Microvesicles were isolated from the conditioned media of 3 cell lines (MDA‐MB‐231, AsPC‐1 and A375) by ultracentrifugation at a range of relative centrifugal forces, and the tissue factor (TF) protein and activity, microvesicle number, size distribution and relative density compared. Also, by expressing TF‐tGFP in cells and isolating the microvesicles, the relative density of TF‐containing microvesicles was established. Nanoparticle tracking analysis (NTA) indicated that the larger‐diameter microvesicles (>200 nm) were primarily sedimented at 100,000g and possessed TF‐dependent thrombin and factor Xa generation potential, while in the absence of factor VII, all microvesicles possessed some thrombin generation capacity. Immuno‐precipitation of TF‐containing microvesicles followed by NTA also indicated the range of these microvesicles to be 200–400 nm. Analysis of the microvesicles by gradient density centrifugation showed that lower‐density (<1.1 g/ml) microvesicles were mainly present in the samples recovered at 100,000g and were associated with TF antigen and activity. Analysis of these fractions by NTA confirmed that these fractions were principally composed of the larger‐diameter microvesicles. Similar analysis of microvesicles from healthy or patient plasma supported those obtained from conditioned media indicating that TF activity was mainly associated with lower‐density microvesicles. Furthermore, centrifugation of healthy plasma, supplemented with TF‐tGFP‐containing microvesicles, resulted in 67% retrieval of the fluorescent microvesicles at 100,000g, but only 26% could be recovered at 20,000g. Pre‐centrifugation of conditioned media or plasma at 10,000g improved the speed and yield of recovered TF‐containing microvesicles by subsequent centrifugation at either 20,000g or 100,000g. In conclusion, TF appears to be associated with low‐density (1.03–1.08 g/ml), larger‐diameter (200–350 nm) microvesicles.

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.

Objective— Increased levels of circulating tissue factor (TF) in the form of microparticles increase the risk of thrombosis. However, any direct influence of microparticle-associated TF on vascular endothelial cell proliferation is not known. In this study, the influence of recombinant and microparticle-associated TF on endothelial cell proliferation and mitogen-activated protein kinase signaling mechanisms was examined. Methods and Results— Incubation of human coronary artery endothelial cells with lipidated recombinant full-length TF, or TF-containing microparticles (50 to 200 pmol/L TF), increased the rate of cell proliferation and induced phosphorylation of extracellular signal regulated kinase 1 in a TF-dependent manner. Inhibition of extracellular signal regulated kinase 1/2 using PD98059 or extracellular signal regulated kinase 1/2 antisense oligonucleotides or inhibition of c-Jun N-terminal kinase reduced recombinant TF-mediated cell proliferation. PD98059 also reduced cell proliferation in response to TF-containing microparticles. Inclusion of FVIIa (5 nmol/L) and FXa (10 nmol/L) or preincubation of cells with an inhibitory anti-FVIIa antibody had no additional influence on TF-mediated cell proliferation. However, preincubation of exogenous TF with a β1-integrin peptide (amino acids 579 to 799) reduced TF-mediated proliferation. Conclusion— High concentrations of recombinant or microparticle-associated TF stimulate endothelial cell proliferation through activation of the extracellular signal regulated kinase 1/2 pathway, mediated through a novel mechanism requiring the interaction of exogenous TF with cell surface β1-integrin and independent of FVIIa.

The mechanisms that regulate the incorporation and release of tissue factors (TFs) into cell-derived microparticles are as yet unidentified. In this study, we have explored the regulation of TF release into microparticles by the phosphorylation of serine residues within the cytoplasmic domain of TF. Wild-type and mutant forms of TF, containing alanine and aspartate substitutions at Ser253 and Ser258, were overexpressed in coronary artery and dermal microvascular endothelial cells and microparticle release stimulated with PAR2 agonist peptide (PAR2-AP). The release of TF antigen and activity was then monitored. In addition, the phosphorylation state of the two serine residues within the released microparticles and the cells was monitored for 150 min. The release of wild-type TF as procoagulant microparticles peaked at 90 min and declined thereafter in both cell types. The TF within these microparticles was phosphorylated at Ser253 but not at Ser258. Aspartate substitution of Ser253 resulted in rapid release of TF antigen but not activity, whereas TF release was reduced and delayed by alanine substitution of Ser253 or aspartate substitution of Ser258. Alanine substitution of Ser258 prolonged the release of TF following PAR2-AP activation. The release of TF was concurrent with phosphorylation of Ser253 and was followed by dephosphorylation at 120 min and phosphorylation of Ser258. We propose a sequential mechanism in which the phosphorylation of Ser253 through PAR2 activation results in the incorporation of TF into microparticles, simultaneously inducing Ser258 phosphorylation. Phosphorylation of Ser258 in turn promotes the dephosphorylation of Ser253 and suppresses the release of TF.