Isolation and characterization of platelet-derived extracellular vesicles

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Aatonen, Maria T. ; Öhman, Tiina ; Nyman, Tuula A. ; Laitinen, Saara ; Grönholm, Mikaela ; Siljander, Pia R.-M. (2014)
  • Publisher: Journal of Extracellular Vesicles
  • Journal: Journal of Extracellular Vesicles, volume 3 (issn: 2001-3078, eissn: 2001-3078)
  • Related identifiers: doi: 10.3402/jev.v3.24692, doi: 10.3402/jev.v3.24692, pmc: PMC4125723
  • Subject: Cytology | microvesicle | extracellular vesicles | nanoparticle tracking analysis | proteomics | Extracellular Vesicles; Microparticle; Microvesicle; Exosome; Platelet; Nanoparticle Tracking Analysis; Transmission Electron Microscopy; Proteomics | Original Research Article | platelet | QH573-671 | transmission electron microscopy | exosome | microparticle

Background: Platelet-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.Methods: Platelets 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.Results: The 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.Conclusions: Although 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.Keywords: extracellular vesicles; microparticle; microvesicle; exosome; platelet; nanoparticle tracking analysis; transmission electron microscopy; proteomics(Published: 6 August 2014)Citation: Journal of Extracellular Vesicles 2014, 3: 24692 - access the supplementary material to this article, please see Supplementary files under Article Tools online. 
  • References (54)
    54 references, page 1 of 6

    1. Elzey BD, Sprague DL, Ratliff TL. The emerging role of platelets in adaptive immunity. Cell Immunol. 2005;238:1 9.

    2. Jenne CN, Urrutia R, Kubes P. Platelets: bridging hemostasis, inflammation, and immunity. Int J Lab Hematol. 2013;35: 254 61.

    3. Bertozzi CC, Schmaier AA, Mericko P, Hess PR, Zou Z, Chen M, et al. Platelets regulate lymphatic vascular development through CLEC-2-SLP-76 signaling. Blood. 2010;116:661 70.

    4. Aatonen M, Gronholm M, Siljander PR. Platelet-derived microvesicles: multitalented participants in intercellular communication. Semin Thromb Hemost. 2012;38:102 13.

    5. Heijnen HF, Schiel AE, Fijnheer R, Geuze HJ, Sixma JJ. Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. Blood. 1999;94:3791 9.

    6. Robertson C, Booth SA, Beniac DR, Coulthart MB, Booth TF, McNicol A. Cellular prion protein is released on exosomes from activated platelets. Blood. 2006;107:3907 11.

    7. Gambim MH, do Carmo Ade O, Marti L, Verissimo-Filho S, Lopes LR, Janiszewski M. Platelet-derived exosomes induce endothelial cell apoptosis through peroxynitrite generation: experimental evidence for a novel mechanism of septic vascular dysfunction. Crit Care. 2007;11:R107.

    8. Pook M, Tamming L, Padari K, Tiido T, Maimets T, Patarroyo M, et al. Platelets store laminins 411/421 and 511/521 in compartments distinct from alpha- or dense granules and secrete these proteins via microvesicles. J Thromb Haemost. 2014;12(4):519 527.

    9. Arraud N, Linares R, Tan S, Gounou C, Pasquet JM, Mornet S, et al. Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration. J Thromb Haemost. 2014;12(5):614 627.

    10. Flaumenhaft R, Dilks JR, Richardson J, Alden E, Patel-Hett SR, Battinelli E, et al. Megakaryocyte-derived microparticles: direct visualization and distinction from platelet-derived microparticles. Blood. 2009;113:1112 21.

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