Aim: 

Apoptosis or programmed cell death involves a series of biochemical events, such the concerted action of several caspases, leading to specific cell morphology characteristics and ultimately cell death. Traditionally, two general pathways have been described for the development of apoptosis: the cell-surface death receptor-dependent extrinsic pathway and the mitochondria-dependent intrinsic pathway. However, recent studies have described a new apoptotic pathway activated by endoplasmatic reticulum (ER) stress, which is mainly produced by a reduction in luminal free Ca²⁺ concentration. Hence, we have investigated 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: 

Treatment of platelets with TPEN (200 mM) reduced thapsigargin and thrombin-evoked Ca²⁺ release from the dense tubular system, the analogue of the ER in platelets; in contrast, TPEN did not modify Ca²⁺ mobilization from the acidic stores induced by 2,5-di-(t-butyl)-1,4-hydroquinone. TPEN also induces time-dependent activation of caspase 3 and phosphatidylserine exposure. TPEN-evoked caspase 3 activation is inhibited by pretreatment with the specific caspase 8 inhibitor z-IETD-fmk (100mM) and slightly reduced by the specific caspase 9 inhibitor z-LEHD-cmk (40 mM); however, catalase was without effect on TPEN-mediated response.

Conclusion: 

Our results demonstrate ER stress-induced apoptosis in human platelets mediated by caspase 8 and 9, and subsequently, caspase 3 activation and does not require hydrogen peroxide generation.

Supported by M.E.C.–FEDER BFU2007-60104/BFI and BFU2007-60091/BFI; J.J.L is supported by a DGI fellowship BFU2004-00165.