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Title: The role of O-linked glycosylation in VWF function
Author: Nowak, Agata Anna
ISNI:       0000 0004 2705 1123
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2011
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During synthesis Von Willebrand Factor (VWF) undergoes O-linked glycosylation (OLG) but the functional significance of this has not been fully explored. In this study the 10 OLG sites in VWF were mutated to alanine, individually and as clusters on either or both sides of the A1 domain: Clus-1 (N-terminal side), Clus-2 (C-terminal side) and double cluster (DC). All mutants demonstrated normal expression, multimeric structure and collagen and heparin binding functions. Furthermore, mass spectroscopy analysis showed that the OLG structures presented on recombinant VWF were similar in structure to those on plasma derived VWF including the presence of a rare disialosyl motif. The variants T1486 (C-terminal side of the A1 domain), Clus-2 and DC were less susceptible to ADAMTS13 proteolysis under static and pseudo-shear stress conditions and showed increased binding to ADAMTS13 when in solution. Using an optimised plate-based ELISA assay, it was demonstrated that mutation of the OLG sites on the N-terminal side of the VWF-A1 domain enhanced sensitivity to ristocetin-mediated binding to recombinant GPIbα. Increased GPIbα binding was also observed with desialylated VWF and this effect was attributed to sialic acid residues on the OLGs. Similar results were obtained using the conventional ristocetin cofactor assay. To further investigate these findings, an in vitro flow assay was optimised to analyse the interaction of VWF and platelets under shear stress. Mutation of the OLG sites on the N-terminal side of the A1 domain, increased platelet rolling velocity but did not significantly alter the dissociation rate of the VWF-A1:GPIbα bond. However, they did decrease the number of transient tethering events as a function of time when VWF was immobilised directly on to the flow surface. Interestingly, when compared with wtVWF the T1255A and Clus-1 variants mediated increased platelet capture to collagen under high shear stress. The data presented in this thesis demonstrates that the OLG clustered at the C-terminal side of the A1 domain alter susceptibility of VWF to ADAMTS13 cleavage. Whilst OLGs clustered at the N-terminal side of A1 modulate its interaction with GPIbα both under static and shear conditions. A unifying hypothesis would be that VWF OLG alter conformational stability around the A1 domain and modulate its interactions with adjacent domains and with other molecules. However different effects may predominate under static and flow conditions. Importantly, VWF-platelet interaction in flow assays depends on the way VWF has been immobilised.
Supervisor: Laffan, Mike ; McKinnon, Tom Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral