Submitted March 12, 2009
Accepted May 31, 2009
Exposure of human megakaryocytes to high shear rates accelerates platelet production
Claire Dunois-Larde, Claude Capron, Serge Fichelson, Thomas Bauer, Elisabeth Cramer-Borde, and Dominique Baruch*
INSERM U765, Paris, France
INSERM U567, Paris, France
Universite Paris Descartes, Paris, France
Service d'Orthopedie, Hopital Ambroise Pare, Boulogne, France
Universite de Versailles-St Quentin, Versailles, France
* Corresponding author; email: dominique.baruch{at}univ-paris5.fr.
Platelets originate from megakaryocytes (MKs) by cytoplasmic elongation into proplatelets. Direct platelet release is not seen in bone marrow hematopoietic islands. It was suggested that proplatelet fragmentation into platelets can occur intravascularly, yet evidence of its dependence on hydrodynamic forces is missing. Therefore, we investigated whether platelet production from MKs could be upregulated by circulatory forces. Human mature MKs were perfused at a high shear rate on von Willebrand factor. Cells were observed in real-time by videomicroscopy, and by confocal and electron microscopy after fixation. Dramatic cellular modifications followed exposure to high shear rates: 30-45% adherent MKs were converted into proplatelets and released platelets within 20 minutes, contrary to static conditions that required several hours, often without platelet release. Tubulin was present in elongated proplatelets and platelets, thus ruling out membrane tethers. By using inhibitors, we demonstrated the fundamental roles of microtubule assembly and MK receptor GPIb. Secretory granules were present along the proplatelet shafts and in shed platelets, as shown by P-selectin labeling. Platelets generated in vitro were functional since they responded to thrombin by P-selectin expression and cytoskeletal reorganization. In conclusion, MK exposure to high shear rates promotes platelet production via GPIb, depending on microtubule assembly and elongation.
