Defining the Molecular Mechanisms through Which the Macrophage Galactose Lectin (MGL) Receptor Regulates Von Willebrand Factor Clearance

• Published in: Blood 2018-11, Vol.132 (Supplement 1), p.980-980
• Main Authors: Ward, Soracha E, O'Sullivan, Jamie M, Aguila Martinez, Sonia, Drakeford, Clive, McKinnon, Thomas Anthony Jude, Chion, Alain, O'Donnell, James S
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2018-99-114775

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 wit