Metabolic isotopic labelling of plasma membrane glycoproteins from normal, Glanzmann's and Bernard-Soulier platelets

Abstract

Normal, Glanzmann and Bernard-Soulier platelets were incubated in homologous plasma with 14C-labeled glucosamine (GLU), galactose (GAL) or mannose (MAN). Time-dependence of sugar uptake by the whole cells, plasma membrane incorporation of the individual sugars, the electrophoretic pattern of the metabolically labeled plasma membrane glycoproteins, as well as the activity of the galactosyl- and mannosyl-transferases were compared. Results obtained show a moderate decrease in MAN uptake by Glanzmann platelets, while uptake of either GLU or GAL was similar to controls. Bernard-Soulier platelets show a significant increase in GLU and MAN uptake, while only a moderate increase is observed for GAL. Plasma membrane incorporation of all three sugars is markedly decreased in Bernard-Soulier, however, only MAN incorporation is decreased in Glanzmann. SDS-PAGE (reduced) of plasma membranes resolves from 10 to 14 labeled glycopeptides. For most of the resolved glycopeptides, marked quantitative differences were found between Glanzmann, Bernard-Soulier and normal platelets. Galactosyl- and mannosyl-transferase activity towards endogenous substrates was found to be greatly diminished in both platelet disorders. Taken as a whole, our results strongly suggest that, instead of the lack of one or two specific glycoproteins, as has been reported, a more general perturbation of glycoprotein synthesis, centered on the glycosylating enzymes, seems to be the molecular basis for the platelet malfunction in both, Glanzmann disease and Bernard-Soulier syndrome.