Extracellular Vesicles Derived From Platelets, Red Blood Cells, and Monocyte-Like Cells Differ Regarding Their Ability to Induce Factor XII-Dependent Thrombin Generation.

Carla Tripisciano,Viktoria Weber,René Weiss,Sobha Karuthedom George,Michael B Fischer

doi:10.3389/fcell.2020.00298

Carla Tripisciano, Viktoria Weber + Show 3 more

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https://doi.org/10.3389/fcell.2020.00298

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Abstract

As transmitters of biological information, extracellular vesicles (EVs) are crucial for the maintenance of physiological homeostasis, but also contribute to pathological conditions, such as thrombotic disorders. The ability of EVs to support thrombin generation has been linked to their exposure of phosphatidylserine, an anionic phospholipid that is normally restricted to the inner leaflet of the plasma membrane but exposed on the outer leaflet during EV biogenesis. Here, we investigated whether EVs of different cellular origin and from different settings, namely platelets and red blood cells from blood bank units and a monocyte-like cell line (THP-1), differ regarding their potential to support factor XII-dependent thrombin generation. EVs were isolated from blood products or THP-1 cell culture supernatants using differential centrifugation and characterized by a combination of flow cytometry, nanoparticle tracking analysis, and Western blotting. Soluble factors co-enriched during the isolation of EVs were depleted from blood-cell derived EV fractions using size exclusion chromatography, while proteins bound to the surface of EVs were degraded by mild protease treatment. We found that platelet-derived and red blood cell-derived EVs supported factor XII-dependent thrombin generation to comparable extents, while monocytic EVs failed to support thrombin generation when added to EV-depleted human plasma. We excluded a major contribution of co-enriched soluble proteins or of proteins bound to the EV surface to the thrombogenicity of blood cell-derived EVs. Our data suggest that the enhanced potential of blood cell-derived EVs to support thrombin generation is rather due to enhanced exposure of phosphatidylserine on the surface of blood cell-derived EVs. Extending these investigations to EVs from other cell types, such as mesenchymal stromal cells, will be crucial for their future therapeutic applications.

Highlights

Extracellular vesicles are sub-cellular fragments originating from the endosomal system or shed from the plasma membrane of virtually all cell types of the human body, generated under physiological or pathological conditions (Deatherage and Cookson, 2012; Sedgwick and D’Souza-Schorey, 2018)
According to flow cytometry (FC), 94.7 ± 0.9%, 90.2 ± 0.5%, and 73 ± 13.2% of all events in the Extracellular vesicles (EVs) gate exposed phosphatidylserine, as indicated by their positive lactadherin staining. 93.5 ± 2.1 and 88.8 ± 0.2%, of platelet-derived EVs (pEVs) and red blood cell-derived EVs (rbcEVs), respectively, carried CD41 or CD235a, while only 23.8 ± 5.4% of monocyte-like cell-derived EVs (mlEVs) stained positive for CD45
To obtain an estimate of phosphatidylserine exposed on the surface of pEVs, rbcEVs, and mlEVs, we calculated the difference between specific lactadherin signals for each vesicle subtype and fluorochrome-labeled reagent controls and found that pEVs and rbcEVs (49.3 ± 2.9) exhibited higher surface expression of phosphatidylserine as compared to mlEVs (16.5 ± 2.0)

Summary

Introduction

Extracellular vesicles are sub-cellular fragments originating from the endosomal system or shed from the plasma membrane of virtually all cell types of the human body, generated under physiological or pathological conditions (Deatherage and Cookson, 2012; Sedgwick and D’Souza-Schorey, 2018). Small EVs (40–130 nm, “exosomes”) originate from the fusion of endosomal multivesicular bodies with the plasma membrane, whereas the formation of large EVs (100–1000 nm; “microvesicles”) involves actin-myosin-based contraction of the cytoskeleton and a rearrangement of plasma membrane phospholipids (D’Souza-Schorey and Clancy, 2012; Hessvik and Llorente, 2017). This phospholipid rearrangement results in the translocation of negatively charged phosphatidylserine from the inner to the outer membrane leaflet, creating a catalytic surface for the attachment and interaction of coagulation factors on the EV surface. The exposure of phosphatidylserine on EVs may further contribute to coagulation by supporting the transformation of TF, the high-affinity receptor for FVII/FVIIa and main physiological initiator of the extrinsic coagulation pathway (Mackman, 2009), from a quiescent form into a biologically active state (Chen and Hogg, 2013; Ansari et al, 2016; Grover and Mackman, 2018)

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