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Title: Mechanisms of microparticle release from human platelets
Author: Wei, Hao
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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Platelets release a variety of extracellular vesicles, including platelet-derived microparticles that expose phosphatidylserine on their surface. This thesis aims to resolve the mechanisms by which pro-thrombotic microparticles are released from platelets, which have not been well-characterised. Experimental methods used include flow cytometry, Western blotting, fluorescence-based microplate assays, confocal microscopy and electron microscopy. Intact cholesterol-rich lipid rafts were found to be required for the calpain-dependent release of microparticles from activated platelets. Microparticle release was prevented when cholesterol was depleted or sequestered from the platelet membrane. Membrane blebbing and scission in this process were independent of influx of hydrophilic ions or the endosomal sorting complex required for transport. It was found that microparticles were also released from platelets undergoing apoptosis, which would progress to secondary necrosis in vitro as they were not cleared. Microparticle release in apoptotic platelets was dependent on Ca2+ entry, intracellular Ca2+ mobilisation and caspase activity, but it was largely independent of calpain. During apoptosis, caspases downregulated microparticle release from platelets in response to pro-coagulant stimuli. This may limit the pro-thrombotic consequences by providing a period of reduced capacity for platelet activation. 2-Aminoethoxydiphenyl borate (2-APB), a non-specific modulator of ion channels, was found to inhibit microparticle release from both activated and apoptotic platelets. In activated platelets, the effect of 2-APB was not related to inhibition of plasma membrane ion channels or calpain activity. In apoptotic platelets, the effect of 2-APB might be associated with inhibition of Ca2+ entry, although the specific target remained unclear. Future studies in identifying the target of 2-APB might provide new insights into how microparticles are released from platelets. Moreover, 2-APB may provide a scaffold for developing a pharmacological inhibitor of microparticle release.
Supervisor: Harper, Matthew Thomas Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Platelets ; Platelet-derived microparticles ; Extracellular vesicles ; Pharmacology ; Blood ; Drug Effects ; Signal Transduction