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. 2014 Aug 6:3.
doi: 10.3402/jev.v3.24692. eCollection 2014.

Isolation and characterization of platelet-derived extracellular vesicles

Maria T Aatonen  1 , Tiina Ohman  2 , Tuula A Nyman  2 , Saara Laitinen  3 , Mikaela Grönholm  1 , Pia R-M Siljander  4
Affiliations

Isolation and characterization of platelet-derived extracellular vesicles

Maria T Aatonen et al. J Extracell Vesicles. .

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.

Keywords: exosome; extracellular vesicles; microparticle; microvesicle; nanoparticle tracking analysis; platelet; proteomics; transmission electron microscopy.

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Figures

Fig. 1
Fig. 1
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.
Fig. 2
Fig. 2
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.
Fig. 3
Fig. 3
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.
Fig. 4
Fig. 4
Size distributions of the total EV populations by NTA and TEM. Vesicle size distributions are shown as percentages of the total EV populations analyzed by NTA (A, 9 independent experiments) and TEM (B, 4 independent experiments). Diameters of vesicles in TEM micrographs were measured manually from 53–81 images/activation and proportioned to a scale bar. At least 400 vesicles were calculated for each condition. Representative images of NTA (insert in A) and the uranyl acetate–stained total EVs induced by Ca2+ ionophore (insert in B, original magnification 4800×). Table comparing the conditions showing statistical significances from A (C). 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.
Fig. 5
Fig. 5
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.
Fig. 6
Fig. 6
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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