Glanzmann thrombasthenia due to a two amino acid deletion in the fourth calcium-binding domain of α(IIb): Demonstration of the importance of calcium-binding domains in the conformation of α(IIb)β3

Abstract

The integrin α(IIb)β3, a calcium-dependent heterodimer, plays a critical role in platelet aggregation. The α(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 α(IIb)β3 on their surface, we found the patient heterozygous for a two amino acid deletion in the fourth α(IIb) calcium- binding domain. When this α(IIb) mutant was expressed in COS-1 cells, we found that the deletion did not interfere with the assembly of α(IIb)β3 heterodimers, but altered their conformation such that they were neither recognized by the heterodimer-specific antibody A2A9 nor able to undergo further intracellular processing ortransport to the cell surface. These results suggest that the calcium-binding domains in α(IIb) play an important role maintaining the overall conformation of α(IIb)β3. To confirm this suggestion, we deleted each of the four 12 amino acid calcium-binding domains in α(IIb) by in vitro mutagenesis and expressed the mutants along with β3 in COS-1 cells. Each construct formed a heterodimer with β3, but none of the heterodimers interacted with A2A9 or underwent further intracellular processing. These data indicate that the calcium-binding domains in α(IIb) are not involved in α(IIb)β3 heterodimer formation, but their presence is required for the intracellular transport of α(IIb)β3 to the cell surface.