Despite the association of cancer-derived circulating tissue factor (TF)-containing microvesicles and hypercoagulable state, correlations with the incidence of thrombosis remain unclear. In this study the upregulation of TF release upon activation of various cancer cell lines, and the correlation with TF and PAR2 expression and/or activity was examined. Microvesicle release was induced by PAR2 activation in seventeen cell lines and released microvesicle density, microvesicle-associated TF activity, and phoshpatidylserine-mediated activity were measured. The time-course for TF release was monitored over 90 min in each cell line. In addition, TF mRNA expression, cellular TF protein and cell-surface TF activities were quantified. Moreover, the relative expression of PAR2 mRNA and cellular protein were analysed. Any correlations between the above parameters were examined by determining the Pearson’s correlation coefficients. TF release as microvesicles peaked between 30–60 min post-activation in the majority of cell lines tested. The magnitude of the maximal TF release positively correlated with TF mRNA (c = 0.717; p < 0.001) and PAR2 mRNA (c = 0.770; p < 0.001) expressions while the percentage increase correlated with PAR2 mRNA (c = 0.601; p = 0.011) and protein (c = 0.714; p < 0.001). There was only a weak correlation between resting TF release, and microvesicle release. However, TF release in resting cells did not significantly correlate with any of the parameters examined. Furthermore, TF mRNA expression correlated with PAR2 mRNA expression (c = 0.745; p < 0.001). In conclusion, our data suggest that TF and PAR2 mRNA, and PAR2 protein are better indicators of the ability of cancer cells to release TF and may constitute more accurate predictors of risk of thrombosis.

The role of tissue factor (TF)-containing microparticles in clot propagation has been established, but the ability of circulating microparticles to initiate coagulation has been disputed. However, TF-bearing microparticles, particularly endothelial-microparticles generated during disease, may interact with extracellular matrices which in turn can localise circulating TF to sites of injury. In order to examine this hypothesis in vitro, microparticles were isolated from human coronary artery endothelial cells transfected to overexpress TF, tumour-necrosis factor (TNF)α-treated cells or non-transfected cells lacking TF. The ability of microparticles to bind collagen-IV, fibronectin and fibrin was examined under static conditions and arterial shear rates (650 s^-1), and also in the presence of inhibitory antibodies against β1-, β3-, α3-and αv-integrins or an anti-TF antibody. TF-microparticles showed increases of up to 43% and 24% in adherence to collagen-IV and fibronectin, respectively, compared to control microparticles under shear flow. Furthermore, TF-containing microparticles, but not the transfected parent cells had increased levels of β1-integrin compared to TF-deficient microparticles. Pre-incubation of microparticles with a β1-integrin-blocking antibody counteracted the additional adhesion of TFmicroparticles compared to control microparticles. Finally, adherence of TF microparticles to collagen-IV or fibronectin resulted in increased TF activity by concentrating TF onto the surface. In conclusion, the presence of TF within microparticles enhances the interactions of endothelial cell-derived microparticles with extracellular matrices in an integrin-dependent manner. Accumulation and localisation of these microparticles in turn results in the enhancement of TF activity. This may be an innate mechanism by which TF-bearing microparticles induce coagulation upon vascular injury. ^* C.E. and M.E.W.C. contributed equally to this study.

Background: The isolation of neuron-derived extracellular vesicles (nEVs) from biofluids offers the potential to discover novel biomarkers to aid in diagnosis and treatment of psychiatric and neurodegenerative diseases. A few studies have used anti-NCAM antibody-bead-based immunocapture to enrich nEVs from plasma, some with little method validation. We therefore examined in detail this method for nEV enrichment. Methods: EVs were isolated from SH-SY5Y cell-conditioned media by precipitation, or from plasma using size exclusion chromatography. EVs were characterised using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and immunoblot analysis. SH-SY5Y-EVs were incubated with anti-NCAM immunocapture beads and examined by flow cytometry, immunoblot analysis and scanning electron microscopy (SEM). Immunocaptured plasma-derived EVs were examined using a sensitive NCAM ELISA, SEM and qPCR for miRNAs. Results: Characterisation of SH-SY5Y-derived and plasma-derived EVs revealed the expected size distributions of EVs using NTA, the presence of EV markers using immunoblot analysis, and a cup-shaped morphology using TEM. Anti-NCAM beads, but not anti-L1CAM or IgG beads, captured NCAM-positive SH-SY5Y-EVs as shown by flow cytometry and immunoblot analysis. Both SH-SY5Y and plasma-derived EVs were visualised on the surface of anti-NCAM immunocapture beads using SEM. A sensitive NCAM ELISA detected NCAM antigen in plasma-derived EVs immunocaptured on anti-NCAM beads. qPCR analysis of plasma-derived EVs detected many miRNAs in total plasma-EVs with high expression of hsa-miR-16-5p, hsa-miR-451a and hsa-miR-126-3p. However, only between two and seven miRNAs were detected in EVs captured on anti-NCAM-beads from three blood donors. Finally, tissue distribution analysis of miRNAs from plasma-derived EVs on anti-NCAM beads revealed that these miRNAs are enriched in tissues or organs such as blood vessels, lung, bone, thyroid and heart, but were not enriched for brain-derived miRNAs. Discussion: This study indicates that anti-NCAM beads can efficiently enrich NCAM-positive EVs from cell culture conditioned media. However, nEV levels in small volumes of plasma are possibly too low to enable efficient anti-NCAM immunocapture for subsequent miRNA analysis. Other neuron-specific markers with high expression levels on nEVs are therefore required for processing patient samples where plasma volumes are low, and to allow efficient isolation of nEVs in clinical studies for subsequent cargo analysis.

The flavoprotein kynurenine 3-monooxygenase (KMO) is localised to the outer mitochondrial membrane and catalyses the synthesis of 3-hydroxykynurenine from L-kynurenine, a key step in the kynurenine pathway (KP) of tryptophan degradation. Perturbation of KP metabolism due to inflammation has long been associated with the pathogenesis of several neurodegenerative disorders, including Huntington’s disease (HD)—which is caused by the expansion of a polyglutamine stretch in the huntingtin (HTT) protein. While HTT is primarily localised to the cytoplasm, it also associates with mitochondria, where it may physically interact with KMO. In order to test this hypothesis, we employed bimolecular fluorescence complementation (BiFC) and found that KMO physically interacts with soluble HTT exon 1 protein fragment in living cells. Notably, expansion of the disease-causing polyglutamine tract in HTT leads to the formation of proteinaceous intracellular inclusions that disrupt this interaction with KMO, markedly decreasing BiFC efficiency. Using confocal microscopy and ultrastructural analysis, we determined KMO and HTT localisation within the cell and found that the KMO-HTT interaction is localized to the outer mitochondrial membrane. These data suggest that KMO may interact with a pool of HTT at the mitochondrial membrane, highlighting a possible physiological role for mitochondrial HTT. The KMO-HTT interaction is abrogated upon polyglutamine expansion, which may indicate a heretofore unrecognized relevance in the pathogenesis of this disorder.