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Activated protein C binds directly to Tie2: possible beneficial effects on endothelial barrier function

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Abstract

Activated protein C (APC) is a natural anticoagulant with strong anti-inflammatory, anti-apoptotic, and barrier stabilizing properties. These cytoprotective properties of APC are thought to be exerted through its pathway involving the binding of APC to endothelial protein C receptor and cleavage of protease-activated receptors. In this study, we found that APC enhanced endothelial barrier integrity via a novel pathway, by binding directly to and activating Tie2, a transmembrane endothelial tyrosine kinase receptor. Binding assays demonstrated that APC competed with the only known ligands of Tie2, the angiopoietins (Angs). APC bound directly to Tie2 (Kd ~3 nM), with markedly stronger binding affinity than Ang2. After binding, APC rapidly activated Tie2 to enhance endothelial barrier function as shown by Evan’s blue dye transfer across confluent cell monolayers and in vivo studies. Blocking Tie2 restricted endothelial barrier integrity. This study highlights a novel mechanism by which APC binds directly to Tie2 to enhance endothelial barrier integrity, which helps to explain APC’s protective effects in vascular leakage-related pathologies.

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Acknowledgements

We thank Dr Anthony Ashton for helpful discussions and The Maternity unit at Royal North Shore Hospital for providing umbilical cords.

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  1. Sutton Arthritis Research Laboratories, Institute of Bone and Joint Research, Kolling Institute of Medical Research, University of Sydney at Royal North Shore Hospital, Level 10, The Kolling Building, St. Leonards, NSW, 2065, Australia

    Nikita Minhas, Meilang Xue & Christopher J. Jackson

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  1. Nikita Minhas
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CJ and MX have patents and commercial interests in molecular variants of APC and skin conditions.

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18_2016_2440_MOESM1_ESM.pptx

Supplementary Fig. 1. APC regulates Tie2 in HUVEC at least partially through EPCR at 4 h. HUVEC were incubated with Biorich medium +2% FCS for 24 h before treatment with APC (1 or 10 µg/ml) for 4 h. For some experiments, HUVEC were pre-incubated in Biorich medium + 2% FCS for 1 h with RCR52 (1 µg/ml), before treatment with APC (1 µg/ml) for 4 h. The cell lysates were analyzed for P-Tie2 by western blotting. Results were normalized with β-actin (PPTX 201 kb)

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Minhas, N., Xue, M. & Jackson, C.J. Activated protein C binds directly to Tie2: possible beneficial effects on endothelial barrier function. Cell. Mol. Life Sci. 74, 1895–1906 (2017). https://doi.org/10.1007/s00018-016-2440-6

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