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

Academic Article
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Overview

authors

  • Bouwens, E. A. M.
  • Stavenuiter, F.
  • Mosnier, Laurent

publication date

  • December 2015

journal

  • Thrombosis and Haemostasis  Journal

abstract

  • 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.

subject areas

  • 3' Untranslated Regions
  • Antigens, CD
  • Biological Availability
  • Caveolae
  • Caveolin 1
  • Cell Compartmentation
  • Cell Membrane
  • Dose-Response Relationship, Drug
  • Endothelial Cells
  • Enzyme Activation
  • Glycosylphosphatidylinositols
  • HEK293 Cells
  • Humans
  • Membrane Microdomains
  • Phosphorylation
  • Protein Binding
  • Protein C
  • Protein Engineering
  • Protein Processing, Post-Translational
  • Proto-Oncogene Proteins c-akt
  • RNA Interference
  • RNA, Small Interfering
  • Receptor, PAR-1
  • Receptors, Cell Surface
  • Recombinant Fusion Proteins
  • Tetradecanoylphorbol Acetate
  • Transduction, Genetic
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Research

keywords

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

International Standard Serial Number (ISSN)

  • 0340-6245

Digital Object Identifier (DOI)

  • 10.1160/th15-01-0079

PubMed ID

  • 26272345
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Additional Document Info

start page

  • 1144

end page

  • 1155

volume

  • 114

issue

  • 6

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