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 Ca(2+) concentration ([Ca(2+)](ER)).


OBJECTIVE
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 Ca(2+) chelator, in human platelets.


METHODS
Changes in cytosolic free Ca(2+) concentration, caspase activity and phosphatidylserine externalization were determined by fluorimetric techniques.


RESULTS
Our results indicate that TPEN reduces the amount of free Ca(2+) releasable by the Ca(2+)-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 H(2)O(2) generation. Activation of caspase-3 and -8 by TPEN was prevented by salubrinal, an agent that prevents ER stress-induced apoptosis.


CONCLUSIONS
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.
López
J J
JJ
Department of Physiology, University of Extremadura, Cáceres, Spain.
Redondo
P C
PC
Salido
G M
GM
Pariente
J A
JA
Rosado
J A
JA
D017209:Apoptosis / Q000187:drug effects*; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism*; D053181:Caspase 8 / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism; D004789:Enzyme Activation; D005029:Ethylenediamines / Q000494:pharmacology*; D006801:Humans; D010718:Phosphatidylserines / Q000378:metabolism; D010766:Phosphorylation; D013917:Thrombin / Q000494:pharmacology
D005029:Ethylenediamines; D010718:Phosphatidylserines; D013917:Thrombin; D053148:Caspase 3; D053181:Caspase 8; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; D002118:Calcium