Alterations in Bcl‐2/Bax protein levels in platelets form part of an ionomycin‐induced process that resembles apoptosis

Abstract

Platelets are physiologically anucleated cells, derived from megakaryocytes, that undergo vesiculation and transformation into small particles when they are stimulated in vitro by ionomycin and other agents. Electron microscopy images suggest a similarity to apoptosis in cells with nuclei, which ends with cell disintegration and formation of apoptotic bodies. By PCR, we have demonstrated mRNA expression of bcl-2, bax, and p53 in highly purified non-stimulated platelets. A side-scatter shift and a decrease in the Bcl-2/Bax protein ratio were observed by flow cytometry analysis after stimulation with ionomycin. The ionomycin-induced modifications were inhibited by the calpain I inhibitor calpeptin and, less effectively, by VAD-cmk, a broad-spectrum caspase inhibitor. However, caspase 3-like activity was very low, with only a twofold increase after ionomycin stimulation, as measured by the cleavage of the fluorogenic peptide substrate DEVD-AMC. Our data indicate that platelets may constitute a natural model for the analysis of cytoplasmic events in apoptosis.

Early increase in DcR2 expression and late activation of caspases in the platelet storage lesion

“…However, the caspase inhibitor z-VAD-fmk does not prevent phosphatidylserine exposure in activated platelets. 9,22,34 In addition, Wolf et al 22 demonstrated that calpain was able to process procaspase-3 and procaspase-9 at specific cleavage sites without generating the active caspase subunits and proposed that calpains, but not caspases, were responsible for apoptosis-like events during platelet activation. 42,43 The role of caspases in platelet Flow cytometry analysis of death receptors and ligands.…”

“…9,26,34 Some of these Bcl-2-related proteins are pro-apoptotic proteins, which are present in healthy cells in a dormant form and behave as stress sensors by monitoring the integrity of vital metabolic processes. 33 One of them is the BH3 domain-only protein Bim 44 that is expressed in many hematopoietic cell types and is sequestered to cytoskeletal structures of healthy cells via association with dynein light chain LC8.…”

“…Effector caspases cleave a limited number of intracellular proteins, leading to the dismantling of the cells that characterizes apoptotic cell death. 18,19 Several observations suggested a role for these apoptotic pathways in the platelet storage lesion: (1) cell shrinkage, release of microvesicules and phosphatidylserine redistribution on the cell surface are observed in both activated plate-lets and apoptotic cells; 8,9,20 (2) human platelets contain caspase-3 and caspase-9, the adaptor molecule Apaf-1 and several proteins of the Bcl-2 family of death regulators; 9,21,22 (3) addition of cytochrome c to cell-free extracts from platelets recapitulates the cytosolic events of apoptosis; 22 (4) exposure of platelets to ionomycin, a calcium ionophore, increases expression of Bax, a pro-apoptotic member of the Bcl-2 family in these cells; 9 and (5) the nucleus was demonstrated not to be an absolute requirement for apoptosis induction. 9,[22][23][24] Based on these observations, we investigated whether proteins involved in apoptosis regulation of nucleated cells could play a role in platelet storage lesion.…”

“…8 Several of the events associated with platelet activation are reminiscent of cell death by apoptosis. 9 Two main pathways have been shown to lead to apoptosis. The intrinsic pathway involves the release of pro-apoptotic molecules such as cytochrome c (cyt c), 10,11 apoptosis-inducing factor (AIF), 12 and Diablo/Smac 13,14 from the mitochondrial intermembrane space under the control of pro-apoptotic and anti-apoptotic proteins of the Bcl-2 family.…”

Bcl-2 family proteins are essential for platelet survival

“…3,[11][12][13] Several studies have demonstrated that the Bcl-2 family proteins (i.e., Bax, Bak, Bcl-X L , and Bcl-2) are also expressed in platelets. 11,12,14,15 The Bcl-2 family of proteins is the key regulators of mitochondria-dependent apoptosis in nucleated cells and consists of both antiapoptotic (Bcl-X L , Bcl-2, Bcl-w, A1, Mcl-1) 16 and proapoptotic (Bak, Bax, Bid, Bim, Bad, Bik, Bmf, Noxa, Puma) 17 members.…”