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Title: The role of cysteine residues and thiol-disulphide exchange in the expression and function of von Willebrand factor
Author: Shapiro, Susan Elizabeth
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Thiol-disulphide reactions are critical to the structure and function of von Willebrand factor (VWF). Intracellular multimerisation is catalysed by intrinsic thiol isomerase activity of CGLC motifs in its propeptide. Conserved CG(L/I)C motifs were identified in the D3, D4 and C1 domains which may possess thiol isomerase activity and contribute to intracellular synthesis of VWF or to extracellular thiol-disulphide exchange. Nine cysteine residues are unpaired in a proportion of plasma VWF molecules and are hypothesised to participate in disulphide-mediated self-association. This thesis aimed to study VWF self-association, the participating individual cysteines and the intrinsic isomerase activity of VWF. To study the unpaired cysteines, point mutants and C domain deletion mutants (where most of these cysteines are located) were made. To study the role of the conserved CG(L/I)C motifs, two complementary types of mutant were made: replacement of D2 domain (known isomerase activity) with either the D3 or D4 domain; and insertion of glycine between vicinal cysteines (known to disrupt isomerase function). Mutants were expressed in HEK293T cells. Mutants which were retained intracellularly were studied by measuring lysate pro-VWF and by EndoH digestion. Secreted mutants were studied by quantifying expression, static collagen binding activity and free thiol content. Mutant VWF function was assessed by measuring its ability to capture platelets when perfused over collagen under shear stress. The lateral self-association of VWF was studied under both static and flow conditions using plasma-derived and recombinant VWF. The results demonstrate that the individual cysteine residues and the C domains of VWF play a crucial role in the folding of VWF during synthesis. Furthermore, the CGLC motif in D3 plays a critical role in the synthesis of VWF. However, extracellular self-association of VWF does not appear to play a significant role in the capture of platelets to collagen under physiological flow conditions.
Supervisor: Laffan, Mike Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available