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.

N-linked glycans within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance

“…29,[32][33][34] In addition, although the molecular mechanisms involved were not elucidated, enhanced VWF clearance has also been associated with loss of specific O-linked glycans. 30,58 In this paper, we demonstrate that complete loss of N-linked glycan expression following PNGase F digestion also results in markedly enhanced macrophage-mediated VWF clearance. Interestingly, recent studies have reported that N-linked glycan expression on factor X also plays a key role in regulating interaction with macrophages.…”

A novel role for von Willebrand factor in the pathogenesis of experimental cerebral malaria

“…Previous studies have demonstrated that these polyclonal anti-human VWF antibodies cross-react with murine VWF and can be used to measure murine plasma VWF:Ag levels. 34,35 After blocking with 3% bovine serum albumin (BSA; Sigma-Aldrich) test samples were then added at appropriate dilutions. Bound murine VWF was detected using horseradish peroxidase-conjugated (HRP) rabbit anti-human VWF antibody (Dako).…”

Common and rare von Willebrand factor (VWF) coding variants, VWF levels, and factor VIII levels in African Americans: the NHLBI Exome Sequencing Project

“…46 Notably, site-directed mutagenesis of Ser1486 to Ala resulted in normal VWF synthesis and multimerization but decreased VWF hemostatic function. 47 On the other hand, the substitution of Ser with Leu at 1486 is predicted to be "benign" at the protein sequence conservation level, while the substitution of Ser1486 with Ala is predicted to be "possibly damaging" by Polyphen2. Therefore, the putative mechanism of effect of the Ser1486Leu variant on lowering VWF:Ag remains to be determined.…”