Structural Analysis of Four Newly Identified Extracellular Membrane-Proximal Missense Mutations in Patients with Glanzmann Thrombasthenia

Background: Glanzmann thrombasthenia (GT), an autosomal recessive inherited platelet disorder, is a moderate to severe bleeding syndrome caused by the absence of platelet aggregation due to quantitative and/or qualitative deficiencies of the αIIbβ3 integrin. We recently identified 41 causative missense mutations of which 24 were novel in a large cohort of 76 GT families (Genoscope project). These mutations mainly localize to the headpiece region of the integrin that has been well studied but 4 mutations although extracellular were proximal to the plasma membrane. We therefore performed molecular modeling of these 4 mutations to obtain new insights into the structure of a poorly understood region of this unique receptor.

Aim: To identify structures or conformations engaged in the stability of the integrin and which are important for maturation and expression.

Results: Of the 4 novel selected mutations, 3 concerned the calf-2 domain of αIIb - Gly792Glu (G823E, nomenclature with leader sequence), Leu924Gln (L955Q) and Thr953Lys (T984K) and one the EGF-3 domain of β3 Gly540Asp (G566D). All of these mutations affected highly conserved amino acids and were predicted to be damaging by in silico analysis (SIFT, Polyphen). None influenced glycosylation or mRNA splicing. They were present either in a homozygous form (β3 G540D) or were heterozygous in association with an identified and proven null mutation. Three were associated with type I GT (<5% αIIbβ3), while the αIIbG792E mutation occurred in a patient with type II GT (with 10% residual αIIbβ3) whose much reduced but partial transport to the surface was confirmed following expression of the recombinant integrin in CHO cells (with pro-αIIb predominating in the cytoplasm). The structural implications of these amino acid substitutions was assessed using PyMol Molecular Graphics System version 1.3 (www.pymol.org) based on the crystallographic data of αIIbβ3 in the bent non-activated state (3fcs PDB file). Amino acids were visualized in the rotamer form showing side change orientations incorporated from the Dunbrack Backbone library with the maximum probability. We first determined that the αIIb calf-2 domain has a β barrel-like structure largely composed of hydrophobic amino acids whose side chains orientate towards the inner cavity. Interestingly, L924Q and T953K substitutions occur at or adjacent to a conserved motif consisting of five polar amino acids central to the β barrel protected from H₂O molecules and involved in H-bond interactions. This particular motif, specific to calf-2, may introduce rigidity close to the membrane. Both L924Q and T953K disrupt the β barrel motif and promote flexibility. G792E is situated between the calf-1 and calf-2 domains in an unstructured connecting loop between two adjacent β sheets. Its replacement by the larger negatively charged Glu introduces steric encumbrance and results in an increase of the angle formed by the two calf domains, probably leading to the straightening of the second distal part of the long arm of αIIb. The β3 G540D substitution is found in the EGF3 domain of β3 that occurs at the axe of the cysteine-rich domain of the β3 arm, facing the αIIb calf-1 and calf-2 domains in the intact integrin. This substitution with the introduction of a charged and larger amino acid results in a weaker link between the two β sheets of EGF-3 and a loss of H-bonds. The result is an increased fragility within the β3 arm structure notably at the site of two stacked aromatic amino acids (H539 and W553) with a moving apart of the β sheets.

Conclusions: We show that 4 novel missense mutations in the extracellular membrane-proximal domains of αIIb and β3 cause conformational changes in domains that control the overall structure of the newly formed integrin. They show how the structure of both domains is under tight quality control and that precisely defined conformations are indispensable for αIIbβ3 maturation.