Download PDF
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)

Also available at
    Permissions and Reprints

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
 

  • References

  • 1 Dahlbäck B, Villoutreix BO. Regulation of blood coagulation by the protein C anticoagulant pathway. Novel insights into structure-function relationships and molecular recognition. Arterioscler Thromb Vasc Biol 2005; 25: 1311-1320.
    PubMedSearch in Google Scholar
  • 2 Rezaie AR. Regulation of the protein C anticoagulant and antiinflammatory pathways. Curr Med Chem 2010; 17: 2059-2069.
    CrossrefPubMedSearch in Google Scholar
  • 3 Bouwens EA, Stavenuiter F, Mosnier LO. Mechanisms of anticoagulant and cytoprotective actions of the protein C pathway. J Thromb Haemost 2013; 11 (Suppl. 01) 242-253.
    CrossrefPubMedSearch in Google Scholar
  • 4 Kerschen EJ, Hernandez I, Zogg M. et al. Activated protein C targets CD8+ dendritic cells to reduce the mortality of endotoxemia in mice. J Clin Invest 2010; 120: 3167-3178.
    CrossrefPubMedSearch in Google Scholar
  • 5 Geiger H, Pawar SA, Kerschen EJ. et al. Pharmacological targeting of the thrombomodulin-activated protein C pathway mitigates radiation toxicity. Nat Med 2012; 18: 1123-1129.
    CrossrefPubMedSearch in Google Scholar
  • 6 Danese S, Vetrano S, Zhang L. et al. The protein C pathway in tissue inflammation and injury: pathogenic role and therapeutic implications. Blood 2010; 115: 1121-1130.
    CrossrefPubMedSearch in Google Scholar
  • 7 Xu J, Ji Y, Zhang X. et al. Endogenous activated protein C signalling is critical to protection of mice from lipopolysaccaride induced septic shock. J Thromb Hae-most 2009; 07: 851-856.
    CrossrefPubMedSearch in Google Scholar
  • 8 Zlokovic BV, Griffin JH. Cytoprotective protein C pathways and implications for stroke and neurological disorders. Trends Neurosci 2011; 34: 198-209.
    CrossrefPubMedSearch in Google Scholar
  • 9 Esmon CT. Protein C anticoagulant system-anti-inflammatory effects. Semin Immunopathol 2012; 34: 127-132.
    CrossrefPubMedSearch in Google Scholar
  • 10 Mosnier LO, Yang XV, Griffin JH. Activated protein C mutant with minimal anticoagulant activity, normal cytoprotective activity, and preservation of thrombin activable fibrinolysis inhibitor-dependent cytoprotective functions. J Biol Chem 2007; 282: 33022-33033.
    CrossrefPubMedSearch in Google Scholar
  • 11 Bock F, Shahzad K, Vergnolle N. et al. Activated protein C based therapeutic strategies in chronic diseases. Thromb Haemost 2014; 111: 610-617.
    Thieme ConnectPubMedSearch in Google Scholar
  • 12 Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway. Blood 2007; 109: 3161-3172.
    CrossrefPubMedSearch in Google Scholar
  • 13 Stearns-Kurosawa DJ, Kurosawa S, Mollica JS. et al. The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex. Proc Natl Acad Sci USA 1996; 93: 10212-10216.
    CrossrefPubMedSearch in Google Scholar
  • 14 Riewald M, Petrovan RJ, Donner A. et al. Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science 2002; 296: 1880-1882.
    CrossrefPubMedSearch in Google Scholar
  • 15 Taylor Jr. FB, Peer GT, Lockhart MS. et al. Endothelial cell protein C receptor plays an important role in protein C activation in vivo. Blood 2001; 97: 1685-1688.
    CrossrefPubMedSearch in Google Scholar
  • 16 Burnier L, Mosnier LO. Novel mechanisms for activated protein C cytoprotective activities involving non-canonical activation of protease-activated receptor 3. Blood 2013; 122: 807-816.
    CrossrefPubMedSearch in Google Scholar
  • 17 Rezaie AR. The occupancy of endothelial protein C receptor by its ligand modulates the PAR-1 dependent signalling specificity of coagulation proteases. IUBMB Life 2011; 63: 390-396.
    CrossrefPubMedSearch in Google Scholar
  • 18 Russo A, Soh UJ, Trejo J. Proteases Display Biased Agonism at Protease-Activated Receptors: Location matters!. Mol Interv 2009; 09: 87-96.
    CrossrefPubMedSearch in Google Scholar
  • 19 Mosnier LO, Sinha RK, Burnier L. et al. Biased agonism of protease-activated receptor 1 by activated protein C caused by non-canonical cleavage at Arg46. Blood 2012; 120: 5237-5246.
    CrossrefPubMedSearch in Google Scholar
  • 20 Lopez-Sagaseta J, Puy C, Tamayo I. et al. sPLA2-V inhibits EPCR anticoagulant and antiapoptotic properties by accommodating lysophosphatidylcholine or PAF in the hydrophobic groove. Blood 2012; 119: 2914-2921.
    CrossrefPubMedSearch in Google Scholar
  • 21 Qu D, Wang Y, Esmon NL. et al. Regulated endothelial protein C receptor shedding is mediated by tumour necrosis factor-alpha converting enzyme/ADAM17. J Thromb Haemost 2007; 05: 395-402.
    CrossrefPubMedSearch in Google Scholar
  • 22 Bouwens EA, Mosnier LO. EPCR encryption induces cellular APC resistance. Blood 2012; 119: 2703-2705.
    CrossrefPubMedSearch in Google Scholar
  • 23 Gu JM, Katsuura Y, Ferrell GL. et al. Endotoxin and thrombin elevate rodent endothelial cell protein C receptor mRNA levels and increase receptor shedding in vivo. Blood 2000; 95: 1687-1693.
    PubMedSearch in Google Scholar
  • 24 Uitte De Willige S, van Marion V, Rosendaal FR. et al. Haplotypes of the EPCR gene, plasma sEPCR levels and the risk of deep venous thrombosis. J Thromb Haemost 2004; 02: 1305-1310.
    CrossrefPubMedSearch in Google Scholar
  • 25 Biguzzi E, Franchi F, Bucciarelli P. et al. Endothelial protein C receptor plasma levels increase in chronic liver disease, while thrombomodulin plasma levels increase only in hepatocellular carcinoma. Thromb Res 2007; 120: 289-293.
    CrossrefPubMedSearch in Google Scholar
  • 26 Kurosawa S, Stearns-Kurosawa DJ, Carson CW. et al. Plasma Levels of Endothelial Cell Protein C Receptor Are Elevated in Patients With Sepsis and Systemic Lupus Erythematosus: Lack of Correlation With Thrombomodulin Suggests Involvement of Different Pathological Processes. Blood 1998; 91: 725-727.
    PubMedSearch in Google Scholar
  • 27 Esmon CT. The endothelial cell protein C receptor. Thromb Haemost 2000; 83: 639-643.
    Thieme ConnectPubMedSearch in Google Scholar
  • 28 Taylor Jr. FB, Stearns-Kurosawa DJ, Kurosawa S. et al. The endothelial cell protein C receptor aids in host defense against Escherichia coli sepsis. Blood 2000; 95: 1680-1686.
    PubMedSearch in Google Scholar
  • 29 Kerschen EJ, Fernandez JA, Cooley BC. et al. Endotoxemia and sepsis mortality reduction by non-anticoagulant activated protein C. J Exp Med 2007; 204: 2439-2448.
    CrossrefPubMedSearch in Google Scholar
  • 30 von Drygalski A, Furlan-Freguia C, Ruf W. et al. Organ-Specific Protection Against Lipopolysaccharide-Induced Vascular Leak Is Dependent on the Endothelial Protein C Receptor. Arterioscler Thromb Vasc Biol 2013; 33: 769-776.
    CrossrefPubMedSearch in Google Scholar
  • 31 Faust SN, Levin M, Harrison OB. et al. Dysfunction of endothelial protein C activation in severe meningococcal sepsis. N Engl J Med 2001; 345: 408-416.
    CrossrefPubMedSearch in Google Scholar
  • 32 Faioni EM, Ferrero S, Fontana G. et al. Expression of endothelial protein C receptor and thrombomodulin in the intestinal tissue of patients with inflammatory bowel disease. Crit Care Med 2004; 32 (05) S266-S270.
    CrossrefPubMedSearch in Google Scholar
  • 33 Moxon CA, Wassmer SC, Milner Jr. DA. et al. Loss of endothelial protein C receptors links coagulation and inflammation to parasite sequestration in cerebral malaria in African children. Blood 2013; 122: 842-851.
    CrossrefPubMedSearch in Google Scholar
  • 34 Fukudome K, Esmon CT. Identification, cloning, and regulation of a novel endothelial cell protein C/activated protein C receptor. J Biol Chem 1994; 269: 26486-26491.
    PubMedSearch in Google Scholar
  • 35 Xu J, Qu D, Esmon NL. et al. Metalloproteolytic release of endothelial cell protein C receptor. J Biol Chem 2000; 275: 6038-6044.
    CrossrefPubMedSearch in Google Scholar
  • 36 Legler DF, Doucey MA, Schneider P. et al. Differential insertion of GPI-anchored GFPs into lipid rafts of live cells. FASEB J 2005; 19: 73-75.
    CrossrefPubMedSearch in Google Scholar
  • 37 Mosnier LO, Zampolli A, Kerschen EJ. et al. Hyper-antithrombotic, non-cytoprotective Glu149Ala-activated protein C mutant. Blood 2009; 113: 5970-5978.
    CrossrefPubMedSearch in Google Scholar
  • 38 Moran P, Caras IW. Fusion of sequence elements from non-anchored proteins to generate a fully functional signal for glycophosphatidylinositol membrane anchor attachment. J Cell Biol 1991; 115: 1595-1600.
    CrossrefPubMedSearch in Google Scholar
  • 39 Bumgarner GW, Zampell JC, Nagarajan S. et al. Modified cell ELISA to determine the solubilisation of cell surface proteins: Applications in GPI-anchored protein purification. J Biochem Biophys Methods 2005; 64: 99-109.
    CrossrefPubMedSearch in Google Scholar
  • 40 Macdonald JL, Pike LJ. A simplified method for the preparation of detergent-free lipid rafts. J Lipid Res 2005; 46: 1061-1067.
    CrossrefPubMedSearch in Google Scholar
  • 41 Lopez-Sagaseta J, Montes R, Puy C. et al. Binding of factor VIIa to the endothelial cell protein C receptor reduces its coagulant activity. J Thromb Haemost 2007; 05: 1817-1824.
    CrossrefPubMedSearch in Google Scholar
  • 42 Fukudome K, Ye X, Tsuneyoshi N. et al. Activation mechanism of anticoagulant protein C in large blood vessels involving the endothelial cell protein C receptor. J Exp Med 1998; 187: 1029-1035.
    CrossrefPubMedSearch in Google Scholar
  • 43 Russo A, Soh UJ, Paing MM. et al. Caveolae are required for protease-selective signalling by protease-activated receptor-1. Proc Natl Acad Sci USA 2009; 106: 6393-6397.
    CrossrefPubMedSearch in Google Scholar
  • 44 Zaitsev S, Kowalska MA, Neyman M. et al. Targeting recombinant thrombomodulin fusion protein to red blood cells provides multifaceted thromboprophylaxis. Blood 2012; 119: 4779-4785.
    CrossrefPubMedSearch in Google Scholar
  • 45 Huang X, Ding WQ, Vaught JL. et al. A soluble tissue factor-annexin V chimeric protein has both procoagulant and anticoagulant properties. Blood 2006; 107: 980-986.
    PubMedSearch in Google Scholar
  • 46 Carnemolla R, Greineder CF, Chacko AM. et al. PECAM targeted oxidant-resistant mutant thrombomodulin fusion protein with enhanced potency in vitro and vivo. J Pharmacol Exp Ther 2013; 347: 339-345.
    CrossrefPubMedSearch in Google Scholar
  • 47 Legler DF, Johnson-Leger C, Wiedle G. et al. The alpha v beta 3 integrin as a tumour homing ligand for lymphocytes. Eur J Immunol 2004; 34: 1608-1616.
    CrossrefPubMedSearch in Google Scholar
  • 48 McHugh RS, Ahmed SN, Wang YC. et al. Construction, purification, and functional incorporation on tumour cells of glycolipid-anchored human B7–1 (CD80). Proc Natl Acad Sci USA 1995; 92: 8059-8063.
    CrossrefPubMedSearch in Google Scholar
  • 49 Huang JH, Getty RR, Chisari FV. et al. Protein transfer of preformed MHC-peptide complexes sensitizes target cells to T cell cytolysis. Immunity 1994; 01: 607-613.
    CrossrefPubMedSearch in Google Scholar
  • 50 Contreras JL, Eckstein C, Smyth CA. et al. Activated protein C preserves functional islet mass after intraportal transplantation: a novel link between endothelial cell activation, thrombosis, inflammation, and islet cell death. Diabetes 2004; 53: 2804-2814.
    CrossrefPubMedSearch in Google Scholar
  • 51 Xue M, Dervish S, Harrison LC. et al. Activated Protein C Inhibits Pancreatic Islet Inflammation, Stimulates T Regulatory Cells, and Prevents Diabetes in Non-obese Diabetic (NOD) Mice. J Biol Chem 2012; 287: 16356-16364.
    CrossrefPubMedSearch in Google Scholar
  • 52 Chen H, Teramura Y, Iwata H. Co-immobilisation of urokinase and thrombomodulin on islet surfaces by poly(ethylene glycol)-conjugated phospholipid. J Control Release 2011; 150: 229-234.
    CrossrefPubMedSearch in Google Scholar
  • 53 Cui W, Angsana J, Wen J. et al. Liposomal Formulations of Thrombomodulin Increase Engraftment After Intraportal Islet Transplantation. Cell Transplant 2010; 19: 1359-1367.
    CrossrefPubMedSearch in Google Scholar
  • 54 Ilmakunnas M, Pesonen EJ, Hockerstedt K. et al. Graft protein C entrapment is associated with reduced phagocyte activation during reperfusion in human liver transplantation. Crit Care Med 2006; 34: 426-432.
    CrossrefPubMedSearch in Google Scholar
  • 55 Kuriyama N, Isaji S, Hamada T. et al. The cytoprotective effects of addition of activated protein C into preservation solution on small-for-size grafts in rats. Liver Transpl 2009; 16: 1-11.
    PubMedSearch in Google Scholar
  • 56 Lee KF, Lu B, Roussel JC. et al. Protective Effects of Transgenic Human Endothelial Protein C Receptor Expression in Murine Models of Transplantation. Am J Transplant 2012; 12: 2363-2372.
    CrossrefPubMedSearch in Google Scholar
  • 57 Kooyman DL, Byrne GW, McClellan S. et al. In vivo transfer of GPI-linked complement restriction factors from erythrocytes to the endothelium. Science 1995; 269: 89-92.
    CrossrefPubMedSearch in Google Scholar
  • 58 Kooyman DL, Byrne GW, Logan JS. Glycosyl phosphatidylinositol anchor. Exp Nephrol 1998; 06: 148-151.
    CrossrefPubMedSearch in Google Scholar
  • 59 Petersen JEV, Bouwens EA, Tamayo I. et al. Protein C system defects inflicted by the malaria parasite protein PfEMP1 can be overcome by a soluble EPCR variant. Thromb Haemost 2015; 114: 1038-1048.
    Thieme ConnectPubMedSearch in Google Scholar