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Isolation and characterization of platelet-derived extracellular vesicles
Maria T Aatonen1, Tiina Ohman2, Tuula A Nyman2
1Division of Biochemistry and Biotechnology, Department of Biosciences, University of Helsinki, Helsinki, Finland.
Journal of Extracellular Vesicles
|August 23, 2014
Summary
Platelet extracellular vesicles (EVs) are released constitutively, but activation pathways significantly alter their quantity and protein content. This heterogeneity invalidates protein normalization and necessitates improved isolation methods for comprehensive analysis.
Area of Science:
- Hematology
- Cell Biology
- Biochemistry
Background:
- Platelet-derived extracellular vesicles (EVs) play roles in hemostasis, immunity, and development.
- Previous studies focused on microparticles, with less analysis of exosomes and EV characterization under varied conditions.
- Obtaining pure EVs free from contaminants has been a significant challenge.
Purpose of the Study:
- To optimize an extracellular vesicle (EV) isolation protocol from platelets.
- To compare the quantity and protein content of EVs generated by different activation pathways.
- To characterize the heterogeneity of platelet-derived EVs.
Main Methods:
- Platelets were isolated and activated using thrombin, collagen, lipopolysaccharide (LPS), or Ca(2+) ionophore.
- Extracellular vesicles (EVs), including microparticles and exosomes, were isolated via differential centrifugation.
- Quantification and size distribution were assessed using nanoparticle tracking analysis (NTA), and protein content was analyzed by proteomics.
Main Results:
- Platelets constitutively release EVs, with activation pathways differentially regulating EV quantity and quality.
- Thrombogenic activation was the most potent physiological EV generator.
- EVs exhibited significant heterogeneity in protein content and cargo depending on the activation method and vesicle subpopulation.
Conclusions:
- Platelet EV release can be modulated, with activation pathways dictating EV number and cargo.
- Activation-dependent variations invalidate the use of protein content for sample normalization.
- Current methods like flow cytometry and differential centrifugation may not capture all EV subpopulations, necessitating novel isolation and differentiation techniques.
