Title:
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Factors affecting the viability of human platelets
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Human platelets die in vitro in a caspase-independent manner with features of necrosis. Little is known about the factors affecting the mode of platelet death nor how death is brought about. This thesis aimed to generate novel tools for the discrimination of live from dead platelets and to evaluate the role of autophagy, temperature and sugar metabolism in the demise of platelets in vitro. Three dyes, 10-nonyl acridine orange, calcein and FM 4-64 were found to be useful for the evaluation of platelet viability. It was found that during in vitro storage dead platelets formed metalloproteinase-dependent aggregates and shed CD42b. Platelets died more slowly at ambient temperature than at 37°C and appeared to arrest in death when kept at 4°C. Cold-stored platelets remained viable for ten days but whereupon transfer to 37°C they died rapidly. Platelets did not rely on exogenous glucose for viability but hypoglycaemia sensitised them to pro-apoptotic stimuli. Inhibitors of glycogen breakdown were toxic to platelets, implicating glycogen in the maintenance of platelet viability in vitro. Autophagy could not be implicated in the loss of platelet viability but data suggested a role for crinophagy in the maintenance of platelet function.
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