Platelet formation is the consequence of caspase activation within megakaryocytes.
Platelets are formed from mature megakaryocytes (MKs) and arise from the development of long and thin cytoplasmic extensions called proplatelets. After platelet release, the senescent MKs (nucleus surrounded by some cytoplasm) undergo cell death by apoptosis. To explore the precise role of apoptosis in proplatelet formation, we grew human MKs from CD34(+) cells and assessed the possible role of caspases. Proteolytic maturation of procaspase-3 and procaspase-9 was detected by immunoblots in maturing MKs as well as in proplatelet-bearing MKs and senescent MKs. Cleavage of caspase substrates such as gelsolin or poly adenosine diphosphate (ADP)-ribose polymerase (PARP) was also detected. Interestingly, activated forms of caspase-3 were detected in maturing MKs, before proplatelet formation, with a punctuate cytoplasmic distribution, whereas a diffuse staining pattern was seen in senescent and apoptotic MKs. This localized activation of caspase-3 was associated with a mitochondrial membrane permeabilization as assessed by the release of cytochrome c, suggesting an activation of the intrinsic pathway. Moreover, these MKs with localized activated caspase-3 had no detectable DNA fragmentation. In contrast, when apoptosis was induced by staurosporine, diffuse caspase activation was seen; these MKs had signs of DNA fragmentation, and no proplatelet formation occurred. The pan-caspase inhibitor z-VAD.fmk as well as more specific inhibitors of caspase-3 and caspase-9 blocked proplatelet formation, whereas an inhibitor of calpeptin had no effect. Overexpression of Bcl-2 also inhibited proplatelet formation in maturing MKs. Thus, localized caspase activation is causal to proplatelet formation. We conclude that proplatelet formation is regulated by a caspase activation limited to only some cellular compartments.
De Botton
Stephane
S
Institut National de la Santé et de la Recherche Médicale U362 and the Centre National de la Recherche Scientifique (Unité Mixte de Recherche 1599), Institut Gustave Roussy, Institut Fédératif de Recherche 54, Villejuif, France.
Sabri
Siham
S
Daugas
Eric
E
Zermati
Yael
Y
Guidotti
Jacques Emmanuel
JE
Hermine
Olivier
O
Kroemer
Guido
G
Vainchenker
William
W
Debili
Najet
N
D018952:Antigens, CD34 / Q000032:analysis; D017209:Apoptosis / Q000187:drug effects; D001792:Blood Platelets / Q000166:cytology*; D053148:Caspase 3; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002462:Cell Membrane / Q000648:ultrastructure; D002463:Cell Membrane Permeability; D003593:Cytoplasm / Q000201:enzymology; D053938:DNA Fragmentation; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006410:Hematopoiesis; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000276:immunology; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008533:Megakaryocytes / Q000166:cytology* / Q000201:enzymology* / Q000648:ultrastructure; D019311:Staurosporine / Q000494:pharmacology
D018952:Antigens, CD34; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; D019311:Staurosporine