Glanzmann Mutations : New Allelic Variants and Role of the β3 Lys253 in the αIIb/β3 Interaction Highlighted by the Lys253Met Substitution.

Abstract

1312 Poster Board I-336 Introduction Glanzmann thrombasthenia (GT) is an autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation. GT results from defects of the platelet fibrinogen receptor αIIbβ3. GT mutations provide useful tools for structure-function relationship studies of αIIbβ3. Patient and methods Genomic DNA from 6 patients has been amplified for αIIb and β3 promoters and exons sequences. PCR products were directly sequenced. Potential RNA processing alterations have been studied in silico by using Genscan, NNSPLICE and ESEFinder online tools. When no RNA splicing anomaly was predicted, the effect of single point mutation on αIIbβ3 expression has been studied by using transiently transfected Cos cells. Finally, structural consequences of amino acid substitutions has been studied using the published model of αIIbβ3 (code PDB 2VDL) and structural modelling. Results 7 new mutations have been characterized. 1 deletion / insertion, 2 single point mutations inducing stop codon and 1 resulting in splicing site disruption were identified. The 3 last identified single point mutations were not predicted to affect normal RNA processing but has been shown to prevent normal expression of mutant αIIbβ3 at the surface of Cos cells. The p.Meth118Arg and p.Gly221Asp substitutions that induce both important steric hindrance and charge modifications, are located inside the β-I domain of β3. So they should deeply alter the proper folding of the β-I domain, preventing the complex expression at the platelet surface. On the other hand, the p.Lys253Met protrudes from the β-I domain toward the αIIb β-propeller. A structural model of the Met253 β-I mutant has been done. An estimation of the direct electrostatic and desolvation free energies of interaction between the β-I domain surface and the αIIb β-propeller indicated that rather than the presence of a methionine, it is the lost of the Lys253 which is responsible for the complex expression defect. Conclusion Seven new GT mutations have been identified and the p.Lys253Met substitution helped to define a key role of the Lys253 in the αIIb β-propeller / β3 β-I domains interaction. Disclosures No relevant conflicts of interest to declare.