A Gln747→Pro Substitution in the αIIb Subunit Is Responsible for a Moderate αIIbβ3Deficiency in Glanzmann Thrombasthenia

Abstract

To clarify a molecular defect responsible for moderate αIIbβ3 deficiency, we examined two unrelated patients, MT and MS, suffering from type II and type I Glanzmann thrombasthenia (GT), respectively. Sequence analysis of polymerase chain reaction (PCR) fragments derived from platelet mRNA showed a single A→C substitution at nucleotide (nt) 2334 leading to a Gln747→ Pro in αIIb in both patients. Allele-specific restriction enzyme analysis (ASRA) of genomic DNA demonstrated that patient MT was homozygous for the Gln747→Pro substitution and patient MS was compound heterozygous for this substitution and for an RNA splice mutation at the consensus sequence of the splice acceptor site of exon 18 (AG→AA). Furthermore, ASRA showed that, among 17 unrelated Japanese GT patients, this Gln747→Pro substitution was detected in 4 patients, including MT and MS (homozygous, 2 patients; heterozygous, 2 patients). Cotransfection of Pro747αIIb and β3 constructs into 293 cells resulted in moderate reduction in the amount of αIIbβ3 within the transfected cells as well as on the cell surface. However, Pro747αIIbβ3 bound the ligand mimetic monoclonal antibody (MoAb) PAC-1 after activation of αIIbβ3 by the MoAb PT25-2, suggesting that the mutant αIIbβ3 possesses the ligand-binding function. The association between the mutant proαIIb and β3 was not disturbed. Surface labeling and pulse chase study showed that the Gln747→Pro substitution moderately impaired both intracellular transport of the αIIbβ3 heterodimers to the Golgi apparatus and endoproteolytic cleavage of proαIIb into heavy and light chains. By contrast, replacement of Gln747 with Ala by mutagenesis did not impair αIIbβ3expression on the cell surface. These results suggest that the presence of Pro, rather than the absence of Gln, at amino acid residue 747 on αIIb is responsible for moderate αIIbβ3 deficiency.© 1998 by The American Society of Hematology.