Abstract Number: PB1035
Meeting: ISTH 2021 Congress
Background: Activated platelets are known to release the chemokines CCL5 and CXCL4, which can be deposited onto the endothelial cells inducing monocyte arrest.
Aims: In this study, we aimed to elucidate the fate of CCL5 and CXCL4 after endothelial deposition.
Methods: HUVECs and the endothelial cell line EA.hy926 were incubated with CCL5 or CXCL4 for up to 120 minutes and analyzed with light-, confocal- or stimulated emission depletion (STED) microscopy. To quantify internalization, whole cell lysates and organelle-fractionated cells were analyzed using ELISA. Monocyte arrest was evaluated using laminar flow leukocyte adhesion assays.
Results: Both CCL5 and CXCL4 were rapidly internalized in endothelial cells (<10 min). Whereas CXCL4 remained partly presented on the cell surface, all of the CCL5 was internalized. Endocytosis was dependent on dynamin and clathrin, as internalization was blocked by specific inhibitors. Cell surface proteoglycans, chemokine binding polysaccharides, had a less definite role in the internalization of CCL5 and CXCL4. Combined incubation of CCL5 and CXCL4 with endothelial cells did not influence the internalization or the localization of either of the chemokines. Localization studies by confocal and super-resolution microscopy suggested that both CCL5 and CXCL4 partly have a nuclear localization. This was supported by cell fractionation, which revealed a relatively high nuclear accumulation of both CCL5 and CXCL4. Internalization of chemokines appears less in cells with an inflammatory phenotype, and monocyte-arrest was higher to endothelial cells, when incubated with CCL5 and CXCL4.
Conclusions: In summary, endothelial cells rapidly and actively internalize CCL5 and CXCL4 by clathrin and dynamin-dependent endocytosis, where the chemokines appear to be directed to the nucleus. These findings introduce a potential novel, non-canonical role of alpha-granule released chemokines in the cross-talk of activated platelets and endothelial cells, which could have implications for the mechanisms in which leukocytes are attracted to sites of inflammation.
To cite this abstract in AMA style:Dickhout A, van Zandvoort MA, R Koenen R. Rapid Internalization and Nuclear Translocation of CCL5 and CXCL4 in Endothelial Cells [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 1). https://abstracts.isth.org/abstract/rapid-internalization-and-nuclear-translocation-of-ccl5-and-cxcl4-in-endothelial-cells/. Accessed September 23, 2021.
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