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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

Protein yields of platelet MPs and EXOs. Platelets (125×106) were activated and MPs and EXOs were pelleted as described in Fig. 1. Protein concentrations were measured with µBCA assay from 9 donors. Total protein yields are shown as means with standard deviation and range (A and B). Since the total protein yields showed a large variation among donors, fold changes normalized to control conditions were calculated as in Fig. 2 (C). Statistical significances of the fold changes were determined by t-test (paired two-sample for means, two-way) assuming unequal variances. P-values of less than 0.05 (*), less than 0.01 (**) and less than 0.001 (***) were considered significant.
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Figure 0005: Protein yields of platelet MPs and EXOs. Platelets (125×106) were activated and MPs and EXOs were pelleted as described in Fig. 1. Protein concentrations were measured with µBCA assay from 9 donors. Total protein yields are shown as means with standard deviation and range (A and B). Since the total protein yields showed a large variation among donors, fold changes normalized to control conditions were calculated as in Fig. 2 (C). Statistical significances of the fold changes were determined by t-test (paired two-sample for means, two-way) assuming unequal variances. P-values of less than 0.05 (*), less than 0.01 (**) and less than 0.001 (***) were considered significant.

Mentions: To confirm the EV-generating potency of the activators measured by NTA, total protein was analyzed from the EV pellets. The protein yields of MPs and EXOs also varied depending on donors, and the variation was even more dramatic than when EV number was measured (Fig. 5A and B). Therefore, fold changes from each respective donor were again calculated by comparing each activation in relation to control. Fold changes of the total protein concentrations of MPs and EXOs induced by TC, Ca2+ ionophore and LPS paralleled the order of potency of the various activators seen by NTA (Fig. 5), but the increase in the Ca2+ ionophore–induced MPs and EXOs was substantially smaller compared to the differences seen by NTA (for the calculated value of MPs, see Fig. 2C). Next, protein/vesicle ratios were calculated to compare the activators for their ability to induce protein-packing intoEVs. Despite the smaller number of vesicles formed by TC and LPS, the EVs contained more protein, whereas the MPs and the EXOs induced by Ca2+ ionophore were strikingly protein-poor (Fig. 5D–E). Interestingly, EXOs, but not MPs, of unactivated platelets were also enriched in their protein content compared to the stimulated platelet-derived EXOs (Fig. 5E and Supplementary Fig. 4). Overall, in the 9 analyzed donors, there was either very little (Ca2+ionophore MP, R=−0.419, p-value 0.262, Ca2+ionophore EXO, R=−0.656, p-value 0.055, LPS MP R=0.678, p-value 0.045) or no correlation between the total protein and the vesicle concentration by NTA. Altogether, protein packaging to EVs differed depending on the inducing agonist, and the Ca2+ ionophore–induced EVs were quantitatively protein-poor in comparison to the platelet EVs formed by either TC or LPS activation, or constitutively in time.


Isolation and characterization of platelet-derived extracellular vesicles.

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

Protein yields of platelet MPs and EXOs. Platelets (125×106) were activated and MPs and EXOs were pelleted as described in Fig. 1. Protein concentrations were measured with µBCA assay from 9 donors. Total protein yields are shown as means with standard deviation and range (A and B). Since the total protein yields showed a large variation among donors, fold changes normalized to control conditions were calculated as in Fig. 2 (C). Statistical significances of the fold changes were determined by t-test (paired two-sample for means, two-way) assuming unequal variances. P-values of less than 0.05 (*), less than 0.01 (**) and less than 0.001 (***) were considered significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 0005: Protein yields of platelet MPs and EXOs. Platelets (125×106) were activated and MPs and EXOs were pelleted as described in Fig. 1. Protein concentrations were measured with µBCA assay from 9 donors. Total protein yields are shown as means with standard deviation and range (A and B). Since the total protein yields showed a large variation among donors, fold changes normalized to control conditions were calculated as in Fig. 2 (C). Statistical significances of the fold changes were determined by t-test (paired two-sample for means, two-way) assuming unequal variances. P-values of less than 0.05 (*), less than 0.01 (**) and less than 0.001 (***) were considered significant.
Mentions: To confirm the EV-generating potency of the activators measured by NTA, total protein was analyzed from the EV pellets. The protein yields of MPs and EXOs also varied depending on donors, and the variation was even more dramatic than when EV number was measured (Fig. 5A and B). Therefore, fold changes from each respective donor were again calculated by comparing each activation in relation to control. Fold changes of the total protein concentrations of MPs and EXOs induced by TC, Ca2+ ionophore and LPS paralleled the order of potency of the various activators seen by NTA (Fig. 5), but the increase in the Ca2+ ionophore–induced MPs and EXOs was substantially smaller compared to the differences seen by NTA (for the calculated value of MPs, see Fig. 2C). Next, protein/vesicle ratios were calculated to compare the activators for their ability to induce protein-packing intoEVs. Despite the smaller number of vesicles formed by TC and LPS, the EVs contained more protein, whereas the MPs and the EXOs induced by Ca2+ ionophore were strikingly protein-poor (Fig. 5D–E). Interestingly, EXOs, but not MPs, of unactivated platelets were also enriched in their protein content compared to the stimulated platelet-derived EXOs (Fig. 5E and Supplementary Fig. 4). Overall, in the 9 analyzed donors, there was either very little (Ca2+ionophore MP, R=−0.419, p-value 0.262, Ca2+ionophore EXO, R=−0.656, p-value 0.055, LPS MP R=0.678, p-value 0.045) or no correlation between the total protein and the vesicle concentration by NTA. Altogether, protein packaging to EVs differed depending on the inducing agonist, and the Ca2+ ionophore–induced EVs were quantitatively protein-poor in comparison to the platelet EVs formed by either TC or LPS activation, or constitutively in time.

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