Investigating the clearance of VWF A-domains using site-directed PEGylation and novel N-linked glycosylation
Background: Previous studies have demonstrated that the A1A2A3 domains of von Willebrand factor (VWF) play a key role in regulating macrophage-mediated clearance in vivo. In particular, the A1-domain has been shown to modulate interaction with macrophage low-density lipoprotein receptor-related protein-1 (LRP1) clearance receptor. Furthermore, N-linked glycans within the A2-domain have been shown to protect VWF against premature LRP1-mediated clearance. Importantly, however, the specific regions within A1A2A3 that enable macrophage binding have not been defined.
Objective and methods: To address this, we utilized site-directed PEGylation and introduced novel targeted N-linked glycosylation within A1A2A3-VWF and subsequently examined VWF clearance.
Results: Conjugation with a 40-kDa polyethylene glycol (PEG) moiety significantly extended the half-life of A1A2A3-VWF in VWF-/- mice in a site-specific manner. For example, PEGylation at specific sites within the A1-domain (S1286) and A3-domain (V1803, S1807) attenuated VWF clearance in vivo, compared to wild-type A1A2A3-VWF. Furthermore, PEGylation at these specific sites ablated binding to differentiated THP-1 macrophages and LRP1 cluster II and cluster IV in-vitro. Conversely, PEGylation at other positions (Q1353-A1-domain and M1545-A2-domain) had limited effects on VWF clearance or binding to LRP1.Novel N-linked glycan chains were introduced at N1803 and N1807 in the A3-domain. In contrast to PEGylation at these sites, no significant extension in half-life was observed with these N-glycan variants.
Conclusions: These novel data demonstrate that site specific PEGylation but not site specific N-glycosylation modifies LRP1-dependent uptake of the A1A2A3-VWF by macrophages. This suggests that PEGylation, within the A1- and A3-domains in particular, may be used to attenuate LRP1-mediated clearance of VWF.
CommentsThis is the peer reviewed version of the following article:, Fazavana J. et al. Investigating the clearance of VWF A-domains using site-directed PEGylation and novel N-linked glycosylation. J Thromb Haemost. 2020;18(6):1278-1290, which has been published in final form at https://doi.org/10.1111/jth.14785 This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.
Published CitationFazavana J. et al. Investigating the clearance of VWF A-domains using site-directed PEGylation and novel N-linked glycosylation. J Thromb Haemost. 2020;18(6):1278-1290
Publication Date28 February 2020
- Irish Centre for Vascular Biology
- School of Pharmacy and Biomolecular Sciences
- Vascular Biology
- Health Professions Education
- Immunity, Infection and Inflammation
- Accepted Version (Postprint)