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O-linked glycans protect VWF
clearance.14,15,19,20 To examine whether MGL may also play a role, we investigated binding for a number of type 1C (VWF-R1205H, R1205C, R1205S, S2179F) and type 2B (VWF-V1316M and -R1450E) variants. No evidence of enhanced MGL binding was observed for VWF-V1316M or any of the type 1C variants (Online Supplementary Figure S3). Interestingly, significantly reduced MGL bind- ing was seen for VWF-R1450E compared to wild-type recombinant VWF. We hypothesise that this change in binding is due to conformational effects within the A1 domain affecting O-linked glycosylation during post- translational modification and/or accessibility of specific OLG for the MGL interaction.
Platelet-von Willebrand factor sialylation and MGL interaction
Platelet α-granules contain approximatey 20% of the total VWF present in platelet-rich plasma.47,48 Previous studies have demonstrated that platelet-derived (plt)- VWF has altered glycosylation compared to pdVWF.48 In particular, plt-VWF does not express ABO blood group determinants and is hypo-sialylated.49,50 Importantly, these glycosylation differences influence susceptibility to ADAMTS-13 cleavage.43 Using lectin-binding ELISA, we confirmed that the quantitative reduction in plt-VWF sia- lylation was predominantly attributable to a specific reduction in N-linked sialylation (Figure 7A, B). As a result of this decreased N-sialylation, terminal galactose
A
expression was significantly increased on plt-VWF com- pared to pd-VWF (Figure 7C). Critically, despite the sig- nificant reduction in N-linked sialylation, we observed no increase in MGL binding for plt-VWF (Figure 7D). Moreover, in vivo clearance of plt-VWF in VWF-/- mice was similar to that of pd-VWF (Figure 7E). Cumulatively, these novel data further support our hypothesis that O- linked sialylation on VWF plays a key role in protecting VWF against MGL-mediated clearance.
Discussion
Recent studies have demonstrated that complex glycan structures, which account for 20% of total VWF monomeric mass, play a key role in regulating the half- life of VWF in vivo.3,25,51 In addition, a number of lectin receptors have been shown to bind VWF.3 Critically, however, the relative importance of these receptors in modulating physiological and pathological VWF clear- ance has not been defined. Moreover, the particular VWF glycan determinants involved in modulating interactions with specific lectin receptors remain unclear. In this study, using a series of in vivo and in vitro methodologies, we demonstrated that both murine homologs of the MGL receptor bind to VWF and contribute to the physi- ological clearance of endogenous murine VWF. Consequently, combined inhibition of both mMGL1 and
Figure 5. Role of MGL in modulating pathological enhanced clearance of α2-3 Neu-VWF. (A) To investigate the importance of MGL in regulating the enhanced clearance of von Willebrand fac- tor (VWF) with reduced O-linked sialylation, purified human plas- ma-derived (pd)VWF was treated with α2-3 neuraminidase (α2-3 Neu-VWF). In vivo clearance was then assessed in VWF-/- mice for α2-3 Neu-VWF in the presence or absence of combined mMGL1 and mMGL2 inhibition and compared to that of wild type pdVWF. At each time point, residual circulating VWF concentration was determined by an enzyme-linked immunosorbent assay for VWF:antigen (VWF:Ag). All results are plotted as percentage residual VWF:Ag levels relative to the amount injected. Data are represented as mean ± standard error of mean (SEM). In some cases, the SEM cannot be seen because of its small size. (B) To assess the relative roles of MGL and ASGPR in modulating the pathological increased clearance following removal of α2-3 sialy- lation, in vivo clearance studies were also performed in dual VWF-/-Asgr1-/- knockout mice in the presence or absence of com- bined mMGL1 and mMGL2 inhibition.
B
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