Defining the mechanisms through which von willebrand factor sialic acid expression regulates in vivo clearance

Abstract

Von Willebrand Factor (VWF) is a complex plasma sialoglycoprotein which plays a key role in maintaining normal haemostasis. While the biosynthesis, structure, and functions of VWF are well characterised, the molecular mechanisms regarding VWF clearance remain poorly understood. Nonetheless, enhanced VWF clearance is of major importance in the pathophysiology underlying von Willebrand disease (VWD). The programme of work detailed in this thesis examined the role of VWF glycosylation and lectin receptors in modulating circulatory clearance. Previous studies have shown that loss of terminal sialic acid causes enhanced VWF clearance through the Asialoglycoprotein receptor (ASGPR). Importantly however a number of other lectin clearance receptors have also been implicated in regulating the clearance of hyposialylated glycoproteins. In initial studies, the specific importance of N- vs O-linked sialic acid in protecting against VWF clearance was investigated. Subsequently, a potential role for contributions by additional lectin receptors in modulating the reduced half-life of hyposialylated VWF was also studied. α2-3-linked sialic acid accounts for less than 20% of the total sialic acid expressed on VWF and is predominantly present on O-linked glycans. Nevertheless, specific digestion with α2-3 neuraminidase (α2-3Neu-VWF) was sufficient to cause markedly enhanced VWF clearance. Interestingly, in vivo clearance experiments in dual VWF-/-/Asgr1-/- mice demonstrated enhanced clearance of α2-3Neu-VWF even in the absence of the ASGPR. Collectively these findings suggest that O-linked glycan structures on VWF play an important role in protecting against premature clearance, and that other lectin receptors besides ASGPR are important in regulating this clearance. The macrophage galactose-type lectin (MGL) is a C-type lectin that binds to glycoproteins expressing terminal N-acetylgalactosamine or galactose residues. Importantly, the markedly enhanced clearance of hyposialylated VWF in VWF-/-/Asgr1-/- mice was significantly attenuated in the presence of an anti-MGL inhibitory antibody. Furthermore, dose-dependent binding of human VWF to purified recombinant human MGL was confirmed using surface plasmon resonance. Additionally, plasma VWF:Ag levels were significantly elevated in MGL1-/- mice compared with controls. Together, these findings identify MGL as a novel macrophage lectin receptor involved in VWF clearance in vivo. Importantly, these studies also demonstrate that MGL is important in regulating the clearance of both wild-type and hyposialylated VWF. Previous studies have reported that as glycoproteins age in plasma, there is progressive loss of terminal sialylation from N-linked glycans. Interestingly, in further studies we have shown that VWF ageing is also associated with hyposialylation. This loss of terminal sialic acid residues is modulated through neuraminidase enzymes present in normal plasma. Resulting in exposure of sub-terminal galactose, driving clearance through the ASGPR and MGL receptors. Interestingly, inhibition of plasma neuraminidases was associated with a significant prolongation in VWF half-life and a secondary increase in endogenous murine plasma VWF levels. These exciting data suggest that neuraminidase inhibition may offer a novel mechanism through which to increase plasma VWF levels in patients with VWD.