Cheung, Yam Fung Hilaire 
ORCID: 0000-0003-0917-5918
(2023).
Integrating phosphoinositide metabolism, platelet functional data and computational modelling to decipher GPVI-signalling in platelet activation.
University of Birmingham.
Ph.D.
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Cheung2023PhD.pdf
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Abstract
Cardiovascular diseases are the leading cause of mortality worldwide, and platelets are critical in their pathophysiology. Current antiplatelet therapies come with a risk of bleeding, so treatments targeting new pathways that preserve haemostasis are needed. Two potential inter-related candidates being investigated in this thesis are collagen and fibrin(ogen) receptor GPVI, which has a major role in thrombosis but a minor one in haemostasis, and phosphoinositides and their associated kinase/phosphatases, which are involved in Ca\(^{2+}\) mobilisation and regulation of pleckstrin homology (PH) domains-containing proteins.
The key to efficient GPVI and phosphoinositides targeting is understanding the interconnections between the proteins and lipid intermediates. In this thesis, I have developed an ion chromatography-mass spectrometry (IC-MS) based method to differentiate the phosphoinositides’ positional isomers, particularly PtdIns(3,4)P\(_2\) and PtdIns(3,5)P\(_2\). This is followed by the development of a mathematical model that can simulate phosphoinositides metabolism upon GPVI activation. Our model predicts the effect of phosphatidylinositol 4-kinase A (PI4KA) inhibitor GSK-A1 on inositol triphosphate (InsP\(_3\)) and Ca\(^{2+}\) mobilisation, demonstrating its role on PtdIns(4,5)P\(_2\) resynthesis to sustain downstream signalling.
I further investigate the role of tyrosine kinase inhibitors, integrin αIIbβ3 and GPVI antagonists in GPVI activation and platelet aggregation. The results show that collagen-related peptide (CRP)-induced sustained tyrosine phosphorylation is reversed upon inhibition of GPVI signalling but not aggregation in light transmission aggregometry (LTA), and that platelet disaggregation depends on the choice of agonist and the presence of shear based on published data. I have developed a dimer and tetramer of the GPVI-blocking nanobody, nanobody 2 (Nb2). My results show that Nb2 dimer Nb2-2 is a more potent antagonist than Nb2 against GPVI, while Nb2 tetramer Nb2-4 acts as a potent GPVI agonist at the nanomolar range making it the first agonist for GPVI in human platelets of known stoichiometry. We have further developed an ordinary differential equation (ODE) based model to illustrate the relation of ligand valency with GPVI clustering and activation. Overall, this suggests that dimerisation of GPVI alone does not induce activation of human platelets, and a tetravalent but not a divalent ligand induces measurable GPVI clustering and activation. The dimeric ligand Nb2-2 has the potential to be developed into a viable antithrombotic. The tetravalent nanobody is the first agonist of known stoichiometry for GPVI and could be used to enable the comparison of results between laboratories and a clinically defined test for the study of platelet function.
| Type of Work: | Thesis (Doctorates > Ph.D.) | ||||||||||||||||||
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| Award Type: | Doctorates > Ph.D. | ||||||||||||||||||
| Supervisor(s): |
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| Licence: | All rights reserved | ||||||||||||||||||
| College/Faculty: | Colleges (2008 onwards) > College of Medical & Dental Sciences | ||||||||||||||||||
| School or Department: | Institute of Cardiovascular Sciences | ||||||||||||||||||
| Funders: | European Commission | ||||||||||||||||||
| Subjects: | Q Science > QD Chemistry Q Science > QP Physiology Q Science > QR Microbiology |
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| URI: | http://etheses.bham.ac.uk/id/eprint/13697 |
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