Identification of a region in glycoprotein IIIa involved in subunit association with glycoprotein IIb: further lessons from Iraqi-Jewish Glanzmann thrombasthenia

The most frequent mutation causing Glanzmann thrombasthenia in IraqI-Jews ( IJ-1) is an 11-bp deletion in exon 13 of the glycoprotein (GP) IIIa gene. T his deletion predicts a frameshift that results in the elimination of the C 406-C655 disulfide bond and a premature termination codon shortly before th e transmembrane domain. To determine the contribution of each of these alte rations to the thrombasthenic phenotype, Chinese hamster ovary or baby hams ter kidney cells were cotransfected with normal GPIIb complementary DNA (cD NA) and the following GPIIIa cDNAs: normal, cDNA bearing IJ-1 mutation, 201 1T >A mutated cDNA predicting C655S (single-letter amino acid codes) substi tution, and 2019A >T mutated cDNA predicting Stop657. Elimination of the C4 06-C655 disulfide bond by C655S substitution did not affect GPIIb/IIIa surf ace expression or binding of the transfected cells to immobilized fibrinoge n, whereas elimination of the transmembrane and cytoplasmic domains in IJ-1 and Stop657 mutants prevented both surface expression and binding of the t ransfected cells to immobilized fibrinogen. Immunohistochemical staining an d immunoprecipitation demonstrated that the elimination of amino acids 657- 762 in IJ-1 and Stop657 prevented intracellular GPIIb/IIIa complex formatio n, and differential immunofluorescence staining of GPIIIa and cellular orga nelles suggested that the truncated uncomplexed GPIIIa protein was retained in the endoplasmic reticulum. Because the use of GPIIIa Stop693 and normal GPIIb cDNAs yielded GPIIb/IIIa complex formation, though with lower effici ency, it is suggested that amino acids 657-692 of GPIIIa are essential for the intracellular association of GPIIb and GPIIIa. (C) 2001 by The American Society of Hematology.