Elucidating mechanisms of genetic cross-disease associations: an integrative approach implicates protein C as a causal pathway in arterial and venous diseases

Genome-wide association studies have identified many individual genetic loci associated with multiple complex traits and common diseases. There are, however, few examples where the molecular basis of such pleiotropy has been elucidated. To address this challenge, we describe an integrative approach, focusing on the p.Ser219Gly (rs867186 A>G) variant in the PROCR gene (encoding the endothelial protein C receptor, EPCR), which has been associated with lower coronary artery disease (CAD) risk but higher venous thromboembolism (VTE) risk. In a phenome scan of 12 cardiometabolic diseases and 24 molecular factors, we found that PROCR-219Gly associated with higher plasma levels of zymogenic and activated protein C as well as coagulation factor VII. Using statistical colocalization and Mendelian randomization analyses, we uncovered shared genetic etiology across activated protein C, factor VII, CAD and VTE, identifying p.S219G as the likely causal variant at the locus. In a recall-by-genotype study of 52 healthy volunteers stratified by p.S219G, we detected 2.5-fold higher soluble EPCR levels and 1.2-fold higher protein C levels in plasma per effect allele, suggesting the allele induces EPCR shedding from the membrane of endothelial cells. Finally, in cell adhesion assays, we found that increasing concentrations of activated protein C, but not soluble EPCR, reduced leukocyte–endothelial cell adhesion, a marker for vascular inflammation. These results support a role for protein C as a causal factor in arterial and venous diseases, suggesting that PROCR-219Gly protects against CAD through anti-inflammatory mechanisms while it promotes VTE risk through pro-thrombotic mechanisms. Overall, our study illustrates a multi-modal approach that can help reveal molecular underpinnings of cross-disease associations.

A meta-analysis of the endothelial protein C receptor rs867186 genotype in malaria

BACKGROUND : During P. falciparum infection, the binding of P. falciparum erythrocyte membrane protein 1 (PfEMP1) to endothelial cells (EC) results in the sequestration of pRBC. Several receptors located on the endothelial cells, including intercellular adhesion molecule 1 (ICAM-1), CD36, and endothelial protein C receptor (EPCR), contribute to PfEMP1 adhesion to the microvasculature. PfEMP1, expressed on the surface of parasitized red blood cells (pRBC), is composed of cysteine-rich interdomain regions (CIDR) and Duffy binding-like (DBL) domains. CIDRα1 competitively binds to EPCR with activated protein C (APC) and impairs cytoprotective and anticoagulant effects by APC, which plays important roles in severe malaria (SM) pathogenesis such as cerebral malaria (CM) and severe malaria anemia (SMA). The strategy to inhibit EPCR binding to pRBC while concomitantly strengthen its binding to APC may be crucial in restoring disrupted protein C (PC) system’s function. The purpose of this study is to evaluate the association between malaria severity and the EPCR genotypes as well as with soluble EPCR (sEPCR), and the study also addresses the physiological relevance of EPCR genetic polymorphism. RESULTS : In this study, we conducted a meta-analysis on the eligible studies by comparing the frequency of EPCR rs867186-GG versus rs867186-GA and -AA genotype in SM, mild malaria (MM) or uncomplicated malaria (UM) patients and healthy individuals from Thailand, Uganda, Benin, Tanzania, and Ghana. We also determined the relationship between rs867186 genotype and sEPCR levels. Our results showed that the genotype rs867186-GG is higher in MM/UM than in SM patients. SM patients carrying the rs867186-GG genotype have higher plasma soluble EPCR (sEPCR) levels than in rs867186-AG and rs867186-AA carriers. MM/UM patients carrying the rs867186-AG genotype have significantly higher level of sEPCR compared to those carrying rs867186-AA. Similarly, the rs867186-GG is associated with high sEPCR level in healthy individuals. CONCLUSIONS : This meta-analysis demonstrates that pRBCs and EPCR interactions are associated with malaria severity, and treatments that block their binding via PfEMP1 CIDRα1 could be a potential therapy for SM.

A meta-analysis of the endothelial protein C receptor rs867186 genotype in malaria

BACKGROUND : During P. falciparum infection, the binding of P. falciparum erythrocyte membrane protein 1 (PfEMP1) to endothelial cells (EC) results in the sequestration of pRBC. Several receptors located on the endothelial cells, including intercellular adhesion molecule 1 (ICAM-1), CD36, and endothelial protein C receptor (EPCR), contribute to PfEMP1 adhesion to the microvasculature. PfEMP1, expressed on the surface of parasitized red blood cells (pRBC), is composed of cysteine-rich interdomain regions (CIDR) and Duffy binding-like (DBL) domains. CIDRα1 competitively binds to EPCR with activated protein C (APC) and impairs cytoprotective and anticoagulant effects by APC, which plays important roles in severe malaria (SM) pathogenesis such as cerebral malaria (CM) and severe malaria anemia (SMA). The strategy to inhibit EPCR binding to pRBC while concomitantly strengthen its binding to APC may be crucial in restoring disrupted protein C (PC) system’s function. The purpose of this study is to evaluate the association between malaria severity and the EPCR genotypes as well as with soluble EPCR (sEPCR), and the study also addresses the physiological relevance of EPCR genetic polymorphism. RESULTS : In this study, we conducted a meta-analysis on the eligible studies by comparing the frequency of EPCR rs867186-GG versus rs867186-GA and -AA genotype in SM, mild malaria (MM) or uncomplicated malaria (UM) patients and healthy individuals from Thailand, Uganda, Benin, Tanzania, and Ghana. We also determined the relationship between rs867186 genotype and sEPCR levels. Our results showed that the genotype rs867186-GG is higher in MM/UM than in SM patients. SM patients carrying the rs867186-GG genotype have higher plasma soluble EPCR (sEPCR) levels than in rs867186-AG and rs867186-AA carriers. MM/UM patients carrying the rs867186-AG genotype have significantly higher level of sEPCR compared to those carrying rs867186-AA. Similarly, the rs867186-GG is associated with high sEPCR level in healthy individuals. CONCLUSIONS : This meta-analysis demonstrates that pRBCs and EPCR interactions are associated with malaria severity, and treatments that block their binding via PfEMP1 CIDRα1 could be a potential therapy for SM.

Meta-analysis of the endothelial protein C receptor rs867186 genotype in malaria

The interaction between the P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of parasitized red blood cells (pRBC) and the endothelial cells (EC) receptors during P. falciparum infection results in the sequestration of pRBC from blood circulation. The amount of sequestration is determined by specific interactions among PfEMP1 and several host adhesion receptors, including intercellular adhesion molecule 1 (ICAM-1), CD36, and endothelial protein C receptor (EPCR). PfEMP1 is composed of multiple domains such as the cysteine-rich inter domain region (CIDR) and Duffy binding –like (DBL) domains. CIDRα1 competitively binds to EPCR with activated protein C (APC) and impair cytoprotective and anticoagulant effects by APC, which plays an important role in severe malaria (SM) pathogenesis such as cerebral malaria (CM) and severe malaria anemia (SMA). The strategy to inhibit EPCR binding to pRBC while to concomitantly strengthen its binding to APC may be crucial in restoring impaired protein C (PC) system’s function. The purpose of this study is to evaluate the association between severity of malaria and the EPCR genotypes as well as the soluble EPCR (sEPCR), and the study also addresses the physiological relevance of EPCR genetic polymorphism. In this study, we conducted meta-analysis on the eligible studies by comparing the frequency of EPCR rs867186-GG versus rs867186- GA and -AA genotype in SM, mild malaria (MM) or uncomplicated malaria (UM) patients and healthy individuals from Thailand, Uganda, Benin, Tanzania, and Ghana. We also determined the relationship between rs867186 genotype and sEPCR levels. Our results showed that the gene type of rs867186-GG is higher in MM/UM than in SM patients. SM patients carrying the rs867186-GG genotype have higher plasma soluble EPCR (sEPCR) levels than in rs867186-AG and rs867186-AA carriers. A significant difference is seen with the higher plasma sEPCR expression among MM/UM patients carrying the rs867186-AG genotype compared to those carrying rs867186-AA. Similarly, the rs867186-GG is associated with sEPCR level in healthy individuals. In conclusion, this meta-analysis demonstrates that pRBCs and EPCR interactions are associated with malaria severity, and treatments that block pRBC binding to EPCR via PfEMP1 CIDRα1 could be a potential therapy for SM.

Endothelial protein C receptor polymorphisms and risk of sepsis in a Chinese population

Objective To examine the potential relationship of EPCR polymorphisms and the risk of sepsis in a Chinese population. Methods Snapshot SNP genotyping assays and DNA sequencing methods were used to detect polymorphisms of the EPCR gene, rs2069948C/T (2532C/T) and rs867186A/G (6936A/G), in 64 patients with sepsis and in 113 controls. Soluble EPCR (sEPCR) was measured by ELISA. Results There were significant differences in the allele and genotype frequencies of EPCR gene rs2069948C/T and allele frequencies of rs867186A/G between male and female patients and controls. Females carrying rs2069948 C/T genotype or T allele and males carrying rs867186 A allele were associated with a significantly increased risk of sepsis. Plasma sEPCR levels of sepsis patients were higher than controls and showed no correlation with EPCR gene polymorphisms. Conclusions EPCR polymorphisms may be associated with increased risk of sepsis, but this has no effect on the release of sEPCR in patients with sepsis.

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

SummaryThe 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.

Disruption of EPCR-Dependent Functions By Plasmodium Falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Can be Rescued By a Soluble EPCR Variant

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.