title: In vivo analysis of the role of O-glycosylations of von Willebrand factor.
abstract: The objective of this project was to study the function of O-glycosylations in von Willebrand factor (VWF) life cycle. In total, 14 different murine Vwf cDNAs mutated on one or several O-glycosylations sites were generated: 9 individual mutants, 2 doublets, 2 clusters and 1 mutant with all 9 murine glycosylation sites mutated (Del-O-Gly). We expressed each mutated cDNA in VWF deficient-mice by hydrodynamic injection. An immunosorbent assay with Peanut Agglutinin (PNA) was used to verify the O-glycosylation status. Wild-type (WT) VWF expressed by hepatocytes after hydrodynamic injection was able to bind PNA with slightly higher affinity than endothelial-derived VWF. In contrast, the Del-O-Gly VWF mutant did not bind PNA, demonstrating removal of O-linked glycans. All mutants displayed a normal multimeric pattern. Two mutants, Del-O-Gly and T1255A/T1256A, led to expression levels 50% lower than those induced by WT VWF and their half-life in vivo was significantly reduced. When testing the capacity of each mutant to correct the bleeding time of VWF-deficient mice, we found that S1486A, T1255A, T1256A and the doublet T1255A/T1256A were unable to do so. In conclusion we have shown that O-glycosylations are dispensable for normal VWF multimerization and biosynthesis. It also appears that some O-glycosylation sites, particularly the T1255 and T1256 residues, are involved in the maintenance of VWF plasma levels and are essential for normal haemostasis. As for the S1486 residue, it seems to be important for platelet binding as demonstrated in vitro using perfusion experiments.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale U770, Le Kremlin-Bicêtre, France.
authors.forename: Idinath
authors.initials: I
authors.lastname: Badirou
authors.forename: Mohamad
authors.initials: M
authors.lastname: Kurdi
authors.forename: Paulette
authors.initials: P
authors.lastname: Legendre
authors.forename: Julie
authors.initials: J
authors.lastname: Rayes
authors.forename: Marijke
authors.initials: M
authors.lastname: Bryckaert
authors.forename: Caterina
authors.initials: C
authors.lastname: Casari
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Lenting
authors.forename: Olivier D
authors.initials: OD
authors.lastname: Christophe
authors.forename: Cecile V
authors.initials: CV
authors.lastname: Denis
mesh_terms: D000818:Animals; D001760:Bleeding Time; D019556:COS Cells; D002522:Chlorocebus aethiops; D006031:Glycosylation; D006207:Half-Life; D051379:Mice; D009154:Mutation; D019887:Peanut Agglutinin; D055503:Protein Multimerization; D013927:Thrombosis / Q000209:etiology; D014842:von Willebrand Diseases / Q000378:metabolism*; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
chemical_list: D019887:Peanut Agglutinin; D014841:von Willebrand Factor