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Thromb Haemost 2015; 114(06): 1144-1155
DOI: 10.1160/TH15-01-0079
Coagulation and Fibrinolysis
Schattauer GmbH

Cell painting with an engineered EPCR to augment the protein C system

Eveline A. M. Bouwens
1   Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA
,
Fabian Stavenuiter
1   Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA
,
Laurent O. Mosnier
1   Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA
› Author Affiliations Financial support: This work was supported by an American Heart Association Western States Affiliate postdoctoral fellowship (E. A. M. B.) and National Institutes of Health (NHLBI) grant HL104165 (L. O. M.).
Further Information

Publication History

Received: 27 January 2015

Accepted after major revision: 28 June 2015

Publication Date:
30 November 2017 (online)

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Summary

The protein C (PC) system conveys beneficial anticoagulant and cytoprotective effects in numerous in vivo disease models. The endothelial protein C receptor (EPCR) plays a central role in these pathways as cofactor for PC activation and by enhancing activated protein C (APC)-mediated protease-activated receptor (PAR) activation. During inflammatory disease, expression of EPCR on cell membranes is often diminished thereby limiting PC activation and APC’s effects on cells. Here a caveolae-targeting glycosylphosphatidylinositol (GPI)-anchored EPCR (EPCR-GPI) was engineered to restore EPCR’s bioavailability via “cell painting.” The painting efficiency of EPCR-GPI on EPCR-depleted endothelial cells was time- and dose-dependent. The EPCR-GPI bioavailability after painting was long lasting since EPCR surface levels reached 400 % of wild-type cells after 2 hours and remained > 200 % for 24 hours. EPCR-GPI painting conveyed APC binding to EPCR-depleted endothelial cells where EPCR was lost due to shedding or shRNA. EPCR painting normalised PC activation on EPCR-depleted cells indicating that EPCR-GPI is functional active on painted cells. Caveolin-1 lipid rafts were enriched in EPCR after painting due to the GPI-anchor targeting caveolae. Accordingly, EPCR painting supported PAR1 and PAR3 cleavage by APC and augmented PAR1-dependent Akt phosphorylation by APC. Thus, EPCR-GPI painting achieved physiological relevant surface levels on endothelial cells, restored APC binding to EPCR-depleted cells, supported PC activation, and enhanced APC-mediated PAR cleavage and cytoprotective signalling. Therefore, EPCRGPI provides a novel tool to restore the bioavailability and functionality of EPCR on EPCR- depleted and -deficient cells.

Keywords

Endothelial protein C receptor - glycosylphosphatidylinositol anchors - protease-activated receptors - protein C - vascular endothelium
 

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