Dissecting the biochemical architecture and morphological release pathways of the human platelet extracellular vesiculome

Abstract

Platelet extracellular vesicles (PEVs) have emerged as potential mediators in intercellular communication. PEVs exhibit several activities with pathophysiological importance and may serve as diagnostic biomarkers. Here, imaging and analytical techniques were employed to unveil morphological pathways of the release, structure, composition, and surface properties of PEVs derived from human platelets (PLTs) activated with the thrombin receptor activating peptide (TRAP). Based on extensive electron microscopy analysis, we propose four morphological pathways for PEVs release from TRAP-activated PLTs: (1) plasma membrane budding, (2) extrusion of multivesicular α-granules and cytoplasmic vacuoles, (3) plasma membrane blistering and (4) "pearling" of PLT pseudopodia. The PLT extracellular vesiculome encompasses ectosomes, exosomes, free mitochondria, mitochondria-containing vesicles, "podiasomes" and PLT "ghosts". Interestingly, a flow cytometry showed a population of TOM20LC3 PEVs, likely products of platelet mitophagy. We found that lipidomic and proteomic profiles were different between the small PEV (S-PEVs; mean diameter 103 nm) and the large vesicle (L-PEVs; mean diameter 350 nm) fractions separated by differential centrifugation. In addition, the majority of PEVs released by activated PLTs was composed of S-PEVs which have markedly higher thrombin generation activity per unit of PEV surface area compared to L-PEVs, and contribute approximately 60% of the PLT vesiculome procoagulant potency.

Mode of induction of platelet-derived extracellular vesicles is a critical determinant of their phenotype and function

“…In previous studies investigating the ability of PDEVs to support thrombin generation, it was shown that size altered their procoagulant properties where smaller vesicles (142 ± 12 nm) stimulated less thrombin generation than a larger population (173 ± 15 nm) 4,20 . The current study demonstrates that not only size is important for the negatively-charged phospholipid exposure profiles and procoagulant activity, but the stimuli that triggered PDEVs generation.…”

Extracellular Vesicles in Herpes Viral Spread and Immune Evasion

“…Exosomes, on the other hand, are enriched in tetraspanins (CD9, CD63 and CD81, among others), which are frequently used as exosomal markers ( Andreu and Yañez-Mo, 2014 ) and also in endosomal markers such as ALIX and TSG101 ( Kowal et al, 2016 ; Willms et al, 2016 ). Although the presence of PS exposed in exosomes has been postulated ( Thery et al, 2009 ; Colombo et al, 2014 ; De Paoli et al, 2018 ) other studies question that exosomes expose PS just after secretion from cells ( Lai R.C. et al, 2016 ; Skotland et al, 2017 ), remaining this point to be fully clarified.…”

The source of cell-free mitochondrial DNA in trauma and potential therapeutic strategies

“…Flow cytometry is a fast, sensitive and specific test for quantifying mitochondria and microparticles. Specific markers for membrane-encapsulated mitochondria can be used in conjunction with cell-permeable mitochondrial stains such as Mitotracker to measure the cell-type origin [ 22 , 57 , 58 ]. For free mitochondria, outer membrane proteins such as TOM20 or TOM70 can be targeted for labelling [ 22 ].…”