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S.E. Ward et al.
mMGL2 resulted in a 3-fold increase in murine plasma VWF levels which was attributable to a significant decrease in clearance rate. Importantly, the magnitude of the increased in vivo VWF levels associated with com- bined MGL inhibition was greater than that reported fol- lowing inhibition of other VWF clearance receptors in mice (~2.5 fold vs. ~1.5 fold), suggesting that MGL plays an important role in regulating physiological clearance of VWF.
To further investigate how MGL interacts with VWF, we first investigated the roles of specific VWF domains. Our data demonstrate that the A1A2A3 domains of VWF are predominantly responsible for modulating MGL binding. Furthermore, studies using isolated A domains showed that the A1 domain plays a critical role in regu- lating the MGL interaction. Interestingly, the binding of both full-length and A1A2A3-VWF to MGL was marked- ly enhanced in the presence of ristocetin, suggesting that the MGL-binding site in A1 may not be fully accessible in normal globular VWF. This concept is in keeping with findings of previous studies that showed significantly increased VWF binding to macrophages in the presence of ristocetin, botrecetin or shear stress.14,16 From a biolog- ical perspective, these data suggest that any VWF circu- lating in an ‘active’ GpIb binding conformation will be cleared rapidly by macrophage MGL, which may be important in minimizing thrombotic risk. Importantly, our data further show that C-terminal A3-CK-VWF also
A
binds MGL. Although the binding was less than that observed with N-terminal D’A3-VWF, this observation suggests that additional MGL-recognition sites beyond the A1 domain may contribute to the MGL interaction.
Mass spectrometry studies have demonstrated signifi- cant and site-specific heterogeneity in the carbohydrate structures expressed on human pdVWF.34-37 Nevertheless, the majority of both the N- and O-linked glycans are capped with negatively-charged sialic acid residues. In this study, we demonstrated that specific loss of α2-3 linked sialylation from the O-linked glycans of VWF causes enhanced MGL binding in vitro, and causes markedly enhanced MGL-mediated clearance in vivo. In contrast, removal of α2-6 linked sialylation, which con- stitutes most of the total sialic acid expressed on human VWF and, in particular, the vast majority of the sialyla- tion on N-glycans, has minimal effect on MGL binding and/or clearance. Our data further suggest that the two O-linked glycan clusters located either side of the A1 domain play a key role in regulating binding to MGL. Previous studies have demonstrated that these O-glycan clusters have significant effects on local VWF conforma- tion.52,53 Further studies will be required to determine the molecular mechanisms through which these specific O- glycans regulate MGL-mediated VWF binding and clear- ance. Nevertheless, our findings demonstrate that MGL contributes to physiological VWF clearance by binding to exposed Gal residues on O-linked carbohydrate struc-
Figure 6. ASGPR in combination with MGL modulates the increased clearance α2-3,6,8,9 Neu-VWF. (A) To investi- gate whether MGL plays a role in the enhanced clearance of von Willebrand factor (VWF) from which both the N- and O-sialylation had been removed purified human plasm- derived (pd)VWF was treated with α2-3,6,8,9 neu- raminidase (α2-3,6,8,9 Neu-VWF). In vivo clearance of α2- 3,6,8,9 Neu-VWF was then assessed in VWF-/- mice 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 α2-3,6,8,9 sialylation, 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. *P<0.05, **P<0.01, ns: not significant.
B
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haematologica | 2022; 107(3)