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. 2019 Apr 16;10:1780. doi: 10.1038/s41467-019-09607-x

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© The Author(s) 2019

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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Fig. 1 — Characterization of human blood from influenza-infected patients. a–d Blood from influenza-infected patients was fixed after intravenous collection and stained as described in Methods. In all cases DNA was assessed by DAPI. a Many platelets appeared similar to those observed in control blood from healthy donors with a size of 2–5 µm. b Some platelets from influenza-infected patients had undergone spreading with a distinctive distribution of CD41. c Platelets and platelet microparticles associated with DNA (arrow). d Spread platelets were found surrounding released DNA with distinct DNA content in their center. Of note, blood from influenza-infected patients and controls in (a–d) was not permeabilized. Since formaldehyde can cause certain levels of permeabilization as a function of cross-linking positive staining in control samples for H4 and MPO do not necessarily indicate activation. e–g Levels of proteins related to DNA release in the plasma of influenza-infected patients assessed by ELISA. Source data are provided as a Source Data file. e neutrophil elastase, f myeloperoxidase (MPO), and g histone nucleosome core. The graphs represent the average ± SD of healthy donors (n = 15) and influenza-infected patients (n = 18); significance for (e–g) was assessed by Mann–Whitney U test, star symbol (*) indicates p < 0.0001. h, i To synchronize time of influenza presence as a function of infection and quantify the released DNA, we treated blood from human donors for 30 min with sucrose-purified infectious influenza (WSN/33) at constant rotation and 37 °C. Influenza was used at 1 pfu to 100 platelets. h Representative images of blood from 3 donors with influenza and one (out of 3) healthy control and i their quantitation. Of note, in certain cases the DNA is not entirely covered with platelets, suggesting differences in kinetics of interaction and/or a physiological relationship that needs further in vivo characterization. Data in graph is represented as average ± SD of n = 3 different donors; significance was assessed by unpaired t-test (two-tailed value) and star symbol (*) indicates p = 0.0019, df = 4