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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

Concentration of EVs induced by three platelet agonists. Platelets (250×106 platelets/ml) were activated by thrombin and collagen (TC) co-stimulus, LPS or Ca2+ ionophore, and supernatants (total EVs, EXOs) obtained by the differential centrifugations were measured by NTA from 9 donors. Concentrations (108 vesicles/ml) of the formed total EVs and EXOs are shown from 5 different conditions as means with standard deviation and range (A and C). Values for MPs were calculated by subtracting the EXO concentrations from the total EV concentrations (C). Due to the large variability of EV concentrations between donors, fold changes were calculated by comparing each activation to its time-matched control (ctrl 30 min for TC and Ca2+ ionophore, ctrl 3 h for LPS) (B). 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 0.01 (**) and less than 0.001 (***) were considered significant.
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Figure 0002: Concentration of EVs induced by three platelet agonists. Platelets (250×106 platelets/ml) were activated by thrombin and collagen (TC) co-stimulus, LPS or Ca2+ ionophore, and supernatants (total EVs, EXOs) obtained by the differential centrifugations were measured by NTA from 9 donors. Concentrations (108 vesicles/ml) of the formed total EVs and EXOs are shown from 5 different conditions as means with standard deviation and range (A and C). Values for MPs were calculated by subtracting the EXO concentrations from the total EV concentrations (C). Due to the large variability of EV concentrations between donors, fold changes were calculated by comparing each activation to its time-matched control (ctrl 30 min for TC and Ca2+ ionophore, ctrl 3 h for LPS) (B). 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 0.01 (**) and less than 0.001 (***) were considered significant.

Mentions: Next, EVs were produced from platelets by different agonists, thrombin and collagen co-stimulation (TC), Ca2+ ionophore and LPS, and isolated by differential centrifugations as detailed in Fig. 1. The platelet-derived EV populations were analyzed with NTA and compared with their time-matched controls. NTA-analyses were only performed in the supernatants containing total EVs or the MP-depleted EXOs to avoid pellet suspension-induced aggregate formation of EVs. As expected by the previously published results, Ca2 + ionophore induced the greatest number of platelet EVs, 14.5×of control (142±11×108 EVs/ml, Fig. 2A). Among the physiologically relevant agonists, surprisingly, the thrombotic activation (TC) only induced approx. 1.5×increase of the mean total EV concentration, and the LPS-induced EV generation hardly differed from its control (Fig. 2B). Depending on the agonist 1.5–4 fold more MPs than EXOs were formed under all conditions, as calculated by subtracting the remaining EXO concentrations from the total EVs. NTA analysis of the EXO fraction showed a 2- and a 10-fold increase for TC- and Ca2+ ionophore-activation, respectively, whereas no increase of EXOs was observed by LPS (Fig. 2B).


Isolation and characterization of platelet-derived extracellular vesicles.

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

Concentration of EVs induced by three platelet agonists. Platelets (250×106 platelets/ml) were activated by thrombin and collagen (TC) co-stimulus, LPS or Ca2+ ionophore, and supernatants (total EVs, EXOs) obtained by the differential centrifugations were measured by NTA from 9 donors. Concentrations (108 vesicles/ml) of the formed total EVs and EXOs are shown from 5 different conditions as means with standard deviation and range (A and C). Values for MPs were calculated by subtracting the EXO concentrations from the total EV concentrations (C). Due to the large variability of EV concentrations between donors, fold changes were calculated by comparing each activation to its time-matched control (ctrl 30 min for TC and Ca2+ ionophore, ctrl 3 h for LPS) (B). 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 0.01 (**) 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 0002: Concentration of EVs induced by three platelet agonists. Platelets (250×106 platelets/ml) were activated by thrombin and collagen (TC) co-stimulus, LPS or Ca2+ ionophore, and supernatants (total EVs, EXOs) obtained by the differential centrifugations were measured by NTA from 9 donors. Concentrations (108 vesicles/ml) of the formed total EVs and EXOs are shown from 5 different conditions as means with standard deviation and range (A and C). Values for MPs were calculated by subtracting the EXO concentrations from the total EV concentrations (C). Due to the large variability of EV concentrations between donors, fold changes were calculated by comparing each activation to its time-matched control (ctrl 30 min for TC and Ca2+ ionophore, ctrl 3 h for LPS) (B). 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 0.01 (**) and less than 0.001 (***) were considered significant.
Mentions: Next, EVs were produced from platelets by different agonists, thrombin and collagen co-stimulation (TC), Ca2+ ionophore and LPS, and isolated by differential centrifugations as detailed in Fig. 1. The platelet-derived EV populations were analyzed with NTA and compared with their time-matched controls. NTA-analyses were only performed in the supernatants containing total EVs or the MP-depleted EXOs to avoid pellet suspension-induced aggregate formation of EVs. As expected by the previously published results, Ca2 + ionophore induced the greatest number of platelet EVs, 14.5×of control (142±11×108 EVs/ml, Fig. 2A). Among the physiologically relevant agonists, surprisingly, the thrombotic activation (TC) only induced approx. 1.5×increase of the mean total EV concentration, and the LPS-induced EV generation hardly differed from its control (Fig. 2B). Depending on the agonist 1.5–4 fold more MPs than EXOs were formed under all conditions, as calculated by subtracting the remaining EXO concentrations from the total EVs. NTA analysis of the EXO fraction showed a 2- and a 10-fold increase for TC- and Ca2+ ionophore-activation, respectively, whereas no increase of EXOs was observed by LPS (Fig. 2B).

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