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Isolation and characterization of platelet-derived extracellular vesicles.

Aatonen MT, Ohman T, Nyman TA, Laitinen S, Grönholm M, Siljander PR - J Extracell Vesicles (2014)

Bottom Line: Therefore, we optimized an EV isolation protocol and compared the quantity and protein content of EVs induced by different agonists.Ca(2+) ionophore generated a large population of protein-poor and unselectively packed EVs.These activation-dependent variations render the use of protein content in sample normalization invalid.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry and Biotechnology, Department of Biosciences, University of Helsinki, Helsinki, Finland.

ABSTRACT

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 Ca(2+) 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. Ca(2+) 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.

No MeSH data available.


Related in: MedlinePlus

Flow chart of the isolation of platelets and platelet-free EV subpopulations. Platelets were isolated from whole blood using an iodixanol gradient, as described in the Materials and methods section. After platelet activation to induce vesiculation, platelets and cell remnants were removed by the detailed differential centrifugations and the obtained MP and EXO pellets were stored –80°C for further analyses. Alternatively, the supernatants containing total EVs or EXOs were freshly used for EM- and NTA-analyses. F1, platelet suspension; F2, purified platelets; F3, leukocytes/erythrocytes/granulocytes; PGE1, prostaglandin E1; ACD, acidic citrate dextrose.
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Figure 0001: Flow chart of the isolation of platelets and platelet-free EV subpopulations. Platelets were isolated from whole blood using an iodixanol gradient, as described in the Materials and methods section. After platelet activation to induce vesiculation, platelets and cell remnants were removed by the detailed differential centrifugations and the obtained MP and EXO pellets were stored –80°C for further analyses. Alternatively, the supernatants containing total EVs or EXOs were freshly used for EM- and NTA-analyses. F1, platelet suspension; F2, purified platelets; F3, leukocytes/erythrocytes/granulocytes; PGE1, prostaglandin E1; ACD, acidic citrate dextrose.

Mentions: Venous blood was drawn from healthy volunteers who had given their informed consent according to the Declaration of Helsinki and had not taken any medication during the prior 7 days. Blood counts were analyzed with Coulter T-540 (Beckman Coulter, Inc., CA, USA). Blood was collected in the laboratory at the same time in the morning before breakfast, using an 18-gauge needle with a free-flowing technique to prevent platelet activation. The first 3 ml of blood was discarded and the next 25 ml was taken into 5 ml of acidic citrate dextrose (ACD, 39 mM citric acid, 75 mM sodium citrate, 135 mM [D]-glucose, pH 4.5). A detailed flow chart of the isolation procedure, modified from Birschmann et al. (27), is displayed in Fig. 1. Within 20 min of collection, platelet-rich plasma (PRP) was obtained by centrifugation at 200×g for 12 min at room temperature (RT) without a brake (Eppendorf 5810R with A-4-81 rotor, Eppendorf AG, Hamburg, Germany). PRP was transferred into a polypropylene tube and 1/10 volume ACD and 100 ng/ml prostaglandin E1 (PGE1, Sigma-Aldrich, St. Louis, MO, USA) was added to prevent platelet activation during isolation. PRP was centrifuged at 900×g for 15 min at RT and plasma was aspirated. Platelets were suspended in 6 ml of Hepes-NaCl2 buffer (10 mM Hepes, 0.85% NaCl, pH 7.4) and layered on a discontinuous 10–17% iodixanol gradient (OptiPrep, Axis-Shield plc., Dundee, Scotland) in Hepes/NaCl buffer (27). The gradient was centrifuged at 300×g for 20 min at RT without a brake and the platelet fraction was collected (Fig. 1). Platelets were centrifuged at 900×g for 15 min at RT and the resulting pellet was washed once and resuspended with Ca2+-free Tyrode-Hepes buffer (137 mM NaCl, 0.3 mM NaH2PO4, 3.5 mM Hepes, 5.5 mM [D]-glucose, pH 7.35). Platelet concentration was measured with Coulter T-540.


Isolation and characterization of platelet-derived extracellular vesicles.

Aatonen MT, Ohman T, Nyman TA, Laitinen S, Grönholm M, Siljander PR - J Extracell Vesicles (2014)

Flow chart of the isolation of platelets and platelet-free EV subpopulations. Platelets were isolated from whole blood using an iodixanol gradient, as described in the Materials and methods section. After platelet activation to induce vesiculation, platelets and cell remnants were removed by the detailed differential centrifugations and the obtained MP and EXO pellets were stored –80°C for further analyses. Alternatively, the supernatants containing total EVs or EXOs were freshly used for EM- and NTA-analyses. F1, platelet suspension; F2, purified platelets; F3, leukocytes/erythrocytes/granulocytes; PGE1, prostaglandin E1; ACD, acidic citrate dextrose.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125723&req=5

Figure 0001: Flow chart of the isolation of platelets and platelet-free EV subpopulations. Platelets were isolated from whole blood using an iodixanol gradient, as described in the Materials and methods section. After platelet activation to induce vesiculation, platelets and cell remnants were removed by the detailed differential centrifugations and the obtained MP and EXO pellets were stored –80°C for further analyses. Alternatively, the supernatants containing total EVs or EXOs were freshly used for EM- and NTA-analyses. F1, platelet suspension; F2, purified platelets; F3, leukocytes/erythrocytes/granulocytes; PGE1, prostaglandin E1; ACD, acidic citrate dextrose.
Mentions: Venous blood was drawn from healthy volunteers who had given their informed consent according to the Declaration of Helsinki and had not taken any medication during the prior 7 days. Blood counts were analyzed with Coulter T-540 (Beckman Coulter, Inc., CA, USA). Blood was collected in the laboratory at the same time in the morning before breakfast, using an 18-gauge needle with a free-flowing technique to prevent platelet activation. The first 3 ml of blood was discarded and the next 25 ml was taken into 5 ml of acidic citrate dextrose (ACD, 39 mM citric acid, 75 mM sodium citrate, 135 mM [D]-glucose, pH 4.5). A detailed flow chart of the isolation procedure, modified from Birschmann et al. (27), is displayed in Fig. 1. Within 20 min of collection, platelet-rich plasma (PRP) was obtained by centrifugation at 200×g for 12 min at room temperature (RT) without a brake (Eppendorf 5810R with A-4-81 rotor, Eppendorf AG, Hamburg, Germany). PRP was transferred into a polypropylene tube and 1/10 volume ACD and 100 ng/ml prostaglandin E1 (PGE1, Sigma-Aldrich, St. Louis, MO, USA) was added to prevent platelet activation during isolation. PRP was centrifuged at 900×g for 15 min at RT and plasma was aspirated. Platelets were suspended in 6 ml of Hepes-NaCl2 buffer (10 mM Hepes, 0.85% NaCl, pH 7.4) and layered on a discontinuous 10–17% iodixanol gradient (OptiPrep, Axis-Shield plc., Dundee, Scotland) in Hepes/NaCl buffer (27). The gradient was centrifuged at 300×g for 20 min at RT without a brake and the platelet fraction was collected (Fig. 1). Platelets were centrifuged at 900×g for 15 min at RT and the resulting pellet was washed once and resuspended with Ca2+-free Tyrode-Hepes buffer (137 mM NaCl, 0.3 mM NaH2PO4, 3.5 mM Hepes, 5.5 mM [D]-glucose, pH 7.35). Platelet concentration was measured with Coulter T-540.

Bottom Line: Therefore, we optimized an EV isolation protocol and compared the quantity and protein content of EVs induced by different agonists.Ca(2+) ionophore generated a large population of protein-poor and unselectively packed EVs.These activation-dependent variations render the use of protein content in sample normalization invalid.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry and Biotechnology, Department of Biosciences, University of Helsinki, Helsinki, Finland.

ABSTRACT

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 Ca(2+) 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. Ca(2+) 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.

No MeSH data available.


Related in: MedlinePlus