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. 2017 Jul 26;7:6522. doi: 10.1038/s41598-017-03262-2

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Thrombin generation by platelet-derived vesicle fractions. The thrombogenicity of isolated EVs was studied with a thrombin generation assay. Aliquots of EV suspensions (protein content as indicated in the figure) were incubated with vesicle-free plasma, and the thrombin-dependent cleavage of a fluorogenic substrate was quantified over time as described in the Methods section. (a) Thrombin generation after a 60 min incubation of vesicle-free plasma with increasing amounts of platelet-derived EV fractions I and II in comparison to vesicles isolated with an exosome isolation kit (EXkit). Vesicle-free plasma served as control (n = 8); (b) Representative curve of dose-dependent kinetics of thrombin generation induced by EV fraction I under the same conditions as shown in panel a; (c,d) An increase of the centrifugal force from 1,500 g to 2,500 g to remove residual platelets prior to EV isolation did not affect the amount of thrombin generation nor the lag phase to the onset of thrombin generation (n = 6); (e,f) Effect of phosphatidylserine blockade on thrombin generation. Pre-incubation of EV fraction I with annexin V to mask phosphatidylserine decreased thrombin generation in a dose-dependent manner (n = 6); asterisks denote P values ≤0.05.