N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine induces apoptosis through the activation of caspases-3 and-8 in human platelets. A role for endoplasmic reticulum stress

Background: Apoptosis or programmed cell death involves a number of biochemical events, including the activation of caspases, which lead to specific cell morphology changes and ultimately cell death. Traditionally, two apoptotic pathways have been described: the cell-surface death receptor-dependent extrinsic pathway and the mitochondria-dependent intrinsic pathway. Alternatively, apoptosis has been reported to be induced by endoplasmic reticulum (ER) stress, which is mainly induced by a reduction in intraluminal free Ca2+ concentration ([Ca2+](ER)). Objectives: The present study aimed to investigate the development of apoptotic events after ER stress induced by N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), an ER Ca2+ chelator, in human platelets. Methods: Changes in cytosolic free Ca2+ concentration, caspase activity and phosphatidylserine externalization were determined by fluorimetric techniques. Results: Our results indicate that TPEN reduces the amount of free Ca2+ releasable by the Ca2+-mobilizing agonist thrombin. TPEN induced activation of caspase-3, -8 and -9 and subsequent phosphatidylserine externalization. The ability of TPEN to induce phosphatidylserine externalization was smaller than that of thrombin. In addition, TPEN was able to induce phosphorylation of the eukaryotic initiation factor 2 alpha (eIF2 alpha). TPEN-mediated caspase-3 activation requires functional caspase-8, but is independent of H2O2 generation. Activation of caspase-3 and -8 by TPEN was prevented by salubrinal, an agent that prevents ER stress-induced apoptosis. Conclusion: These findings provide experimental evidence for the existence of ER stress-mediated apoptosis in human platelets, a process that might limit platelet life span upon prolonged stimulation with agonists.