Intact N-glycopeptide analysis of human platelets reveals a Glycostructure important for platelet function.

Glycosylation is an important posttranslational modification in platelets, and the glycosylation pattern is critical for platelet function. To date, the exploration of the roles of various glycoforms in specific platelet functions is largely lacking. In this study, a global analysis of intact N-glycopeptides in human platelets was performed to map all the glycopeptides, glycosites and glycans of platelets. The glycopeptides were enriched by the ZIC- hydrophilic interaction chromatography method and then analyzed by Liquid Chromatography-Tandem Mass Spectrometry analysis. A total of 1,425 intact glycopeptides belonging to 190 N-glycoproteins from human platelets were identified. Moreover, 358 glycans modified 328 glycosites from those glycoproteins. Functional analysis revealed that these glycoproteins are involved mainly in processes and pathways related to platelet adhesion. Among the proteins in these adhesion-related annotations, von Willebrand factor, thrombospondin 1and glycoprotein V were found to contain a possible Lewis y structure, and this finding was further verified by immunoprecipitation assays. As a blood group-related antigen, Lewis y was previously reported to exist in human platelets, but its function remains unclear. Since the glycosylation of von Willebrand factor, thrombospondin 1 and glycoprotein V is involved in platelet-collagen adhesion, the importance of Lewis y on platelet function was evaluated by adhesion assays, which demonstrated that the blockade of Lewis y on platelets decreased the adhesion of platelets to collagen I under both static and flow conditions.