Summary
In this study the uptake of tissue factor (TF)-positive microparticles by endothelial
cells and the recycling of the TF component were examined. Human dermal blood endothelial
cells (HDBEC) were incubated with microparticles derived from cancer cell lines for
up to 6 hours. Measurement of HDBEC cell surface TF antigen revealed two distinct
peaks at 30 and 180–240 minutes post-incubation with TF-positive, but not TF-deficient
microparticles. However, only the second peak was concurrent with high TF activity
as determined by a chromogenic thrombin-generation assay. Annexin V-labelling of HDBEC
showed phosphatidylserine exposure following 90 minutes incubation with microparticles,
which explains the high TF activity associated with the second antigen peak. Analysis
of TF mRNA levels revealed no de novo expression of TF mRNA in response to microparticles,
and pre-incubation of cells with cycloheximide did not prevent the appearance of TF.
However, blocking endocytosis with a dynamin inhibitor prolonged the disappearance
and prevented the reappearance of TF antigen on the cell surface. Incubation of HDBEC
with microparticles containing TF-GFP revealed the early co-localisation of TF with
Rab4 and Rab5, followed by co-localisation with the late endosomal/trans-Golgi network
marker Rab9, and the recycling endosome marker Rab11. siRNAmediated suppression of
Rab11 reduced the reappearance of TF on the cell surface. These data suggest a mechanism
by which TF-containing microparticles are internalised by endothelial cells and the
TF moiety recycled to the cell surface. Together with the exposure of phosphatidylserine,
this is capable of inducing a substantial increase in the procoagulant potential of
the surface of endothelial cells.
Keywords
Tissue factor - microparticles - endothelial cells - procoagulant activity - recycling
- endosome
