Two Different β3 Cysteine Substitutions Alter αIIbβ3 Maturation and Result in Glanzmann Thrombasthenia

 

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Summary

We report the defects responsible for Glanzmann thrombasthenia in two patients showing traces of abnormally migrating platelet β3 in immunoblotting. Using PCR-SSCP and direct sequencing, we identified a novel homozygous mutation in exon 10 of the β3 gene of patient 1 which gave a C⁴⁵⁷ to Y amino acid substitution. A C⁵⁴² to R substitution in β3 of patient 2 was previously reported by us. These cysteines are present in EGF-domains 1 and 3 respectively of β3. We therefore constructed mutants carrying substitutions on cysteine residues in each of the first three EGF domains of β3, C⁴⁵⁷, C⁴⁹⁵ and C⁵⁴² respectively. Transient expression of these mutants in COS-7 cells, including the C⁵⁴² and C⁵⁴⁷ double mutant, proved that disulfide disruption directly affects cell surface expression of the integrin. We then showed by metabolic (³⁵S) labeling and Endo-H glycosidase treatment that these substitutions strongly affected complex maturation within the cell.

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