Abstract 402

The site of platelet production in the human body is still unclear, but several evidence, including the recent demonstration by our team that shear stress in vitro accelerates platelet formation, favour the hypothesis of an intravascular location of the platelet release process. Therefore we have undertaken the following study to compare the effect of two cell types from the human bone marrow microenvironment, stromal medullary cells and endothelial cells, on the final steps of megakaryocyte (MK) maturation and platelet production. Firstly, supporting the goal of our study, we show that entire mature MK can be encountered in the lumen of normal bone marrow sinusoids and therefore enter the circulation of flowing blood; Secondly, we have used a new microcapillary device coated with von Willebrand factor and were able to confirm by videomicroscopy that exposure of human mature MK to shear forces accelerates proplatelet extension and facilitates platelet liberation from the tips of proplatelets. During this process, the nuclear lobes of polyploid MK tend to separate and to form distinct proplatelet units ready to deliver platelets: this observation might explain why massive senescent MK nuclei are only rarely found in human tissues, bone marrow or lung. Then, we used flow cytometry, optical and electron microscopy, to document and visualise the interaction of human medullary stromal cells (HS5), murine medullary stromal cells (MS5), and human endothelial cells (HUVEC) with the final steps of human MK maturation, namely proplatelet formation. MK were grown from umbilical cord blood CD34+ cells in the presence of Stem Cell Factor (SCF) and thrombopoietin and co-cultured with stromal cells between day 10 and day 13 of culture. Stromal cells virtually completely inhibited proplatelet formation from MK, whereas endothelial cells showed no effect and allowed the emission of proplatelets. EM and flow cytometry confirmed that stromal cells blocked proplatelet and platelet formation and showed that this was accompanied with a considerable development of demarcation membranes which coincided with a 60% increase of CD41 expression by MK. In parallel, stromal cells induced a reduction of apoptosis signs with a reduction of annexin V fixation by maturing MK. Since the step of proplatelet formation is accompanied with cytoplasmic apoptotic signs, among which increased phosphatidyl serine exposure, this result is in accordance with the observed blockade of proplatelet extension. Stromal cell secretion include several growth factors, namely SCF and GMCSF. MK were cultured in the presence of stromal cell culture supernatants, and with these growth factors either individually or mixed together, but this had no effects on proplatelet production, suggesting that physical contacts between the two cell types are necessary. In conclusion, our data show that human bone marrow microenvironment (stromal cells) has an inhibitory effect on proplatelet and platelet formation whereas the vascular microenvironment (endothelial cells) is permissive. This may explain the absence of proplatelets in the bone marrow parenchyma and be an additional evidence that platelet formation and release has to mainly occur in the intravascular compartment.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.

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