Abstract
Endothelial cells (ECs) are major modulators of hemostasis by expressing and releasing pro- and anticoagulant mediators into the circulation. Previous studies showed that cultured ECs release procoagulant mediators into cell culture supernatants as evidenced by the reduction of viscoelastic clotting time. This effect was reversed with an anti-tissue factor antibody. Here, we aimed to investigate whether tissue factor (TF) was released by endothelial-derived extracellular vesicles (EVs) and which portion of the released vesicles displays the most prominent procoagulant properties. After stimulation of ECs with tumor-necrosis factor-α (TNF-α) the supernatants of EC cultures were subjected to differential centrifugation steps to collect larger and smaller EVs which were then characterised by nanoparticle tracking analysis (NTA) and flow cytometry. Mixed with fresh human blood and analysed by thromboelastometry EVs exerted a significant procoagulant stimulus, which could be partly reversed by addition of an anti-TF antibody. Moreover, TF activity was confirmed in the centrifuged fractions. In summary, our results provide evidence of the procoagulant potential of smaller and larger endothelial-derived EV fractions detected by thromboelastometry. The observed effect is most likely due to the release of TF-bearing EVs of different dimensions, which are released upon TNF-α stimulation of endothelial cell cultures.
Highlights
Endothelial cells (ECs) are major modulators of hemostasis by expressing and releasing pro- and anticoagulant mediators into the circulation
We have previously shown a tissue factor (TF)-dependent reduction of clotting time (CT) in the otherwise non-activated ROTEM assay when adding tumor-necrosis factor-α (TNF-α)-stimulated endothelial cell culture supernatants to whole blood from healthy volunteers[18]
We found that both Annexin V+/TF+ and single positive particles were significantly elevated in P14 TNF-α-stimulated samples compared to the respective unstimulated controls while no significant change could be detected in P100 samples (Fig. 2D)
Summary
Endothelial cells (ECs) are major modulators of hemostasis by expressing and releasing pro- and anticoagulant mediators into the circulation. Previous studies showed that cultured ECs release procoagulant mediators into cell culture supernatants as evidenced by the reduction of viscoelastic clotting time. This effect was reversed with an anti-tissue factor antibody. In the classical coagulation cascade model of secondary haemostasis, the initiation of coagulation is triggered via contact activation with negatively charged surfaces (intrinsic pathway) or tissue factor (extrinsic pathway). EVs are small vesicles, consisting of a lipid bilayer membrane which contain proteins, and coding as well as non-coding RNA and DNA5 Based on their size and the site of cellular origin, three different types of EVs are distinguished: (I) Shedding microvesicles which directly bleb from the plasma membrane (
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