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

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.
• 2 Rezaie AR. Regulation of the protein C anticoagulant and antiinflammatory pathways. Curr Med Chem 2010; 17: 2059-2069.
• 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.
• 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.
• 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.
• 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.
• 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.
• 8 Zlokovic BV, Griffin JH. Cytoprotective protein C pathways and implications for stroke and neurological disorders. Trends Neurosci 2011; 34: 198-209.
• 9 Esmon CT. Protein C anticoagulant system-anti-inflammatory effects. Semin Immunopathol 2012; 34: 127-132.
• 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.
• 11 Bock F, Shahzad K, Vergnolle N. et al. Activated protein C based therapeutic strategies in chronic diseases. Thromb Haemost 2014; 111: 610-617.
• 12 Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway. Blood 2007; 109: 3161-3172.
• 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.
• 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.
• 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.
• 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.
• 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.
• 18 Russo A, Soh UJ, Trejo J. Proteases Display Biased Agonism at Protease-Activated Receptors: Location matters!. Mol Interv 2009; 09: 87-96.
• 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.
• 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.
• 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.
• 22 Bouwens EA, Mosnier LO. EPCR encryption induces cellular APC resistance. Blood 2012; 119: 2703-2705.
• 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.
• 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.
• 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.
• 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.
• 27 Esmon CT. The endothelial cell protein C receptor. Thromb Haemost 2000; 83: 639-643.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 35 Xu J, Qu D, Esmon NL. et al. Metalloproteolytic release of endothelial cell protein C receptor. J Biol Chem 2000; 275: 6038-6044.
• 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.
• 37 Mosnier LO, Zampolli A, Kerschen EJ. et al. Hyper-antithrombotic, non-cytoprotective Glu149Ala-activated protein C mutant. Blood 2009; 113: 5970-5978.
• 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.
• 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.
• 40 Macdonald JL, Pike LJ. A simplified method for the preparation of detergent-free lipid rafts. J Lipid Res 2005; 46: 1061-1067.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 58 Kooyman DL, Byrne GW, Logan JS. Glycosyl phosphatidylinositol anchor. Exp Nephrol 1998; 06: 148-151.
• 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.