Endothelial Cell-derived Extracellular Vesicles Size-dependently Exert Procoagulant Activity Detected by Thromboelastometry

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.

Extracellular vesicles in cardiovascular homeostasis and disease

“…Progression of the coagulation cascade requires the presence and activation of several enzymes that aid in the generation of thrombin, and many of the rate limiting enzymatic reactions begin at the surface of EVs [24]. Tissue factor, a key enzyme that initiates the coagulation cascade, is found on subpopulations of EVs derived from vascular SMCs [25*], endothelial cells [26], monocytes/macrophages [27], and platelets [28]. Further, the charged phospholipid profile (e.g., enrichment of phosphatidylserine) characteristic of many EV populations may also aid in the generation of thrombin [28].…”

Use of Human Umbilical Vein Endothelial Cells (HUVEC) as a Model to Study Cardiovascular Disease: A Review

“…HUVEC exposed to high glucose concentrations increased EMV production and induced changes in their composition and functionality [162,165], contributing to endothelial dysfunction development and progression. Moreover, TNF-α stimulation of HUVEC caused changes in EVs protein [163,[166][167][168][169], and microRNA expression [168][169][170]. These changes induced functional alterations in the recipient cells, suggesting that EC-derived EVs have a vital mediator role in CV homeostasis [140,[163][164][165][166][167][168][169][170].…”

Endothelial–Vascular Smooth Muscle Cells Interactions in Atherosclerosis

“…EVs have been reported to function in post-plaque rupture responses, which promote tissue factor, a rate-limiting enzyme, to initiate the coagulation cascade. Both ECs ( 46 ) and VSMCs ( 47 ) can release TF-loaded VEs; however, it remains unknown how EC and VSMC talk to each other to control the proper release of the same factor. Notably, one of the cargoes carried by EC-derived EVs is miRNA, a discussion of which follows below.…”