Abstract
Introduction
Although the biological mechanisms underpinning VWF clearance remain poorly understood, accumulating data has shown that increased clearance is a common pathophysiology in type 1 VWD patients. Moreover, enhanced clearance has been implicated in the etiology of types 2 and 3 VWD. Previous studies have shown that loss of terminal sialic acid from VWF glycans results in enhanced clearance. Furthermore, ST3Gal-IV knockout mice exhibit reduced plasma VWF levels due to rapid in vivo clearance. Initial studies suggested that clearance of hyposialylated VWF was mediated via the asialoglycoprotein receptor (ASGPR). However, we recently reported a novel role for the Macrophage Galactose Lectin (MGL) in regulating clearance of VWF (Ward et al, Blood 2018). In this study, we sought to elucidate the molecular mechanisms through which MGL interacts with human VWF.
Methods
Recombinant VWF variants, truncated A1A2A3 and isolated A1, A2 and A3 domains were expressed in HEK293T cells. Human plasma derived (pdVWF) was purified from commercial VWF concentrates as before. VWF glycoforms were generated by digestion with exoglycosidases. VWF-MGL interaction was assessed using plate binding ELISA assays. In vivo clearance of VWF variants was assessed in both Asgr1+/+VWF-/-and Asgr1-/-VWF-/-mice in the presence/absence of anti-MGL blocking antibodies.
Results
Previous studies have shown 80% of total sialic acid on VWF is expressed on N glycans in α2-6 linkage. In contrast, the other 20% of VWF sialylation is α2-3 linked on O linked glycans. Removal O linked sialylation (with α2-3 neuraminidase) was sufficient to increase VWF clearance in VWF-/- and Asgr1-/-VWF-/-mice (half life = 9.0 ± 1 mins and 8.3 ± 2mins). Interestingly, in vivo clearance of α2-3 Neu VWF in VWF-/-andAsgr1-/-VWF-/-mice was almost as rapid as that of asialo-VWF (digested with α2-3,6,8,9 neuraminidase). Critically, the increased clearance of α2-3 Neu VWF in VWF-/- is attenuated in the presence of MGL blocking antibodies (120mins residual α2-3 Neu VWF is 10.6% ±2% , compared to 10.2% ±1% for that of pdVWF). Plate-binding studies confirmed that α2-3 Neu VWF and α2-3,6,8,9 Neu VWF demonstrated enhanced binding to MGL compared to pdVWF (155% and 134% versus 100%; p =0.017 and 0.006). In keeping with in vivo clearance data, removal of α2-6 linked sialic acid from N glycans did not further promote binding to MGL. Finally, although PNGase removal of VWF N glycans did not affect MGL binding, treatment with O glycosidase significantly attenuated binding (p<0.0001). All together, these data demonstrate that O-linked sialylation plays a critical role in protecting VWF from MGL mediated clearance.
The mature VWF monomer contains 10 O linked glycans, with eight clustered around the A1 domain. To further investigate the role of these glycans in regulating MGL interaction, site directed mutagenesis was used to generate VWF molecules lacking either O-linked cluster 1 (T1248, T1255, T1256, T1263) or O-linked cluster 2 (T1468, T1477, S1486, T1487) respectively. Binding of Δcluster 1 VWF to MGL was similar to that of wild-type VWF. In contrast, binding of the Δcluster 2 VWF variant to MGL was markedly reduced (84 % vs 40%; p=0.0027). O linked glycan structures are known to play important roles in maintaining glycoprotein conformation. To further define the mechanism through which these O glycan structures influence MGL mediated clearance, binding of a truncated A1A2A3 fragment and isolated recombinant A1, A2 and A3 domains were examined. Interestingly, MGL-binding to full length rVWF and A1A2A3 were both significantly increased in the presence of ristocetin. Interestingly, no binding of isolated A2 or A3 domains to MGL was observed, while A1 domain binding to MGL was seen both in the presence and absence of ristocetin.
Conclusions
In conclusion, these findings define a novel role for O linked sialylation in protecting VWF from MGL mediated clearance. Our data further demonstrate that the cluster of O linked glycans located at the C-terminal end of the A1 domain play a specific role in regulating VWF clearance via MGL, and that conformation of A1A2A3 may be important in determining accessibility of these glycans for the clearance receptor. Further studies will be required to determine whether abnormalities in these O-glycan determinants may be important in the pathophysiology of VWD, particularly in patients with type 1C VWD who lack VWF gene coding mutations.
Disclosures
O'Donnell: Bayer: Research Funding, Speakers Bureau; Novo Nordisk: Research Funding, Speakers Bureau; Leo Pharma: Speakers Bureau; Octapharma: Speakers Bureau; CSL Behring: Consultancy; Daiichi Sankyo: Consultancy; Pfizer: Consultancy, Research Funding; Baxter: Research Funding, Speakers Bureau; Shire: Research Funding, Speakers Bureau.
Functional Characterization of Brain Tumor-Initiating Cells: Implications for Preclinical Models and Drug Development
