The ability to readily identify dead platelets is invaluable to studies examining the means of their death, factors affecting their lifespan and their means of clearance by phagocytes. The aim of the present work was to develop a vital staining procedure for the rapid and objective discrimination of live from dead platelets that accrued in citrated platelet rich plasma (cPRP) incubated at 37°C for several days. By transmission electron microscopy it was noted that platelet death was morphologically similar to necrosis and associated with aggregate formation. The vital dyes calcein-AM and FM 4–64 were found to robustly report the death of platelets and indicated that the aggregates which formed during incubation were populated exclusively by dead platelets. Additionally, platelet death was associated with the shedding of CD42b. Microscopic and cytometric analyses of incubated cPRP indicated that shedding of CD42b and aggregate formation by dead platelets were completely inhibited by the metalloproteinase inhibitor GM6001. Automated counting of platelets incubated in the presence of GM6001 revealed that death did not lead to a loss in cellularity. It is proposed that calcein-AM and FM4–64 are effective as vital stains for the reliable assessment of platelet viability and that platelet aggregation can occur by a novel mechanism dependent upon platelet death and metalloproteinase activity.

Factors affecting platelet survival are poorly understood. To explore the hypothesis that platelet lifespan correlates with the lifespan of a key housekeeping process we subjected human platelets to in vitro incubation at 37°C for 24 h to several days under hypoglycaemic conditions. Viability was assessed both by microscopy and flow cytometry using calcein-AM and/or FM4-64. In keeping with previous data we found that, under control conditions platelets died at a linear rate during 120 h of incubation. Hypoglycaemia did not affect the death rate but did lead to an increase in the frequency of platelets unable to accumulate the mitochondrial potentiometric dye 10-Nonyl Acridine Orange (NAO) and promoted platelet death in response to the pro-apoptotic molecule BH3I-2′. Hypoglycaemia led to an increase in intraplatelet calcium that could be prevented 2-aminoethoxydiphenylborate (2-APB), a store operated calcium channel (SOCC) blocker. However, this agent was unable to rescue the platelets’ ability to accumulate NAO. These data suggest that extracellular glucose is utilised by platelets for calcium homeostasis and maintenance of mitochondrial integrity and that hypoglycaemia primes platelets for death.

The factors controlling the lifespan of platelets both in vivo and in vitro are poorly understood. What is known is that platelet aging in vivo leads to a reduction in the platelets’ responsiveness to physiological agonists and that younger platelets are more haemostatically active than older ones. Under in vitro and ex vivo conditions platelets also lose function and ultimately die for reasons that are unclear, by mechanisms that share some similarity with those used by nucleated cells for programmed cell death. The consequences of platelet death in vitro include the formation of a novel platelet-platelet interaction that occurs between dead but not viable platelets and the shedding of the collagen receptor, GPVI and CD42b, a component of the von Willebrand receptor complex. Both of these phenomena appear to be regulated by metalloproteinase activity. In addition to these observations it is becoming increasingly clear that platelets execute a novel form of programmed cell death in response to agonists such as collagen and thrombin suggesting that their death is intimately associated with effective haemostasis. Finally, platelets must be removed from circulation. It is unclear how senescent platelets are removed, but monocyte-macrophages are an important determinant of thrombus resolution possibly due to the phagocytosis of effete, activated platelets.

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 or how death is brought about. The research programme described in 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 thedemise of platelets in vitro . Two dyes, calcein and FM4-64 were found to beuseful 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.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.