[Characterisation of enucleated cells apoptosis: human platelets and erythrocytes]

Apoptosis is a common mechanism of programmed cell death in virtually all nucleated cells. In spite of the fact that platelets and erythrocytes are the only enucleated cells in mammals they contain most of the apoptosis machinery of other cells and undergo similar apoptotic processes as nucleated cells except those connected with nuclear and chromatin transformation. Here we compare the mechanisms of platelet and erythrocytes apoptosis induced by different stimuli namely, stimulation ofthrombin and collagen receptor (T/C), inhibitor of BclX family proteins (ABT-373) for platelets, tert-butylhydroperoxide (tBH) and calcium ionophore (A-23187) for erythrocytes. Induction of platelet apoptosis by both methods (T/C and ABT-373) lead to strong phosphoetydilserine (PS) externalization, loss of the mitochondrial membrane potential (DeltaPsim), proteolytic cleavage of some cytoskeletal and regulatory proteins, and microparticle (MP) formation. However, there are clear differences between mechanisms of platelet apoptosis induced by TC and ABT-373. T/C induced apoptotic reaction is very fast (reach the maximum at 5 min), whereas ABT-373 induced reaction is more prolonged (first apoptotic evidence appears only after 30 min and reach the maximum after 3 hours). MP formation is much more pronounced in T/C than in ABT-373 stimulated platelets, whereas caspase 3 activation is much more stronger in ABT-373 than in T/C stimulated platelets. The main differences between these two apoptotic pathways are connected with aIIbp3 integrin, which activation appears only after T/C stimulation. For tBH experiments on erythrocytes we established optimal conditions (0.25x1012 cells/L, and strong, 1500 RPM stirring) for elucidation of apoptotic processes and found two independent ways of erythrocytes apoptotic processes; calcium independent, connected with met hemoglobin (metHb) formation (tBH stimulation), and calcium dependent pathway (A-23187 stimulation). Erythrocytes apoptosis induced by tBH is characterized by formation ofmetHb, cell shrinkage, fast (95 % during 3 hours) PS externalization, yield of hemoglobin, probably by vesicle (MP) formation. These cells are transformed to stomatocytes, become highly rigid, and could not be lysed even in pure water. All these reactions are calcium independent. Whereas increase of intracellular calcium concentration by A-23187 connected with formation of exinocytes, less pronounced (17 % during 3 h, 35 % during 15 h) PS externalization and rigidity (lysed in 50 mOsm buffer).