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

Abstract

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.

Trial registration: ClinicalTrials.gov NCT00638794.

Keywords: Malaria; PfEMP1; endothelial cells; endothelial protein C receptor.

Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest

Figures

Figure 1.. The CIDRα1.1 domain of PfEMP1 binds EPCR and inhibits APC binding.

A) Solid phase binding assay of the CIDRα1.1 domain to sEPCR. The Bmax of individual experiments was set to 100%. B) Binding APC to sEPCR in the presence of CIDRα1.1. CIDRα1.1 was incubated with sEPCR for 30 min before addition of APC (50 nM). APC binding in the absence of CIDRα1.1 was considered to represent 100% binding. C) Binding of CIDRα1.1 to endothelial EPCR on EA.hy926 and EPCR knockdown (EPCRKD) EA.hy926 cells expressing <20% EPCR analyzed by on cell western. CIDRα1.1 binding was expressed in arbitrary fluorescence units (FL) reported by the Odyssey (K. Counts). D) APC binding (50 nM) to EA.hy926 endothelial cells in presence of CIDRα1.1. APC binding in the absence of CIDRα1.1 was considered to represent 100% binding. All data points represent the mean ± SD (n=3).

Figure 2.. CIDRα1.1 inhibits cellular functions of EPCR.

A) The effect of CIDRα1.1 on EPCR-dependent enhancement of PC activation. Thrombin-mediated PC activation was determined on EA.hy926 cells in presence of EPCR-binding CIDRα1.1 (50 nM), non-EPCR-binding CIDRα3.5 (50 nM), and the EPCR blocking antibody RCR-252 (20 μg/ml). PC activation in the absence of CIDR or antibody was set to 100%. B) The effect of CIDRα1.1 on EPCR-dependent PAR1 cleavage by APC. APC (100 nM)-mediated PAR1 cleavage as a percentage of the total endogenously expressed PAR1 on the surface of EA.hy926 cells was determined in the presence and absence of CIDRα1.1 (100 nM). C) The effect of CIDRα1.1 on EPCR-dependent protection of endothelial barrier function by APC. APC (50 nM)-mediated protection of thrombin (2 nM)-induced barrier disruption was determined on confluent EA.hy926 cell layers in the presence of EPCR-binding CIDRα1.1 and non-EPCR-binding CIDRα3.1 control. The reduction of impedance induced by thrombin was set as 100% barrier disruption. All data points represent the mean ± SD (A, B: n=3, C: n=6-8). P-values were calculated using: A) and C) 1-way Anova and Dunnett’s Multiple Comparison Test, and for B) Student’s t-test. * denotes p<0.05, *** denotes p<0.001.

Figure 3.. CIDRα1.1 binds similarly to wild type sEPCR and mutated E86A-sEPCR.

A) Solid phase binding assay of the CIDRα1.1 domain to E86A-sEPCR. The Bmax of individual experiments was set to 100%. B) Binding APC to sEPCR in the presence of CIDRα1.1 and E86A-sEPCR. CIDRα1.1 (50 nM) was incubated with E86A-sEPCR and sEPCR for 30 min before addition of APC (50 nM). APC binding in the absence of CIDRα1.1 was considered to represent 100% binding. All data points represent the mean ± SD (n=3).

Figure 4.. E86A-sEPCR prevents cytoadhesion of P. falciparum-infected erythrocytes to primary brain endothelial cells.

Erythrocytes were infected with the P. falciparum laboratory strain FCR3 expressing the IT4VAR20 PfEMP1 that contains the CIDRα1.1 domain. Infected erythrocytes (IE) were allowed to adhere to primary microvascular brain endothelial cells for 1 hour in the presence of various concentrations of wt-sEPCR or E86A-sEPCR. Adhesion proportion of parasite-IE in the absence of sEPCR following a standardized wash was considered to represent 100% adhesion. All data points represent the mean ± SD (n=3).

Figure 5.. E86A-sEPCR allows PC activation and APC endothelial barrier protective effects in the presence of CIDRα1.1

A) The effect of E86A-sEPCR on CIDRα1.1-mediated inhibition of EPCR-dependent enhancement of PC activation. Thrombin-mediated PC activation was determined on EA.hy926 cells in presence of EPCR-binding CIDRα1.1 (50 nM) and E86A-sEPCR. PC activation in the absence of CIDRα1.1 but in the presence of E86A-sEPCR (500 nM) was set to 100%. B) The effect of E86A-sEPCR on CIDRα1.1-mediated inhibition of EPCR-dependent PAR1 cleavage by APC. APC (100 nM)-mediated PAR1 cleavage of endogenous expressed PAR1 on the surface of EA.hy926 cells was determined in the presence and absence of CIDRα1.1 (100 nM) and/or E86A-sEPCR (400 nM). C) The effect of E86A-sEPCR on CIDRα1.1-mediated inhibition of EPCR-dependent protection of endothelial barrier function by APC. APC (25 nM)-mediated protection of thrombin (2 nM)-induced barrier disruption was determined on confluent EA.hy926 cell layers in the presence and absence of EPCR-binding CIDRα1.1 (12.5 nM) and/or E86A-sEPCR (75 nM). The reduction of impedance induced by thrombin was set as 100% barrier disruption. All data points represent the mean ± SD (n=3). P-values were calculated using: A) and C) 1-way ANOVA and Dunnett’s Multiple Comparison Test, B) Student’s t-test. * denotes p<0.05, ** denotes p<0.005, *** denotes p<0.001.

Figure 6.. Schematic model of acquired PC system deficiency due to IE binding to EPCR and restoration of cellular EPCR function by E86A-sEPCR

A) Binding of the CIDRα1 domain of PfEMP1 to EPCR prevents the interaction of PC and APC with EPCR resulting in a loss of cellular EPCR function. The inability of EPCR to promote protein C activation to generate APC (not shown) and to facilitate APC-mediated PAR1 activation to induce cytoprotective activities causes an acquired functional PC system deficiency. B) Although wt-sEPCR binds the CIDRα1 domain of PfEMP1 with high affinity and increased endogenous sEPCR levels have been implicated to protect against severe symptoms of P. falciparum infection, the competition of wt-sEPCR at high concentrations with APC binding to cellular EPCR may counteract the intended restoration PC and APC binding to cellular EPCR. C) The non-(A)PC binding sEPCR variant, E86A-sEPCR, overcomes the limitation of wt-sEPCR and provides a viable decoy strategy for PfEMP1 to enable cellular EPCR functions. Consequently, E86A-sEPCR prevents binding of the CIDRα1 domain of PfEMP1 to cellular EPCR and allows cellular EPCR function to promote protein C activation and to facilitate APC-mediated PAR1 activation to induce cytoprotective activities without competition for APC binding to cellular EPCR.