PLATELET-ASSOCIATED TISSUE FACTOR EXPRESSION: INSIGHTS INTO THE MEGAKARYOCYTE-PLATELET AXIS.
Tesi di Dottorato
Data di Pubblicazione:
2014
Citazione:
PLATELET-ASSOCIATED TISSUE FACTOR EXPRESSION: INSIGHTS INTO THE MEGAKARYOCYTE-PLATELET AXIS / L. Facchinetti ; tutor: M. Camera ; coordinatore: A. Panerai. DIPARTIMENTO DI SCIENZE FARMACOLOGICHE E BIOMOLECOLARI, 2014 Jan 16. 26. ciclo, Anno Accademico 2013. [10.13130/facchinetti-laura_phd2014-01-16].
Abstract:
Tissue factor is the main activator of the blood coagulation cascade and for this reason levels of TF are not easily detectable in cells in contact with blood under physiological conditions. By contrast, in pathological conditions endothelial cells and monocytes can be induced to express TF. Induced-TF is present in atherosclerotic plaques (vessel wall-derived TF) and several studies in the past have documented its role in the plaque thrombogenicity. At the end of the last century it was reported that, under physiological conditions, TF positive microparticles (MPs) circulate in the blood (blood-borne TF) and their number further increases in pathological conditions. These microparticles, which arise mainly from activated endothelial cells and monocytes, could fuse with other cells conferring them the ability to activate coagulation.
Giesen et al. in 2000 showed the presence of TF in human platelets through this mechanism. Since then several papers have documented the presence of TF in human platelets by using different approaches. Despite this in-depth characterization, the platelet-associated TF is matter of controversy by some authors that, failing to identify TF in platelets, argue that the published data are artifacts, thus generating an ongoing debate.
To date two main “cellular entities” may be responsible for the presence of TF in platelets: TF-positive MPs derived from different activated cell types, as proposed by Nemerson’s group, and megakaryocytes that transferring TF mRNA to platelets make them autonomous in the synthesis of the protein. In this regard, ten years ago our group provided the evidence that human megakaryocytes contain TF mRNA. The platelet transcriptome derives from megakaryocytes through finely tuned mechanisms. The direct evidence that megakaryocytes transfer TF mRNA to platelets, however, is still missing; similarly, it has never been investigated whether megakaryocytes express TF protein that can be transferred to platelets; finally, the contribution of platelet TF to clot formation has never been assessed. To test these hypothesis we took advantage of a well-characterized human megakaryoblastic cell line, Meg-01, able to differentiate into megakaryocytes and to release platelets in vitro. This approach allowed us to analyze TF mRNA and protein expression during the differentiation from megakaryoblasts to megakaryocytes and in the released platelets in the complete absence of any other “contaminating” cell that might be a source of microparticles. The expression of TF protein by Meg-01 and Meg-derived platelet (Meg-platelets) was confirmed in human megakaryocytes differentiated from CD34+ cells. Finally, TF mRNA was silenced in Meg-01 megakaryoblasts showing that megakaryocytes, Meg-platelets and MPs were almost devoid of TF.
Using a combination of cell biology, flow cytometry, confocal microscopy and biochemical analyses here we show evidence for the first time that functionally active TF is expressed in human megakaryocytes, and that they transfer this protein to platelets and to microparticles (MPs). TF downregulation in megakaryoblasts by lentiviral shRNA particles almost completely abolished its expression in megakaryocytes, platelets and MPs. Furthermore, TF silencing leads to a decrease of the thrombin generation by megakaryocytes which is reflected on a decrease in thrombin generation by platelets, suggesting that the platelet-associated TF may contribute to the haemostatic capacity of whole blood.
Taken together all these data shoe the evidence that human platelets and MPs carry a megakaryocyte-derived TF which is functionally active and able to trigger thrombin generation.
Finally, this in vitro model may be used to investigate the modification in platelet transcriptome an
Tipologia IRIS:
Tesi di dottorato
Keywords:
tissue factor ; platelets ; megakaryocytes
Elenco autori:
L. Facchinetti
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