Protein C system defects inflicted by the malaria parasite protein PfEMP1 can be overcome by a soluble EPCR variant

Jens E. V. Petersen; Eveline A. M. Bouwens; Ibai Tamayo; Louise Turner; Christian W. Wang; Monique Stins; Thor G. Theander; José Hermida; Laurent O. Mosnier; Thomas Lavstsen

doi:10.1160/TH15-01-0018

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2015; 114(05): 1038-1048
DOI: 10.1160/TH15-01-0018

Blood Cells, Inflammation and Infection

Schattauer GmbH

Protein C system defects inflicted by the malaria parasite protein PfEMP1 can be overcome by a soluble EPCR variant

Jens E. V. Petersen

¹Centre for Medical Parasitology, Department of International Health, Immunology & Microbiology, University of Copenhagen and Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark

,

Eveline A. M. Bouwens

²Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA

,

Ibai Tamayo

³Thrombosis and Haemostasis Laboratory, Center for Applied Medical Research, University of Navarra, Pamplona, Spain

,

Louise Turner

¹Centre for Medical Parasitology, Department of International Health, Immunology & Microbiology, University of Copenhagen and Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark

,

Christian W. Wang

¹Centre for Medical Parasitology, Department of International Health, Immunology & Microbiology, University of Copenhagen and Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark

,

Monique Stins

⁴Department of Neurology, Division NeuroImmunology, Johns Hopkins SOM and Department Molecular biology and Immunology, Malaria Research Institute, Bloomberg SPH, Baltimore, Maryland, USA

,

Thor G. Theander

¹Centre for Medical Parasitology, Department of International Health, Immunology & Microbiology, University of Copenhagen and Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark

,

José Hermida^§

³Thrombosis and Haemostasis Laboratory, Center for Applied Medical Research, University of Navarra, Pamplona, Spain

,

Laurent O. Mosnier^§

²Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA

,

Thomas Lavstsen^§

¹Centre for Medical Parasitology, Department of International Health, Immunology & Microbiology, University of Copenhagen and Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark

› Author Affiliations

Abstract

Summary

The Endothelial Protein C receptor (EPCR) is essential for the anticoagulant and cytoprotective functions of the Protein C (PC) system. Selected variants of the malaria parasite protein, Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) associated with severe malaria, including cerebral malaria, specifically target EPCR on vascular endothelial cells. Here, we examine the cellular response to PfEMP1 engagement to elucidate its role in malaria pathogenesis. Binding of the CIDRα1.1 domain of PfEMP1 to EPCR obstructed activated PC (APC) binding to EPCR and induced a loss of cellular EPCR functions. CIDRα1.1 severely impaired endothelial PC activation and effectively blocked APC-mediated activation of protease-activated receptor-1 (PAR1) and associated barrier protective effects of APC on endothelial cells. A soluble EPCR variant (E86A-sEPCR) bound CIDRα1.1 with high affinity and did not interfere with (A)PC binding to cellular EPCR. E86A-sEPCR used as a decoy to capture PfEMP1, permitted normal PC activation on endothelial cells, normal barrier protective effects of APC, and greatly reduced cytoadhesion of infected erythrocytes to brain endothelial cells. These data imply important contributions of PfEMP1-induced protein C pathway defects in the pathogenesis of severe malaria. Furthermore, the E86A-sEPCR decoy provides a proof-of-principle strategy for the development of novel adjunct therapies for severe malaria.

Keywords

Malaria - endothelial cells - endothelial protein C receptor - PfEMP1

Full Text

References

References
1 Carme B, Bouquety JC, Plassart H.. Mortality and sequelae due to cerebral malaria in African children in Brazzaville, Congo. Am J Trop Med Hyg 1993; 48: 216-221.
2 Birbeck GL, Molyneux ME, Kaplan PW. et al. Blantyre Malaria Project Epilepsy Study (BMPES) of neurological outcomes in retinopathy-positive paediatric cerebral malaria survivors: a prospective cohort study. Lancet Neurol 2010; 9: 1173-1181.
3 Moxon CA, Heyderman RS, Wassmer SC. Dysregulation of coagulation in cerebral malaria. Mol Biochem Parasitol 2009; 166: 99-108.
4 Storm J, Craig AG. Pathogenesis of cerebral malaria--inflammation and cytoadherence. Front Cell Infect Microbiol 2014; 4: 100.
5 Lavstsen T, Turner L, Saguti F. et al. Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children. Proc Natl Acad Sci USA 2012; 109: E1791-1800.
6 Turner L, Lavstsen T, Berger SS. et al. Severe malaria is associated with parasite binding to endothelial protein C receptor. Nature 2013; 498: 502-505.
7 Rowe JA, Claessens A, Corrigan RA. et al. Adhesion of Plasmodium falciparum-infected erythrocytes to human cells: molecular mechanisms and therapeutic implications. Exp Rev Mol Med 2009; 11: e16.
8 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.
9 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; 5: 1817-1824.
10 Bouwens EA, Stavenuiter F, Mosnier LO. Mechanisms of anticoagulant and cytoprotective actions of the protein C pathway. J Thromb Haemost 2013; 11 (Suppl 1) 242-253.
11 Vatsyayan R, Kothari H, Mackman N. et al. Inactivation of Factor VIIa by Antithrombin In Vitro, Ex Vivo and In Vivo: Role of Tissue Factor and Endothelial Cell Protein C Receptor. PloS one 2014; 9: e103505.
12 Mohan Rao LV, Esmon CT, Pendurthi UR. Endothelial cell protein C receptor: a multiliganded and multifunctional receptor. Blood 2014; 124: 1553-1562.
13 Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway. Blood 2007; 109: 3161-3172.
14 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.
15 Esmon CT. The protein C pathway. Chest 2003; 124 (3 Suppl) 26S-32S.
16 Esmon CT. The endothelial protein C receptor. Curr Opin Hematol 2006; 13: 382-385.
17 Burnier L, Mosnier LO. Novel mechanisms for activated protein C cytoprotective activities involving noncanonical activation of protease-activated receptor 3. Blood 2013; 122: 807-816.
18 Mosnier LO, Sinha RK, Burnier L. et al. Biased agonism of protease-activated receptor 1 by activated protein C caused by noncanonical cleavage at Arg46. Blood 2012; 120: 5237-5246.
19 Zlokovic BV, Griffin JH. Cytoprotective protein C pathways and implications for stroke and neurological disorders. Trends Neurosci 2011; 34: 198-209.
20 Mosnier LO, Zlokovic BV, Griffin JH. Cytoprotective-selective activated protein C therapy for ischaemic stroke. Thromb Haemost 2014; 112: 883-892.
21 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.
22 Hemmer CJ, Kern P, Holst FG. et al. Activation of the host response in human Plasmodium falciparum malaria: relation of parasitemia to tumor necrosis factor/cachectin, thrombin-antithrombin III, and protein C levels. Am J Med 1991; 91: 37-44.
23 Mosnier LO. Platelet factor 4 inhibits thrombomodulin-dependent activation of thrombin-activatable fibrinolysis inhibitor (TAFI) by thrombin. J Biol Chem 2011; 286: 502-510.
24 Bouwens EA, Stavenuiter F, Mosnier LO. Cell painting with an engineered EPCR to augment the protein C system. Thromb Haemost. 2015 Epub ahead of print.
25 Mosnier LO, Zampolli A, Kerschen EJ. et al. Hyperantithrombotic, noncytoprotective Glu149Ala-activated protein C mutant. Blood 2009; 113: 5970-5978.
26 Stins MF, Gilles F, Kim KS. Selective expression of adhesion molecules on human brain microvascular endothelial cells. J Neuroimmunol 1997; 76: 81-90.
27 Ye X, Fukudome K, Tsuneyoshi N. et al. The endothelial cell protein C receptor (EPCR) functions as a primary receptor for protein C activation on endothelial cells in arteries, veins, and capillaries. Biochem Biophys Res Comun 1999; 259: 671-677.
28 Schindelin J, Arganda-Carreras I, Frise E. et al. Fiji: an open-source platform for biological-image analysis. Nature Methods 2012; 9: 676-682.
29 Liaw PC, Mather T, Oganesyan N. et al. Identification of the protein C/activated protein C binding sites on the endothelial cell protein C receptor. Implications for a novel mode of ligand recognition by a major histocompatibility complex class 1-type receptor. J Biol Chem 2001; 276: 8364-8370.
30 Preston RJ, Villegas-Mendez A, Sun YH. et al. Selective modulation of protein C affinity for EPCR and phospholipids by Gla domain mutation. FEBS J 2005; 272: 97-108.
31 Otto TD, Rayner JC, Bohme U. et al. Genome sequencing of chimpanzee malaria parasites reveals possible pathways of adaptation to human hosts. Nature Commun 2014; 5: 4754.
32 Craig AG, Grau GE, Janse C. et al. The role of animal models for research on severe malaria. PLoS Pathogens 2012; 8: e1002401.
33 Li W, Zheng X, Gu JM. et al. Extraembryonic expression of EPCR is essential for embryonic viability. Blood 2005; 106: 2716-2722.
34 Zheng X, Li W, Gu JM. et al. Effects of membrane and soluble EPCR on the hemostatic balance and endotoxemia in mice. Blood 2007; 109: 1003-1009.
35 Castellino FJ, Liang Z, Volkir SP. et al. Mice with a severe deficiency of the endothelial protein C receptor gene develop, survive, and reproduce normally, and do not present with enhanced arterial thrombosis after challenge. Thromb Haemost 2002; 88: 462-472.
36 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.
37 Centelles MN, Puy C, Lopez-Sagaseta J. et al. Blocking endothelial protein C receptor (EPCR) accelerates thrombus development in vivo. Thromb Haemost 2010; 103: 1239-1244.
38 Tamayo I, Velasco SE, Puy C. et al. sPLA2-V impairs EPCR-dependent protein C activation and accelerates thrombosis in vivo. J Thromb Haemost 2014; 12: 1921-1927.
39 Cushman M. Inherited risk factors for venous thrombosis. Hematology / the Education Program of the American Society of Hematology American Society of Hematology Education Program 2005; 452-457.
40 Clemens R, Pramoolsinsap C, Lorenz R. et al. Activation of the coagulation cascade in severe falciparum malaria through the intrinsic pathway. Br J Haematol 1994; 87: 100-105.
41 Kerschen EJ, Fernandez JA, Cooley BC. et al. Endotoxemia and sepsis mortality reduction by non-anticoagulant activated protein C. The J Exp Med 2007; 204: 2439-2448.
42 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.
43 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. Arterioscl Thromb Vasc Biol 2013; 33: 769-776.
44 Naka I, Patarapotikul J, Hananantachai H. et al. Association of the endothelial protein C receptor (PROCR) rs867186-G allele with protection from severe malaria. Malaria J 2014; 13: 105.
45 Qu D, Wang Y, Song Y. et al. The Ser219→Gly dimorphism of the endothelial protein C receptor contributes to the higher soluble protein levels observed in individuals with the A3 haplotype. J Thromb Haemost 2006; 4: 229-235.
46 Saposnik B, Reny JL, Gaussem P. et al. A haplotype of the EPCR gene is associated with increased plasma levels of sEPCR and is a candidate risk factor for thrombosis. Blood 2004; 103: 1311-1318.
47 Fukudome K, Kurosawa S, Stearns-Kurosawa DJ. et al. The endothelial cell protein C receptor. Cell surface expression and direct ligand binding by the soluble receptor. J Biol Chem 1996; 271: 17491-17498.
48 Regan LM, Stearns-Kurosawa DJ, Kurosawa S. et al. The endothelial cell protein C receptor. Inhibition of activated protein C anticoagulant function without modulation of reaction with proteinase inhibitors. J Biol Chem 1996; 271: 17499-17503.
49 Liaw PC, Neuenschwander PF, Smirnov MD. et al. Mechanisms by which soluble endothelial cell protein C receptor modulates protein C and activated protein C function. J Biol Chem 2000; 275: 5447-5452.
50 Holash J, Davis S, Papadopoulos N. et al. VEGF-Trap: a VEGF blocker with potent antitumor effects. Proc Natl Acad Sci USA 2002; 99: 11393-11398.
51 Lu G, DeGuzman FR, Hollenbach SJ. et al. A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa. Nature Med 2013; 19: 446-451.
52 Schuepbach RA, Riewald M. Coagulation factor Xa cleaves protease-activated receptor-1 and mediates signaling dependent on binding to the endothelial protein C receptor. J Thromb Haemost 2010; 8: 379-388.
53 Disse J, Petersen HH, Larsen KS. et al. The endothelial protein C receptor supports tissue factor ternary coagulation initiation complex signaling through protease-activated receptors. J Biol Chem 2011; 286: 5756-5767.
54 Kurosawa S, Esmon CT, Stearns-Kurosawa DJ. The soluble endothelial protein C receptor binds to activated neutrophils: involvement of proteinase-3 and CD11b/CD18. J Immunol 2000; 165: 4697-4703.
55 Fink K, Busch HJ, Bourgeois N. et al. Mac-1 directly binds to the endothelial protein C-receptor: a link between the protein C anticoagulant pathway and inflammation?. PloS one 2013; 8: e53103.