Dying to Make Platelets

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Two groups, de Botton et al. and Clarke et al. , showed that platelet formation occurs through a variation of apoptosis involving compartmentalized caspase activation. Megakaryocytes, the platelet precursor cells, undergo a morphological reorganization in which they extend processes called proplatelets, which develop nodes that are eventually released as mature platelets. De Botton et al. found that caspase inhibitors blocked thrombopoietin-stimulated proplatelet formation in cultured human megakaryocytes, as did overexpression of Bcl-2 (an antiapoptotic protein). The authors used Western analysis to demonstrate proteolytic maturation of caspase-3 and caspase-9 and cleavage of caspase substrates during megakaryocyte maturation. Confocal microscopy analysis of immunofluorescently labeled cells indicated that punctate, localized caspase-3 activation occurred before proplatelet formation. Furthermore, early localized caspase-3 activation was associated with mitochondrial release of cytochrome c but not DNA fragmentation or cell death. Clarke et al. found that caspase inhibitors blocked platelet production and proplatelet formation in a human megakaryoblastic cell line that spontaneously generated functional platelets. Fluorescence analysis indicated that the cell bodies of proplatelet-bearing cells contained activated caspases but the proplatelets did not. Cells developing proplatelets showed heterochromatin condensation consistent with early apoptosis. Fas ligation stimulated proplatelet formation, platelet production, and nuclear condensation, and augmented platelet production by primary cultures of mouse megakaryocytes and human bone core explants. The authors used a fluorescent mitochondrial dye to show that proplatelet mitochondria maintained their membrane potentials in cells showing nuclear condensation. Dying platelets cultured in the absence of survival factors, however, showed collapsed mitochondrial membrane potentials and released cytochrome c. Western analysis together with caspase-activating treatments suggested that platelets lacked caspase-9. S. de Botton, S. Sabri, E. Daugas, Y. Zermati, J. E. Guidotti, O. Hermine, G. Kroemer, W. Vainchenker, N. Debili, Platelet formation is the consequence of caspase activation within megakaryocytes. Blood 100 , 1310-1317 (2002). [Online Journal] M. C. H. Clarke, J. Savill, D. B. Jones, B. S. Noble, S. B. Brown, Compartmentalized megakaryocyte death generates functional platelets committed to caspase-independent death. J. Cell Biol . 160 , 577-587 (2003). [Abstract] [Full Text]