Isolation and characterization of platelet‐derived extracellular vesicles

Maria T. Aatonen; Tiina Öhman; Tuula A. Nyman; Saara Laitinen; Mikaela Grönholm; Pia R.‐M. Siljander

doi:10.3402/jev.v3.24692

journal article Open Access Jan 01, 2014

Isolation and characterization of platelet‐derived extracellular vesicles

Maria T. Aatonen Tiina Öhman

Tuula A. Nyman

Saara Laitinen Mikaela Grönholm

Pia R.‐M. Siljander

Journal of Extracellular Vesicles Vol. 3 No. 1 · Wiley

View at Publisher Save 10.3402/jev.v3.24692

Abstract

BackgroundPlatelet‐derived extracellular vesicles (EVs) participate, for example, in haemostasis, immunity and development. Most studies of platelet EVs have targeted microparticles, whereas exosomes and EV characterization under various conditions have been less analyzed. Studies have been hampered by the difficulty in obtaining EVs free from contaminating cells and platelet remnants. Therefore, we optimized an EV isolation protocol and compared the quantity and protein content of EVs induced by different agonists.MethodsPlatelets isolated with iodixanol gradient were activated by thrombin and collagen, lipopolysaccharide (LPS) or Ca2+ ionophore. Microparticles and exosomes were isolated by differential centrifugations. EVs were quantitated by nanoparticle tracking analysis (NTA) and total protein. Size distributions were determined by NTA and electron microscopy. Proteomics was used to characterize the differentially induced EVs.ResultsThe main EV populations were 100–250 nm and over 90% were <500 nm irrespective of the activation. However, activation pathways differentially regulated the quantity and the quality of EVs, which also formed constitutively. Thrombogenic activation was the most potent physiological EV‐generator. LPS was a weak inducer of EVs, which had a selective protein content from the thrombogenic EVs. Ca2+ ionophore generated a large population of protein‐poor and unselectively packed EVs. By proteomic analysis, EVs were highly heterogeneous after the different activations and between the vesicle subpopulations.ConclusionsAlthough platelets constitutively release EVs, vesiculation can be increased, and the activation pathway determines the number and the cargo of the formed EVs. These activation‐dependent variations render the use of protein content in sample normalization invalid. Since most platelet EVs are 100–250 nm, only a fraction has been analyzed by previously used methods, for example, flow cytometry. As the EV subpopulations could not be distinguished and large vesicle populations may be lost by differential centrifugation, novel methods are required for the isolation and the differentiation of all EVs.

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Details

Published: Jan 01, 2014
Vol/Issue: 3(1)
License: View

Authors

M

Maria T. Aatonen

Division of Biochemistry and Biotechnology, Department of Biosciences University of Helsinki Helsinki Finland

T

Tiina Öhman

Institute of Biotechnology, University of Helsinki Helsinki Finland

T

Tuula A. Nyman

Institute of Biotechnology, University of Helsinki Helsinki Finland

S

Saara Laitinen

Finnish Red Cross Blood Service Helsinki Finland

M

Mikaela Grönholm

Division of Biochemistry and Biotechnology, Department of Biosciences University of Helsinki Helsinki Finland

P

Pia R.‐M. Siljander

Division of Biochemistry and Biotechnology, Department of Biosciences University of Helsinki Helsinki Finland; Division of Pharmaceutical Biosciences, Faculty of Pharmacy University of Helsinki Helsinki Finland

Cite This Article

Maria T. Aatonen, Tiina Öhman, Tuula A. Nyman, et al. (2014). Isolation and characterization of platelet‐derived extracellular vesicles. Journal of Extracellular Vesicles, 3(1). https://doi.org/10.3402/jev.v3.24692

Isolation and characterization of platelet‐derived extracellular vesicles

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