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Title: Extended D-E knob-hole interaction sites in fibrin polymerisation, clot formation and clot mechanics
Author: Asquith, Nathan Lee
ISNI:       0000 0004 7655 1562
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2019
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Background: Abnormal thrombus formation and occlusion of a vessel is one of the main events of cardiovascular disease. A main component of the thrombus is the protein fibrin, which is formed by proteolytic cleavage of its precursor fibrinogen by the serine protease thrombin. The cleavage of fibrinogen to fibrin releases fibrinopeptides from the E-region of the molecule which leads to the exposure of peptide sequences termed knobs A and B. The knobs A and B on one fibrin molecule are able to spontaneously interact to binding pockets (termed holes a and holes b) via hydrogen bonds forming half staggered protofibrils. These protofibrils laterally aggregate and form fibrin fibers, providing the clot its strength and stability. Recent molecular dynamic simulations have predicted that there are additional interactions involving amino acids gGlu323 with BLys58, gLys356 with BAsp61, and gAsp297 with BHis67, that surround the binding pocket and provide additional strength and stability to the 'classical' knob-hole contact. In this project I have termed these residues 'extended D-E knob-hole binding sites.' Aim: The aim of this project was to probe the importance of these extended knob-hole interactions in the process of fibrin polymerisation, clot structure and clot mechanics, using recombinant fibrinogen variants with mutations that abolish these electrostatic interactions. Methods: Four recombinant human fibrinogen variants and WT proteins were produced. The following variants with single point mutations in the g-chain of extended knob-hole binding region were produced: gD297N, gE323Q and gK356Q. A triple variant, gDEK (gD297N/gE323Q/gK356Q) with mutations in all residues involved was also produced. Each variant was tested for integrity by circular dichroism and SDS-PAGE. Turbidity and atomic force microscopy were used to study polymerisation kinetics, laser scanning confocal microscopy and scanning electron microscopy were used to study clot structure. Light scattering methods were used to study intrafibrillar protein structure, and clot mechanics was studied using an in-house micro-rheometer. Results: Longitudinal protofibril growth was disrupted for all variants except gK356Q at early stages of polymerisation, but normalised at later time points. Vmax was reduced for all variants. gDEK and gE323Q produced denser clots, whereas gD297N and gK356Q were similar to WT. All variant clots had significantly thinner fibers compared to WT. All variants were slower to lyse, with the exception of gD297N. Clot visco-elastic analysis showed that γDEK was more readily deformable (loss tangent, tanδ), at low frequencies but single mutant variants were unchanged at all frequencies compared to WT. Conclusion: I produced pure and intact recombinant human fibrinogens with mutations at the extended knob-hole binding sites. These data provide clear evidence for the role of extended D-E interactions in supporting the classical knob-hole binding during fibrin formation. Furthermore, the extended D-E interactions were shown to alter clot structure and clot mechanics. Additional studies with these variants in the presence of cells and other vascular components may further elucidate the importance of extended knob-hole interactions in haemostasis and thrombosis.
Supervisor: Ariens, Robert ; Duval, Cedric ; Connell, Simon Sponsor: British Heart Foundation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available