A Gln747→Pro Substitution in the IIb Subunit Is Responsible for a Moderate IIbβ3Deficiency in Glanzmann Thrombasthenia

Abstract

To clarify a molecular defect responsible for moderate IIbβ3 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 IIb 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 Pro747IIb and β3 constructs into 293 cells resulted in moderate reduction in the amount of IIbβ3 within the transfected cells as well as on the cell surface. However, Pro747IIbβ3 bound the ligand mimetic monoclonal antibody (MoAb) PAC-1 after activation of IIbβ3 by the MoAb PT25-2, suggesting that the mutant IIbβ3 possesses the ligand-binding function. The association between the mutant proIIb and β3 was not disturbed. Surface labeling and pulse chase study showed that the Gln747→Pro substitution moderately impaired both intracellular transport of the IIbβ3 heterodimers to the Golgi apparatus and endoproteolytic cleavage of proIIb into heavy and light chains. By contrast, replacement of Gln747 with Ala by mutagenesis did not impair IIbβ3expression on the cell surface. These results suggest that the presence of Pro, rather than the absence of Gln, at amino acid residue 747 on IIb is responsible for moderate IIbβ3 deficiency.© 1998 by The American Society of Hematology.