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Mechanisms of microparticle release from human platelets 10.17863/CAM.47798 Wei, Hao Harper, Matthew Thomas 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. University of Cambridge 2020-01-25 Thesis Doctoral Doctor of Philosophy (PhD) en https://www.repository.cam.ac.uk/handle/1810/300724 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/c3ab5f26-409f-4e60-9efa-39cfaf2e06b9/download 188ffb1cad3bfc70597600424e2080bb https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/643a5c9b-8627-46e4-b2ce-a132363a41b5/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ Platelets Platelet-derived microparticles Extracellular vesicles Pharmacology Blood Drug Effects Signal Transduction