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. 2019 Nov 4;9:15932. doi: 10.1038/s41598-019-52041-8

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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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Molecular mechanisms underlying thrombus formation in inflamed venular microvessels. Thrombus formation in postcapillary venules of the cremaster muscle of WT mice was induced by photochemical injury as detailed in Methods. Panel (A) shows quantitative data for cessation times in animals undergoing intrascrotal stimulation with LPS and treatment with antibodies directed against P-selectin/CD62P, Mac-1/CD11b, vWF, GPIbα, CD40, or CD40L/CD154, with compound GR144053 (inhibitor of GPIIb/IIIa), with DNAse I (NET-degrading enzyme), or vehicle/isotype control antibodies (mean ± SEM for n = 4 per group; *p < 0.05 vs. vehicle/isotype control). Panel (B; scale bar: 10 μm) shows representative confocal microscopy images illustrating the spatial relationship of neutrophils/classical monocytes (Ly-6G/C; blue), platelets (GPIbβ; green) and vWF, CD40, or CD40L/CD154 (red) in thrombi occluding postcapillary venules of the inflamed cremaster muscle of WT mice. The spatial distribution of platelet interactions during thrombus formation in postcapillary venules of the cremaster muscle of WT mice induced by photochemical injury was analyzed by multi-channel in vivo fluorescence microscopy as detailed in Methods. Panel (C) shows quantitative data for animals undergoing intrascrotal stimulation with LPS and treatment with antibodies directed against vWF, GPIbα, CD40, CD40L/CD154, or isotype control antibodies (mean ± SEM for n = 3 per group; *p < 0.05 vs. baseline non-related to neutrophils, ^#p < 0.05 vs. baseline related to neutrophils).