Examination of the up-regulation and transfer of tissue factor to endothelial cells by tumour-derived microparticles

Overexpression of tissue factor and its release as microparticles is a common feature of aggressive cancer. Furthermore, increased levels of tissue factor have been detected in the tumour associated microvascular endothelial cells. The accumulation of active tissue factor on the luminal surface of blood vessels generates a thrombogenic surface which can lead to thrombus formation. Although tissue factor has been shown to be present within the endothelial layer in cancer patients, the source of this tissue factor still remains unknown. It has been shown that tissue factor can be transferred between different types of cells through microparticles. In addition, endothelial cells have been shown to be capable of expressing tissue factor in response to microparticles. Therefore, this study aimed to investigate the source of endothelial cell-associated tissue factor by analysing the transfer of cancer-derived tissue factor by microparticles, to microvascular endothelial cells, in vitro. The study also evaluated the de novo expression of tissue factor within endothelial cells in response to these microparticles. Human dermal blood endothelial cells (HDBEC) were incubated with tissue factor-bearing, or tissue factor-deficient microparticles isolated from the medium of MDA-MB-231 or MCF-7 cell lines respectively. Subsequently, HDBEC-surface tissue factor antigen was quantified over 360 min using a tissue factor-specific ELISA. Surface tissue factor activity was determined using a chromogenic assay and the presence of phosphatidylserine on the HDBEC surface was monitored using annexin V-labelling. Two distinct surface tissue factor antigen peaks were detected at 30 and 120 min following the incubation of the cells with tissue factor-bearing microparticles but not tissue factor-deficient microparticles. However, only the latter peak at 120 min was accompanied with high tissue factor activity. This activity was attributed to the exposure of phosphatidylserineon the endothelial cell surface demonstrated by the increased annexin-V labelling at 90 min. Analysis of tissue factor mRNA, up to 24 h, did not reveal any de novo expression of tissue factor in response to the microparticles. In addition, pre-incubation of HDBEC with a dynamin inhibitor, Dynasore, resulted in the disappearance of the latter tissue factor antigen exposed on the cell surface at 120 min. These data suggest that tumour-derived tissue factor can be transferred to endothelial cells on exposure to tumour derived microparticles. Furthermore, tissue factor-bearing microparticles are internalised by endothelial cells and may then be recycled onto the endothelial cell surface in a highly active form. This in turn may result in a substantial increase in theprocoagulant potential on the surface of endothelial cells leading to thrombosis