A Dinucleotide Deletion Results in Defective Membrane Anchoring and Circulating Soluble Glycoprotein Ibα in a Novel Form of Bernard-Soulier Syndrome

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Abstract
The platelet membrane glycoprotein (GP)Ib-V-IX complex is the receptor for von Willebrand factor and is composed of four membrane-spanning polypeptides: GPIbα, GPIbβ, GPIX, and GPV. A qualitative or quantitative deficiency in the GPIb-V-IX complex on the platelet membrane is the cause of the congenital platelet disorder Bernard-Soulier syndrome (BSS). We describe the molecular basis of a novel variant BSS in a patient in which GPIbα was absent from the platelet surface but present in a soluble form in the plasma. DNA sequence analysis showed a homozygous dinucleotide deletion in the codon for Tyr 508 (TAT) in GPIbα. This mutation (GPIbαΔAT) causes a frame shift that alters the amino acid sequence of GPIbα within its transmembrane region. The hydrophobic nature of the predicted transmembrane region and the cytoplasmic tail at the COOH terminal are altered before reaching a new premature stop codon 38 amino acids short of the wild-type peptide. Although GPIbαΔAT was not detectable on the platelet surface, immunoprecipitation of plasma with specific monoclonal antibodies (MoAbs) identified circulating GPIbα. Transient expression of recombinant GPIbαΔAT in 293T cells also generated a soluble form of the protein. Moreover, when a plasmid encoding GPIbαΔAT was transiently transfected into Chinese hamster ovary (CHO) cells stably expressing the GPβ-IX complex, it failed to be expressed on the cell surface. Thus, a dinucleotide deletion in the codon for Tyr 508 causes a frameshift that alters the amino acid sequence of GPIbα starting within its transmembrane region, changes the hydrophobicity of the normal transmembrane region, and truncates the cytoplasmic domain affecting binding to the cytoskeleton and cytoplasmic proteins. This mutation affects anchoring of the GPIbα polypeptide in platelets and causes the observed BSS phenotype with circulating soluble GPIbα.