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Investigating the clearance of VWF A‐domains using site‐directed PEGylation and novel N‐linked glycosylation
Author(s) -
Fazavana Judicael,
Brophy Teresa M.,
Chion Alain,
Cooke Niamh,
Terraube Virginie,
Cohen Justin,
Parng Chuenlei,
Pittman Debra,
Cunningham Orla,
Lambert Matthew,
O'Donnell James S.,
O'Sullivan Jamie M.
Publication year - 2020
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/jth.14785
Subject(s) - pegylation , lrp1 , glycosylation , chemistry , glycan , von willebrand factor , biochemistry , in vivo , binding site , microbiology and biotechnology , biophysics , polyethylene glycol , biology , ldl receptor , platelet , immunology , glycoprotein , lipoprotein , genetics , cholesterol
Abstract 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.