O-Linked Glycosylation with Sialylated T-Antigen: A Novel Carbohydrate Determinant of von Willebrand Factor Antigen Levels.

Abstract

The glycosylation-profile of von Willebrand factor (VWF) is known to strongly influence its plasma levels. VWF contains several carbohydrate structures, including O-linked glycans that primarily consist of the sialylated T-antigen (NeuAc( α 2–3)Gal( β 1–3)[NeuAc( α 2–6)]GalNAc). It is unknown yet if O-linked carbohydrates affect VWF levels. We therefore developed an immunosorbent-based assay using the lectin Peanut Agglutinin (PNA) to determine O-linked T-antigen on VWF. Control experiments showed that PNA-binding was: (i) specific to T-antigen that was de-sialylated via neuramidase-treatment; (ii) proportional to the number of O-linked glycosylation sites and (iii) independent of the extent of multimerisation. Using this assay, we found a strong correlation between PNA binding and VWF-antigen levels in a series of randomly selected plasma samples (252±125% for VWF<0.5 U/ml (n=15); 131±36% for VWF between 0.5 and 1.5 U/ml (n=32); 92±40% for VWF>1.5 U/ml (n=19); p<0.003). Reduced or increased PNA binding was also observed for patients characterized by increased (liver cirrhosis; n=58) or reduced (von Willebrand disease (VWD)-type 1; n=32) VWF-antigen levels, respectively. VWD-type 1 patients further displayed increased ratios of propeptide over mature VWF-antigen levels (0.38±0.18 versus 0.17±0.03 for patients (n=32) and controls (n=20), respectively; p<0.0001), which is indicative for reduced VWF survival in these patients. Interestingly, a linear relation between PNA binding and propeptide/VWF ratio was observed (Spearman rank= 0.50; p=0.0039), suggesting a potential association between T-antigen glycosylation and VWF survival. Finally, we observed a marked decrease in PNA binding in post-DDAVP samples from various patients (131±45% versus 85±33% in pre- and post DDAVP, respectively; n=18; p=0.0013). Since these patients had distinct underlying pathologies (primary platelet function disorder, hemophilia A or VWD type 1, 2A or 2N), this seems to represent a general phenomenon that is not restricted to VWD. Moreover, it indicates that the O-linked glycosylation profile of VWF stored in endothelial storage-organelles may differ from that of constitutively secreted VWF.