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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

Comparison of the vesicle-inducing capacity of common platelet agonists. The capacity of different activators to induce platelet vesiculation was compared in 6 independent experiments. Platelets (250×106 platelets/ml) were activated and the differential centrifugation–separated supernatants of the isolated vesicle populations (total EVs and EXOs) were measured by NTA. Concentration (108 vesicles/ml) of the formed total EVs (A) and EXOs (B) is shown as mean with standard deviation. Fold changes were calculated by comparing the agonist-induced activation to their time-matched controls. Statistical significances 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 0003: Comparison of the vesicle-inducing capacity of common platelet agonists. The capacity of different activators to induce platelet vesiculation was compared in 6 independent experiments. Platelets (250×106 platelets/ml) were activated and the differential centrifugation–separated supernatants of the isolated vesicle populations (total EVs and EXOs) were measured by NTA. Concentration (108 vesicles/ml) of the formed total EVs (A) and EXOs (B) is shown as mean with standard deviation. Fold changes were calculated by comparing the agonist-induced activation to their time-matched controls. Statistical significances 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: Because of the difference in the number of generated EVs between our NTA-observations and the previous literature, we also measured EV formation by additional platelet activators shown to induce vesiculation. Collagen and the direct glycoprotein VI-activating collagen-related peptide, CRP-XL, have been considered stronger inducers of platelet microvesiculation (32) than thrombin, the thrombin-mimicking hexapeptide TRAP-6 and ADP (29). The order of potency of the agonists in generating total EVs measured by NTA was thrombin > collagen-related peptide (CRP-XL) > TC co-stimulus > collagen, whereas LPS, ADP and TRAP-6 did not differ from the basal level of control samples (Fig. 3A). Also, a slight time-dependent (from 30 min to >3 h) increase in total EVs was observed in the absence of an added agonist (Fig. 3A). To monitor the time-dependent EV formation in the absence of an agonist, a further NTA analysis was undertaken for EV samples from 30 min, 1 h, 3 h, 4 h, 6 h and 8 h incubations, which verified a slight time-dependent increase up to 6 h, followed by a subsequent exponential increase in platelet vesiculation (data not shown). NTA analysis also showed that TC, CRP-XL and thrombin activations increased the number of EXOs, but the results did not reach a statistical difference in the 6 analyzed donors.


Isolation and characterization of platelet-derived extracellular vesicles.

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

Comparison of the vesicle-inducing capacity of common platelet agonists. The capacity of different activators to induce platelet vesiculation was compared in 6 independent experiments. Platelets (250×106 platelets/ml) were activated and the differential centrifugation–separated supernatants of the isolated vesicle populations (total EVs and EXOs) were measured by NTA. Concentration (108 vesicles/ml) of the formed total EVs (A) and EXOs (B) is shown as mean with standard deviation. Fold changes were calculated by comparing the agonist-induced activation to their time-matched controls. Statistical significances 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 0003: Comparison of the vesicle-inducing capacity of common platelet agonists. The capacity of different activators to induce platelet vesiculation was compared in 6 independent experiments. Platelets (250×106 platelets/ml) were activated and the differential centrifugation–separated supernatants of the isolated vesicle populations (total EVs and EXOs) were measured by NTA. Concentration (108 vesicles/ml) of the formed total EVs (A) and EXOs (B) is shown as mean with standard deviation. Fold changes were calculated by comparing the agonist-induced activation to their time-matched controls. Statistical significances 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: Because of the difference in the number of generated EVs between our NTA-observations and the previous literature, we also measured EV formation by additional platelet activators shown to induce vesiculation. Collagen and the direct glycoprotein VI-activating collagen-related peptide, CRP-XL, have been considered stronger inducers of platelet microvesiculation (32) than thrombin, the thrombin-mimicking hexapeptide TRAP-6 and ADP (29). The order of potency of the agonists in generating total EVs measured by NTA was thrombin > collagen-related peptide (CRP-XL) > TC co-stimulus > collagen, whereas LPS, ADP and TRAP-6 did not differ from the basal level of control samples (Fig. 3A). Also, a slight time-dependent (from 30 min to >3 h) increase in total EVs was observed in the absence of an added agonist (Fig. 3A). To monitor the time-dependent EV formation in the absence of an agonist, a further NTA analysis was undertaken for EV samples from 30 min, 1 h, 3 h, 4 h, 6 h and 8 h incubations, which verified a slight time-dependent increase up to 6 h, followed by a subsequent exponential increase in platelet vesiculation (data not shown). NTA analysis also showed that TC, CRP-XL and thrombin activations increased the number of EXOs, but the results did not reach a statistical difference in the 6 analyzed donors.

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