Endothelial cell protein C receptor: a multiliganded and multifunctional receptor

Abstract

Endothelial cell protein C receptor (EPCR) was first identified and isolated as a cellular receptor for protein C on endothelial cells. EPCR plays a crucial role in the protein C anticoagulant pathway by promoting protein C activation. In the last decade, EPCR has received wide attention after it was discovered to play a key role in mediating activated protein C (APC)-induced cytoprotective effects, including antiapoptotic, anti-inflammatory, and barrier stabilization. APC elicits cytoprotective signaling through activation of protease activated receptor-1 (PAR1). Understanding how EPCR-APC induces cytoprotective effects through activation of PAR1, whose activation by thrombin is known to induce a proinflammatory response, has become a major research focus in the field. Recent studies also discovered additional ligands for EPCR, which include factor VIIa, Plasmodium falciparum erythrocyte membrane protein, and a specific variant of the T-cell receptor. These observations open unsuspected new roles for EPCR in hemostasis, malaria pathogenesis, innate immunity, and cancer. Future research on these new discoveries will undoubtedly expand our understanding of the role of EPCR in normal physiology and disease, as well as provide novel insights into mechanisms for EPCR multifunctionality. Comprehensive understanding of EPCR may lead to development of novel therapeutic modalities in treating hemophilia, inflammation, cerebral malaria, and cancer.

Figure 1

EPCR structure. The soluble EPCR molecule (yellow ribbon) with a portion of the protein C Gla domain (green ribbon), and a lipid molecule (the space filling balls in the center). In EPCR, 2 α-helices and an 8-strand β-sheet create a groove that is filled with the phospholipid. Binding of calcium ions (magenta spheres) to the protein C Gla domain exposes the N-terminal ω-loop to interact with EPCR (reproduced from Oganesyan et al., J Biol Chem. 2002;277:24851-24854).

Figure 2

Amino acid homology and binding characteristics of human and murine ligands to EPCR. (A) The amino acid residues in human protein C (hPC) that are involved in binding to human EPCR and the corresponding residues in other proteins were highlighted (light green color). Ability of these proteins to bind either human or murine EPCR is shown relative to human protein C binding to human EPCR, which was assigned 4 checkmarks. The binding properties reported earlier,^,-,,,-, including K_d values, relative amounts of the ligand bound to a constant amount of EPCR, and in vivo displacement of the bound ligand by exogenously administered competing ligand, were used cumulatively in assigning the relative binding. X, no detectable or significant binding; ND, not determined. Both the zymogen and the corresponding active protease ligand interact with EPCR with similar affinities. (B) Amino acid homology of the extracellular region of human and murine EPCR. The 10 residues highlighted with orange boxes are those that, when mutated to alanine, resulted in the loss of APC binding to hEPCR. Residues highlighted in red were shown to be important for proper EPCR conformation. Therefore, when these residues are mutated to alanine, they result in the loss of APC binding and all mAb epitopes.

Figure 3

Various ligands of EPCR. In addition to (A) acting as a cellular receptor for protein C (PC) and APC, (B) EPCR was shown to interact with factor VII (FVII) and factor VIIa (FVIIa), (C) Mac1 on leukocytes, either directly or indirectly through its binding to proteinase 3 (PR3), (D) P falciparum erythrocyte membrane protein 1 (PfEMP1) expressed by P falciparum-infected erythrocyte, and (E) a variant of γδ T-cell receptor induced in response to stress elicited by infection or malignancy. PC/APC, FVII/FVIIa, Mac1, and PfEMP1 bind EPCR at the same or an overlapping site.

Figure 4

The role of EPCR in blood coagulation. In hemostasis, EPCR promotes the activation of protein C (PC) bound to it by thrombin (T):TM complex. The APC then down-regulates thrombin generation by interacting with phospholipids on the membrane and inactivating cofactors factor VIIIa and factor Va. However, in specific therapeutical conditions, such as administration of a high concentration of rFVIIa to treat hemophilic patients with inhibitors or trauma patients, the EPCR anticoagulant function may be diminished as the exogenously administered FVIIa effectively competes with plasma protein C for binding to EPCR and thus displaces protein C from the EPCR, resulting in down-regulation of APC generation. Down-regulation of APC generation could lead to increased thrombin generation as FVa and FVIIIa are relieved from their inactivation by APC. This mechanism may be responsible partly to the hemostatic effect conferred by therapeutic administration of rFVIIa.

Figure 5

The cytoprotective signaling of EPCR-APC. EPCR-bound APC cleaves PAR1, and this PAR1 cleavage specifically activates Rac1 pathway, inhibits the activation of the nuclear factor-κB (NF-κB) pathway, and provides barrier protection. Cross-activation of the S1P1 by sphingosine kinase-1 stimulated by EPCR-APC activation of PAR1 may contribute to the EPCR-APC–mediated barrier protective effect and cell survival. FVIIa bound to EPCR can also cleave PAR1 and provide the barrier protective effect. However, mechanistic details involved in the FVIIa-EPCR–induced barrier protective effect are unknown. In contrast to EPCR-dependent PAR1 signaling, thrombin cleavage of PAR1 leads to RhoA activation and activation of nuclear factor-κB, leading to proinflammatory gene expression and barrier disruption. Various hypotheses were put forth on why EPCR-APC cleavage of PAR1 leads to cytoprotective signaling, whereas thrombin activation of PAR1 leads to proinflammatory response. These hypotheses have been discussed in the text.

Figure 6

Multiple effects of EPCR on cancer. In the vasculature, the EPCR-APC–mediated signaling pathway on the endothelium provides an antimetastatic effect by inhibiting the extravasation of tumor cells through down-regulation of vascular adhesion molecules on the endothelium that are involved in tumor cell adhesion and by enhancing barrier integrity of the endothelium. EPCR on tumor cells may increase the metastatic potential as EPCR-APC signaling promotes tumor cell survival, migration, and invasion. In the tumor compartment, EPCR, in general, promotes tumor growth and burden through its survival benefits. However, in malignant pleural mesothelioma (MPM), EPCR suppresses tumor growth by promoting tumor cell apoptosis and/or inhibiting tumor cell proliferation. At present, it is unclear whether specific receptor(s) present on MPM cells or the pleural microenvironment is responsible for the EPCR-mediated tumor cell apoptosis in MPM. TF, tissue factor; PAR1, protease activated receptor-1.

Figure 7

Role of EPCR in malaria. In malaria, Plasmodium-infected erythrocytes (IEs) express PfEMP1 on the membrane. PfEMP1 binds EPCR in the same region as protein C (PC) and APC; therefore, IEs expressing PfEMP1 compete with PC and APC for EPCR. Such competition reduces PC and APC binding to EPCR and leads to down-regulation of PC activation by the thrombin (T):TM complex and loss of EPCR-APC–induced cytoprotective signaling. This results in increased barrier disruption and proinflammatory responses. Furthermore, binding of IEs to the endothelium, either through PfEMP1-EPCR interaction or other adhesive mechanisms, activates endothelial cells, and the activated endothelial cells release proinflammatory cytokines (C), which induce shedding of EPCR and TM from the cell surface. Loss of EPCR and TM from the cell surface impairs the ability of the endothelium to generate the APC and APC-induced cytoprotective effect. Down-regulation of APC also leads to increased thrombin generation and thrombin-induced proinflammatory reactions.