Endothelial Cell Secretion Combined with Shear Stress Strongly Increase Platelet Production by Human Megakaryocytes,

Abstract 3411

It is well established that megakaryocytes (MK), in order to achieve terminal maturation in the bone marrow, migrate from the osteoblastic niche to the vascular niche, close to medullary sinusoids. It is also admitted that, in order to release platelets, MK extend cytoplasmic extensions in the lumen of the sinusoid followed by platelet release. Therefore, we have investigated the role of endothelial cells combined with shear stress on MK late differentiation steps and platelet production.

To do so, human MK were grown from umbilical cord blood or bone marrow CD34⁺ cells, in the presence of Stem Cell Factor (SCF) and thrombopoietin for 10 days. Then they were co-cultured with human endothelial cells (HUVEC) for 3 days and analysed by video-microscopy, flow cytometry, immunofluorescence and electron microscopy. In these conditions, most MK (>50%) extended numerous and prominent proplatelets: immunofluorescence showed virtually complete unwinding of their cytoplasm, which extended in the form of long proplatelets; this rarely occurred in control MK. Electron microscopy showed that these MK were formed by a coarse chromatin nucleus surrounded by a thin cytoplasmic ring and surrounded by platelet-size territories displaying alpha-granules, mitochondria and canalicular system which coincided to the sections of the proplatelet swellings and to the unwinding of the demarcation membrane system. Another type of experiment of co-culture MK/endothelial cells was conducted in transwells and led to similar results, ie, increase of MK cytoplasmic maturation and proplatelet formation indicating that cell/cell contact was not necessary for up-regulating proplatelet production, rather that (a) soluble product(s) was(ere) secreted from endothelial cells. Flow cytometry, at this step, failed to demonstrate significant changes in the platelet production rate. We then submitted MK co-cultured with endothelial cells to shear stress and examined them by flow cytometry and video microscopy. We could then demonstrate that platelet release was strongly increased (× 3.8 ± 0.9, n=3) when MK had been in contact with endothelial cells compared to control MK. The released platelet-size particles expressed CD41 and, when stimulated by thrombin, were also able to express CD62P. Video-microscopy confirmed that proplatelet and platelet shedding occurred after exposition of mature MK to shear stress.

When examined by video-microscopy, live MK whose DNA had been stained with the fluorescent dye Hoechst 33342 showed that nuclear lobes separated under high shear stress: indeed they became located at opposite poles of the cell, while the cytoplasmic volume extended and elongated, becoming organized in proplatelets which exhibited a succession of platelet size subunits; eventually cytoplasmic scission occurred in parallel with MK nuclear lobe segregation in distinct cell fractions, each carrying proplatelets; proplatelets subsequently fractionated and were then released from the cell core containing the fluorescent nuclear lobe. Thus shear stress leads both to cytoplasm and nucleus fragmentation. This is a dynamic explanation to the fact that entire MK nuclei are rarely found in the human bone marrow. This observation also gives sense to the unique phenomenon of MK polyploidy.

In conclusion, this study indicates that endothelial microenvironment combined with circulatory shear forces are determinant up-regulating factors which increase platelet production. It also shows that shear stress is able to induce nuclear as well as cytoplasmic MK fragmentation, leading to a new anatomical concept of circulating platelet shedding MK subunits.