Alterations in Bcl-2/Bax protein levels in platelets form part of an ionomycin-induced process that resembles apoptosis.
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.
Vanags
D M
DM
Institute of Environmental Medicine, Division of Toxicology, Karolinska Institute, Stockholm, Sweden.
Orrenius
S
S
Aguilar-Santelises
M
M
D017209:Apoptosis / Q000235:genetics; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D019254:Genes, bcl-2; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology*; D008855:Microscopy, Electron, Scanning; D010718:Phosphatidylserines / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger / Q000378:metabolism; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D010718:Phosphatidylserines; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; D015759:Ionomycin; D003546:Cysteine Endopeptidases