title: Glanzmann thrombasthenia due to a two amino acid deletion in the fourth calcium-binding domain of alpha IIb: demonstration of the importance of calcium-binding domains in the conformation of alpha IIb beta 3.
abstract: The integrin alpha IIb beta 3, a calcium-dependent heterodimer, plays a critical role in platelet aggregation. The alpha IIb subunit of the heterodimer contains four highly conserved putative calcium-binding domains in its extracellular portion. During studies of the molecular basis of Glanzmann thrombasthenia in a child of mixed Caucasian background whose platelets expressed little alpha IIb beta 3 on their surface, we found the patient heterozygous for a two amino acid deletion in the fourth alpha IIb calcium-binding domain. When this alpha IIb mutant was expressed in COS-1 cells, we found that the deletion did not interfere with the assembly of alpha IIb beta 3 heterodimers, but altered their conformation such that they were neither recognized by the heterodimer-specific antibody A2A9 nor able to undergo further intracellular processing or transport to the cell surface. These results suggest that the calcium-binding domains in alpha IIb play an important role maintaining the overall conformation of alpha IIb beta 3. To confirm this suggestion, we deleted each of the four 12 amino acid calcium-binding domains in alpha IIb by in vitro mutagenesis and expressed the mutants along with beta 3 in COS-1 cells. Each construct formed a heterodimer with beta 3, but none of the heterodimers interacted with A2A9 or underwent further intracellular processing. These data indicate that the calcium-binding domains in alpha IIb are not involved in alpha IIb beta 3 heterodimer formation, but their presence is required for the intracellular transport of alpha IIb beta 3 to the cell surface.
authors.affiliation: Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, USA.
authors.forename: R B
authors.initials: RB
authors.lastname: Basani
authors.forename: G
authors.initials: G
authors.lastname: Vilaire
authors.forename: S J
authors.initials: SJ
authors.lastname: Shattil
authors.forename: M A
authors.initials: MA
authors.lastname: Kolodziej
authors.forename: J S
authors.initials: JS
authors.lastname: Bennett
authors.forename: M
authors.initials: M
authors.lastname: Poncz
mesh_terms: D000328:Adult; D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D016824:Antigens, Human Platelet / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport; D001792:Blood Platelets / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D008969:Molecular Sequence Data; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013915:Thrombasthenia / Q000235:genetics*; D014162:Transfection
chemical_list: D016824:Antigens, Human Platelet; D000939:Epitopes; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins; D002118:Calcium