Immunofluorescence of the normal αIIbβ3 cell line demonstrated strong αIIb staining on the surface, with αIIb also co-localizing with an ER marker (calnexin) and a Golgi marker (mannosidase II). In contrast, while all three mutants showed αIIb co-localizing in the ER, the G128S mutant showed no co-localization in the Golgi apparatus, and the other two mutants showed only slight co-localization in the Golgi. Surface staining was undetectable in the G128S and S287L mutants and decreased in the G357S mutant. These experiments show that the three β-propeller mutations do not affect the production of pro-αIIb or its stability. Instead, they affect transport from ER to the Golgi, most likely as a result of reducing αIIbβ3 complex formation. Hence, the mutant pro-αIIb appears to be retained in the ER and then degraded.