Comprehensive analysis of platelet glycoprotein Iba ectodomain glycosylation

Background: Platelet glycoprotein (GP) Ib & alpha; is the major ligand-binding subunit of the GPIb-IX-V complex that binds von Willebrand factor. GPIb & alpha; is heavily glycosylated, and its glycans have been proposed to play key roles in platelet clearance, von Willebrand factor binding, and as target antigens in immune thrombocytopenia syndromes. Despite its importance in platelet biology, the glycosylation profile of GPIb & alpha; is not well characterized.Objectives: The aim of this study was to comprehensively analyze GPIb & alpha; amino acid sites of glycosylation (glycosites) and glycan structures.Methods: GPIb & alpha; ectodomain that was recombinantly expressed or that was purified from human platelets was analyzed by Western blot, mass spectrometry glycomics, and mass spectrometry glycopeptide analysis to define glycosites and the structures of the attached glycans.Results: We identified a diverse repertoire of N-and O-glycans, including sialoglycans, Tn antigen, T antigen, and ABO(H) blood group antigens. In the analysis of the re-combinant protein, we identified 62 unique O-glycosites. In the analysis of the endogenous protein purified from platelets, we identified 48 unique O-glycosites and 1 N-glycosite. The GPIb & alpha; mucin domain is densely O-glycosylated. Glycosites are also located within the macroglycopeptide domain and mechanosensory domain. Conclusions: This comprehensive analysis of GPIb & alpha; glycosylation lays the foundation for further studies to determine the functional and structural roles of GPIb & alpha; glycans.