BACKGROUND: Studies of Glanzmann thrombasthenia (GT)-causing mutations has generated invaluable information on the formation and function of integrin áIIbâ(3).OBJECTIVE: To characterize the mutation in four siblings of an Israeli Arab family affected by GT, and to analyze the relationships between the mutant protein structure and its function using artificial mutations.METHODS AND RESULTS: Sequencing disclosed a new A97G transversion in the áIIb gene predicting Asn2Asp substitution at blade 1 of the â-propeller. Alignment with other integrin á subunits revealed that Asn2 is highly conserved. No surface expression of áIIbâ(3) was found in patients' platelets and baby hamster kidney (BHK) cells transfected with mutated áIIb and WT â(3). Although the áIIbâ(3) was formed, the mutation impaired its intracellular trafficking. Molecular dynamics simulations and modeling of the áIIbâ(3) crystal indicated that the Asn2Asp mutation disrupts a hydrogen bond between Asn2 and Leu366 of a calcium binding domain in blade 6, thereby impairing calcium binding that is essential for intracellular trafficking of áIIbâ(3). Substitution of Asn2 to uncharged Ala or Gln partially decreased áIIbâ(3) surface expression, while substitution by negatively or positively charged residues completely abolished surface expression. Unlike áIIbâ(3), áVâ(3) harboring the Asn2Asp mutation was surface expressed by transfected BHK cells, which is consistent with the known lower sensitivity of áVâ(3) to calcium chelation compared with áIIbâ(3).CONCLUSION: The new GT causing mutation highlights the importance of calcium binding domains in the â-propeller for intracellular trafficking of áIIbâ(3). The mechanism by which the mutation exerts its deleterious effect was elucidated by molecular dynamics.
Adolescent Amino Acid Sequence