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Unfolding of the von Willebrand factor A2 domain

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Title: Unfolding of the von Willebrand factor A2 domain
Authors: Lynch, Christopher
Item Type: Thesis or dissertation
Abstract: von Willebrand factor (VWF) is a multidomain glycoprotein that has well-established functions in haemostasis and thrombosis. The lack of a constraining disulphide bond results in unfolding of its VWF A2 domain under rheological forces in the vasculature. This unfolding of the VWF A2 domain is an important aspect of VWF function, as it facilitates the proteolysis and regulation by the plasma metalloprotease ADAMTS13. In this thesis, I investigate the structural elements of the VWF A2 domain that control its transition from folded to unfolded state. Dye binding measurements made during the temperature-induced unfolding of a recombinant VWF A2 domain fragment revealed that its calcium binding site (CBS) and vicinal disulphide bond (VicCC) both stabilise the VWF A2 domain and do so in a cooperative manner. Disruption of the CBS and VicCC in full-length recombinant VWF (FLVWF) results in its spontaneous cleavage by ADAMTS13. Temperature-induced unfolding of the VWF A2 domain was also used to examine the role of N-glycosylation on VWF A2 domain stability. Production and analysis of truncated glycan variants of the VWF A2 domain indicated that the N1574 glycan most likely stabilises the VWF A2 domain through an interaction of its first sugar moiety (GlcNAc) with VWF A2 residue Y1544. Stabilisation provided by N1574-GlcNAc impedes interaction with ADAMTS13. The triad of structural features of the VWF A2 domain, the CBS, VicCC and N1574-glycan, therefore collectively provides folding stability. I have also begun an investigation into the consequence of genetic mutation of residues within the VWF A2 domain. I introduced 34 mutations (that naturally occur and are described within the von Willebrand Disease, VWD, mutation database) into FLVWF, the isolated VWF A2 domain, an A1A2A3 domain fragment and a newly developed VWF A2 FRET construct. Preliminary results suggest that the latter construct may be informative in understanding the molecular basis of a subtype of VWD. Collectively, my findings help to clarify how the functions of VWF are controlled by conformational changes in the VWF A2 domain.
Content Version: Open Access
Issue Date: Feb-2017
Date Awarded: Jun-2017
URI: http://hdl.handle.net/10044/1/48000
DOI: https://doi.org/10.25560/48000
Supervisor: Lane, David
Sponsor/Funder: British Heart Foundation
Funder's Grant Number: FS/14/21/30733
Department: Department of Medicine
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:Medicine PhD theses

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