Abstract
Cells release diverse types of vesicles constitutively or in response to proliferation, injury, inflammation, or stress. Extracellular vesicles (EVs) are crucial in intercellular communication, and there is emerging evidence for their roles in inflammation, cancer, and thrombosis. We investigated the thrombogenicity of platelet-derived EVs, which constitute the majority of circulating EVs in human blood, and assessed the contributions of phosphatidylserine and tissue factor exposure on thrombin generation. Addition of platelet EVs to vesicle-free human plasma induced thrombin generation in a dose-dependent manner, which was efficiently inhibited by annexin V, but not by anti-tissue factor antibodies, indicating that it was primarily due to the exposure of phosphatidylserine on platelet EVs. Platelet EVs exhibited higher thrombogenicity than EVs from unstimulated monocytic THP-1 cells, but blockade of contact activation significantly reduced thrombin generation by platelet EVs. Stimulation of monocytic cells with lipopolysaccharide enhanced their thrombogenicity both in the presence and in the absence of contact activation, and thrombin generation was efficiently blocked by anti-tissue factor antibodies. Our study provides evidence that irrespective of their cellular origin, EVs support the propagation of coagulation via the exposure of phosphatidylserine, while the expression of functional tissue factor on EVs appears to be limited to pathological conditions.
Highlights
IntroductionTissue factor (factor III, CD142) is a 263-amino acid polypeptide consisting of an extracellular N-terminal domain, a transmembrane domain, and a cytoplasmic C-terminus
Active state[8,9,10]
The pro-coagulant activity of Extracellular vesicles (EVs) relies on their exposure of anionic membrane phospholipids, in particular phosphatidylserine, to facilitate the assembly of coagulation complexes at the vesicle surface, while the presence of tissue factor and its potential contribution to the pro-coagulant activity of EVs remain controversial
Summary
Tissue factor (factor III, CD142) is a 263-amino acid polypeptide consisting of an extracellular N-terminal domain, a transmembrane domain, and a cytoplasmic C-terminus. It has a calculated molecular weight of 24.4 kDa, but migrates with an apparent molecular weight of 45–55 kDa in SDS-PAGE due to N-glycosylation at Asn residues 11, 124, and 137. We isolated EV fractions from platelet concentrates from healthy donors to investigate their phosphatidylserine and tissue factor-dependent pro-coagulant activity, and we compared the thrombogenicity of platelet-derived EVs to EVs from non-stimulated and lipopolysaccharide-stimulated monocytic cells
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