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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

Mass spectrometry analysis of platelet MPs and EXOs. Proteomic comparison of MPs and EXOs from 5 different conditions was performed by LTQ Orbitrap XL mass spectrometry from pooled samples of 6 donors to compare the molecular content of different EV subpopulations. Venn diagrams illustrate the common and the unique proteins in MPs (A) and EXOs (B). Common identified proteins for all conditions are listed in (C).
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Figure 0006: Mass spectrometry analysis of platelet MPs and EXOs. Proteomic comparison of MPs and EXOs from 5 different conditions was performed by LTQ Orbitrap XL mass spectrometry from pooled samples of 6 donors to compare the molecular content of different EV subpopulations. Venn diagrams illustrate the common and the unique proteins in MPs (A) and EXOs (B). Common identified proteins for all conditions are listed in (C).

Mentions: To test a hypothesis that the major differences in platelet EV formation by physiological agonists may be more qualitative and not quantitative, we performed a mass spectrometric analysis of the 10 different vesicle populations. Proteomic analysis showed a total of 267 identified proteins for MPs and 194 proteins for EXOs (Supplementary Table I). From the Ca2+ ionophore–induced EVs, 193 unique proteins were identified for MPs and 106 for EXOs, respectively (Fig. 6), which suggested that these EVs represent more of an unselected molecularcompilation of the platelet itself than EVs with a specifically packed content. Venn diagrams of the protein distribution showed that 18 proteins were shared among the different activations within MPs and 19 within the different EXOs, respectively (Fig. 6). From these shared proteins, 13 were common for both MPs and EXOs populations and included common platelet surface proteins such as integrin αIIb and glycoprotein Ib complexes, but also functional proteins such as chemokines (platelet basic protein and platelet factor 4). Multimerin-1 was found exclusively in all the EXO populations. In addition to the agonist-dependent differences, the MP and EXO cargo also changed in the unactivated samples in a time-dependent manner. As a summary, the qualitative protein content of both MPs and EXOs depends on the platelet activating condition inducing the vesicle generation.


Isolation and characterization of platelet-derived extracellular vesicles.

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

Mass spectrometry analysis of platelet MPs and EXOs. Proteomic comparison of MPs and EXOs from 5 different conditions was performed by LTQ Orbitrap XL mass spectrometry from pooled samples of 6 donors to compare the molecular content of different EV subpopulations. Venn diagrams illustrate the common and the unique proteins in MPs (A) and EXOs (B). Common identified proteins for all conditions are listed in (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 0006: Mass spectrometry analysis of platelet MPs and EXOs. Proteomic comparison of MPs and EXOs from 5 different conditions was performed by LTQ Orbitrap XL mass spectrometry from pooled samples of 6 donors to compare the molecular content of different EV subpopulations. Venn diagrams illustrate the common and the unique proteins in MPs (A) and EXOs (B). Common identified proteins for all conditions are listed in (C).
Mentions: To test a hypothesis that the major differences in platelet EV formation by physiological agonists may be more qualitative and not quantitative, we performed a mass spectrometric analysis of the 10 different vesicle populations. Proteomic analysis showed a total of 267 identified proteins for MPs and 194 proteins for EXOs (Supplementary Table I). From the Ca2+ ionophore–induced EVs, 193 unique proteins were identified for MPs and 106 for EXOs, respectively (Fig. 6), which suggested that these EVs represent more of an unselected molecularcompilation of the platelet itself than EVs with a specifically packed content. Venn diagrams of the protein distribution showed that 18 proteins were shared among the different activations within MPs and 19 within the different EXOs, respectively (Fig. 6). From these shared proteins, 13 were common for both MPs and EXOs populations and included common platelet surface proteins such as integrin αIIb and glycoprotein Ib complexes, but also functional proteins such as chemokines (platelet basic protein and platelet factor 4). Multimerin-1 was found exclusively in all the EXO populations. In addition to the agonist-dependent differences, the MP and EXO cargo also changed in the unactivated samples in a time-dependent manner. As a summary, the qualitative protein content of both MPs and EXOs depends on the platelet activating condition inducing the vesicle generation.

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