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Isolation of Platelet-Derived Extracellular Vesicles

  • Maria Aatonen
  • Sami Valkonen
  • Anita Böing
  • Yuana Yuana
  • Rienk Nieuwland
  • Pia SiljanderEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1545)

Abstract

Platelets participate in several physiological functions, including hemostasis, immunity, and development. Additionally, platelets play key roles in arterial thrombosis and cancer progression. Given this plethora of functions, there is a strong interest of the role of platelet-derived (extracellular) vesicles (PDEVs) as functional mediators and biomarkers. Moreover, the majority of the blood-borne EVs are thought to originate from either platelets or directly from the platelet precursor cells, the megakaryocytes, which reside in the bone marrow. To circumvent confusion, we use the term PDEVs for both platelet-derived and/or megakaryocyte-derived EVs. PDEVs can be isolated from blood or from isolated platelets after activation. In this chapter, we describe all commonly used PDEV isolation methods from blood and prepurified platelets.

Key words

Extracellular vesicle Platelet Microparticle Exosome Isolation Size-exclusion chromatography Immunobeads Gradient centrifugation 

Notes

Acknowledgments

Part of this work was funded by the European Metrology Research Programme (EMRP) under the Joint Research Project HLT02 (www.metves.eu). The EMRP is jointly funded by the EMRP participating countries within the European Association of National Metrology Institutes and the European Union (S.V., Y.Y., and R.N.).

Part of this work was funded by the Magnus Ehrnrooth Foundation, the Medicinska Understödsförening Liv och Hälsa r.f., the Otto Malm Foundation, and the Oscar Öflund Foundation (M.A. and P.S.). Part of the work was funded by the SalWe Research Program GET IT DONE Tekes grant nro 3986/31/2013 (S.V., P.S.).

References

  1. 1.
    Flaumenhaft R, Mairuhu AT, Italiano JE (2010) Platelet- and megakaryocyte-derived microparticles. Semin Thromb Hemost 36(8):881–887CrossRefPubMedGoogle Scholar
  2. 2.
    van der Pol E, van Gemert MJ, Sturk A, Nieuwland R, van Leeuwen TG (2012) Single vs. swarm detection of microparticles and exosomes by flow cytometry. J Thromb Haemost 10(5):919–930CrossRefPubMedGoogle Scholar
  3. 3.
    van der Pol E, Coumans FA, Grootemaat AE, Gardiner C, Sargent IL, Harrison P et al (2014) Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J Thromb Haemost 12(7):1182–1192CrossRefPubMedGoogle Scholar
  4. 4.
    Varga Z, Yuana Y, Grootemaat AE, van der Pol E, Gollwitzer C, Krumrey M et al (2014) Towards traceable size determination of extracellular vesicles. J Extracell Vesicles 3:PMCID: PMC3916677. doi: 10.3402/jev.v3.23298 CrossRefGoogle Scholar
  5. 5.
    Gemmell CH, Sefton MV, Yeo EL (1993) Platelet-derived microparticle formation involves glycoprotein IIb-IIIa. Inhibition by RGDS and a Glanzmann’s thrombasthenia defect. J Biol Chem 268(20):14586–14589PubMedGoogle Scholar
  6. 6.
    Siljander P, Farndale RW, Feijge MA, Comfurius P, Kos S, Bevers EM et al (2001) Platelet adhesion enhances the glycoprotein VI-dependent procoagulant response: Involvement of p38 MAP kinase and calpain. Arterioscler Thromb Vasc Biol 21(4):618–627CrossRefPubMedGoogle Scholar
  7. 7.
    Aatonen MT, Ohman T, Nyman TA, Laitinen S, Gronholm M, Siljander PR (2014) Isolation and characterization of platelet-derived extracellular vesicles. J Extracell Vesicles 3:PMCID: PMC4125723. doi: 10.3402/jev.v3.24692 CrossRefGoogle Scholar
  8. 8.
    Sims PJ, Wiedmer T, Esmon CT, Weiss HJ, Shattil SJ (1989) Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane. Studies in Scott syndrome: an isolated defect in platelet procoagulant activity. J Biol Chem 264(29):17049–17057PubMedGoogle Scholar
  9. 9.
    Ray DM, Spinelli SL, Pollock SJ, Murant TI, O'Brien JJ, Blumberg N et al (2008) Peroxisome proliferator-activated receptor gamma and retinoid X receptor transcription factors are released from activated human platelets and shed in microparticles. Thromb Haemost 99(1):86–95PubMedPubMedCentralGoogle Scholar
  10. 10.
    Siljander P, Carpen O, Lassila R (1996) Platelet-derived microparticles associate with fibrin during thrombosis. Blood 87(11):4651–4663PubMedGoogle Scholar
  11. 11.
    Gambim MH, do Carmo-Ade O, Marti L, Verissimo-Filho S, Lopes LR, Janiszewski M (2007) Platelet-derived exosomes induce endothelial cell apoptosis through peroxynitrite generation: experimental evidence for a novel mechanism of septic vascular dysfunction. Crit Care 11(5):107CrossRefGoogle Scholar
  12. 12.
    Brown GT, McIntyre TM (2011) Lipopolysaccharide signaling without a nucleus: kinase cascades stimulate platelet shedding of proinflammatory IL-1beta-rich microparticles. J Immunol 186(9):5489–5496CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Yuana Y, Osanto S, Bertina RM (2012) Use of immuno-magnetic beads for direct capture of nanosized microparticles from plasma. Blood Coagul Fibrinolysis 23(3):244–250CrossRefPubMedGoogle Scholar
  14. 14.
    Holmberg A, Blomstergren A, Nord O, Lukacs M, Lundeberg J, Uhlen M (2005) The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures. Electrophoresis 26(3):501–510CrossRefPubMedGoogle Scholar
  15. 15.
    Green NM (1990) Avidin and streptavidin. Methods Enzymol 184:51–67CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Maria Aatonen
    • 1
  • Sami Valkonen
    • 1
    • 2
  • Anita Böing
    • 2
  • Yuana Yuana
    • 2
  • Rienk Nieuwland
    • 2
  • Pia Siljander
    • 1
    Email author
  1. 1.Division of Biochemistry and Biotechnology, Faculty of Biological and Environmental SciencesUniversity of HelsinkiHelsinkiFinland
  2. 2.Laboratory of Experimental Clinical Chemistry, Vesicle Observation CenterAcademic Medical Centre of the University ofAmsterdamAmsterdamThe Netherlands

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