Cell painting with an engineered EPCR to augment the protein C system.
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, EPCR-GPI provides a novel tool to restore the bioavailability and functionality of EPCR on EPCR- depleted and -deficient cells.
Eveline A M
EA
Bouwens
Fabian
F
Stavenuiter
Laurent O. Mosnier, Department of Molecular and Experimental Medicine (MEM-180), The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037, USA, Tel.: +1 858 784 8220, Fax: +1 858 784 2243, E-mail: lmosnier@scripps.edu.
Laurent O
LO
Mosnier
D020413:3' Untranslated Regions / Q000235:genetics; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D001682:Biological Availability; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1 / Q000378:metabolism; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000378:metabolism*; D000074283:Endothelial Protein C Receptor; D004789:Enzyme Activation; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism*; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D044463:Receptor, PAR-1 / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014161:Transduction, Genetic
D020413:3' Untranslated Regions; D015703:Antigens, CD; D051242:Caveolin 1; D000074283:Endothelial Protein C Receptor; D017261:Glycosylphosphatidylinositols; C478370:PROCR protein, human; D011486:Protein C; D034741:RNA, Small Interfering; D044463:Receptor, PAR-1; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C494918:AKT1 protein, human; D051057:Proto-Oncogene Proteins c-akt; D013755:Tetradecanoylphorbol Acetate
Endothelial protein C receptor; glycosylphosphatidylinositol anchors; protease-activated receptors; protein C; vascular endothelium