Abstract
Introduction: The endothelial protein C receptor (EPCR) is essential for the functions of the protein C system. EPCR enhances the activation of protein C and facilitates the activation of protease activated receptors 1 and 3 by activated protein C (APC) that are required for its cytoprotective activities. Recently EPCR was implicated in the pathogenesis of cerebral malaria due to Plasmodium falciparum infection. Cerebral malaria results >500,000 deaths annually, and survivors often suffer neurological impairments. Expression of P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) on the surface of infected erythrocytes (IE) allows for IE sequestration on microvascular endothelial cells in the brain, which causes blood-brain barrier dysfunction, vascular leakage, edema, local thrombosis, and inflammation that ultimately results in coma and death. A subset of PfEMP1 variants, associated with cerebral malaria, was found to contain a Cysteine-rich Inter Domain Region (CIDR) subtype alpha 1 (CIDRα1) that binds with high affinity to EPCR. Here we determined the effects of CIDRα1 on EPCR-dependent functions of the protein C system. Moreover, we demonstrate that disruption of EPCR’s cellular functions by CIDRα1.1 can be effectively rescued by soluble E86A-EPCR that is devoid of (A)PC binding.
Materials and Methods: EPCR binding FCR3 IT4VAR20 CIDRα1.1 and non-EPCR binding controls FCR3 IT4VAR15 CIDRα3.5, and Dd2 Dd2VAR01 CIDRa3.1 were produced in insect cells. Protein C and APC were plasma-derived and E86A- and wt-soluble EPCR (sEPCR) were produced in HEK293 cells. Cellular EPCR functions were determined on the EA.hy926 endothelial cells and endothelial permeability was determined by TER using the iCelligence (ACEA). IE adhesion assays were done on HBMECs using the P. falciparum FCR3 strain expressing IT4VAR20 PfEMP1.
Results: CIDRα1.1 bound to sEPCR with an apparent Kd of 4.3 nM and was a competitive inhibitor of APC binding to sEPCR. Dose-dependent binding of CIDRα1.1 to EA.hy926 cells but not to EPCR knockdown EA.hy926 cells indicated that EPCR was the main receptor for CIDRα1.1 binding on these cells. CIDRα1.1 inhibited APC binding to EA.hy926 cells with an IC50~35 nM. CIDRα1.1 inhibited protein C activation on EA.hy926 by >75% similar to the anti-EPCR blocking antibody rcr252, whereas control CIDRα3.5 did not affect protein C activation. Furthermore, CIDRα1.1 reduced APC-mediated PAR1 cleavage on EA.hy926 cells >3-fold and accordingly abrogated APC-mediated protection against thrombin-induced barrier disruption. Control CIDRα3.1 did not affect APC-mediated barrier protection.
To determine whether CIDRα1.1 inhibited APC binding to EPCR by direct competition or by steric hindrance, binding of CIDRα1.1 to E86A-sEPCR that does not bind APC was determined. Remarkably, CIDRα1.1 bound E86A-sEPCR with similar affinity compared to wt-sEPCR. Therefore, the ability of E86A-sEPCR to compete for CIDRα1.1 binding to cellular EPCR was determined. E86A-sEPCR dose-dependently restored APC binding to wt-sEPCR in the presence of CIDRα1.1. Protein C activation on EA.hy926 cells in the presence of CIDRα1.1 was partially restored at 5 nM and completely restored at 50 nM E86A-sEPCR. Likewise, E86A-sEPCR enhanced PAR1 cleavage by APC in the presence of CIDRα1.1 >4-fold and abrogated inhibition of APC’s barrier protective effects by CIDRα1.1. Finally, cytoadhesion of IE expressing full-length CIDRα1.1-containing PfEMP1 to HBMECs was inhibited ~70% by both E86A- and wt-sEPCR (IC50~30 nM).
Conclusions: The binding of CIDRα1.1 to EPCR greatly compromised the ability of EPCR to enhance protein C activation and to facilitate APC-mediated PAR1 cleavage and induction of endothelial barrier protective effects. Based on the well-documented neuroprotective effects of the protein C system in the brain it is likely that the PfEMP1-induced loss of EPCR-dependent functions provide a seminal contribution to the mortality and neurological damages associated with cerebral malaria. Although both E86A- and wt-sEPCR can compete for CIDRα1.1 binding to cellular EPCR, E86A-sEPCR does not bind (A)PC and therefore does not interfere with (A)PC binding to cellular EPCR. In summary, these results provide novel insights into the possible pathogenesis of cerebral malaria and present a proof-of-concept strategy for the development of novel adjunct therapies for cerebral malaria.
Disclosures
No relevant conflicts of interest to declare.
Cytoadherence Properties of Plasmodium knowlesi-Infected Erythrocytes
