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Objective To investigate the effects of protein C receptor (PROCR) on the maturation and immune functions of mouse bone marrow-derived dendritic cells (BMDCs) induced by whole glucan particle (WGP). Methods The GSE2197 dataset was downloaded from the Gene Expression Omnibus (GEO) database of the National Center of Biotechnology Information (NCBI). By analyzing the microarray data of this dataset, differentially expressed genes (DEGs) were identified. Immune-related genes were obtained from the Immunology Database and Analysis Portal (IMMPORT) and intersected with DEGs to acquire immune-related differential genes. Key genes and relevant signaling pathways were identified and analyzed using specialized bioinformatics algorithms, including R packages. PCR was used to detect the differential genes in BMDCs cultured with or without WGP. In vitro, we extracted bone marrow from the tibias and fibulas of mice and induced the bone marrow cells to differentiate into BMDCs using FMS-like tyrosine kinase 3 ligand (Flt-3L). After induction, we transfected these BMDCs and subsequently stimulated them with WGP. Flow cytometry was utilized to analyze the expression of surface molecules CD40, CD80, CD86, major histocompatibility complex I/II(MHC-I/II), and programmed cell death 1-ligand 1(PD-L1) to evaluate the immunophenotype of BMDCs. ELISA was used to measure the secretion levels of cytokines interleukin 12p40(IL-12p40), IL-6 and IL-10 in the supernatants. In T cell experiments, CD8 + and CD4 + T cells were isolated from the lymph nodes and spleens of OT-I and OT-II mice using magnetic bead sorting kits. These T cells were co-cultured with specific antigen ovalbumin (OVA) and the previously prepared BMDCs. Finally, flow cytometry was used to assess T cell proliferation and differentiation to evaluate the role of BMDCs in antigen-specific T cell responses. Results Analysis revealed that the PROCR gene was among the immune-related genes differentially expressed in WGP-induced BMDCs compared to CpG-stimulated BMDCs(GSE2197). Knockdown of the PROCR gene resulted in reduced surface expression of MHC-I and increased secretion of IL-10 in WGP-stimulated BMDCs. Additionally, the ability of BMDCs to drive CD8 + T cells to produce interferon γ (IFN-γ) was significantly weakened. Conclusion PROCR regulates the expression of MHC-I and the secretion of IL-10 during the maturation of BMDCs induced by WGP, which in turn affects the proliferation and differentiation of CD8 + IFN-γ + T cells in BMDCs.

Abstract: Chronic kidney disease (CKD) is a major contributor to morbidity and mortality in sickle cell disease (SCD). Anemia, induced by chronic persistent hemolysis, is associated with the progressive deterioration of renal health, resulting in CKD. Moreover, patients with SCD experience acute kidney injury (AKI), a risk factor for CKD, often during vaso-occlusive crisis associated with acute intravascular hemolysis. However, the mechanisms of hemolysis-driven pathogenesis of the AKI-to-CKD transition in SCD remain elusive. Here, we investigated the role of increased renovascular rarefaction and the resulting substantial loss of the vascular endothelial protein C receptor (EPCR) in the progressive deterioration of renal function in transgenic SCD mice. Multiple hemolytic events raised circulating levels of soluble EPCR (sEPCR), indicating loss of EPCR from the cell surface. Using bone marrow transplantation and super-resolution ultrasound imaging, we demonstrated that SCD mice overexpressing EPCR were protective against heme-induced CKD development. In a cohort of patients with SCD, plasma sEPCR was significantly higher in individuals with CKD than in those without CKD. This study concludes that multiple hemolytic events may trigger CKD in SCD through the gradual loss of renovascular EPCR. Thus, the restoration of EPCR may be a therapeutic target, and plasma sEPCR can be developed as a prognostic marker for sickle CKD., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Citrus species among the most important and widely consumed fruit in the world due to Vitamin C, essential oil glands, and flavonoids. Highly variable simple sequence repeats (SSR) markers are one of the most informative and versatile molecular markers used in perennial tree genetic research. SSR survey of Citrus sinensis and Citrus maxima were identified perfect SSRs spanning nine chromosomes. Furthermore, we categorized all SSR motifs into three major classes based on their tract lengths. We designed and validated a class I SSRs in the C. sinensis and C. maxima genome through electronic polymerase chain reaction (ePCR) and found 83.89% in C. sinensis and 78.52% in C. maxima SSRs producing a single amplicon. Then, we selected extremely variable SSRs (> 40 nt) from the ePCR-verified class I SSRs and in silico validated across seven draft genomes of citrus, which provided us a subset of 84.74% in C. sinensis and 77.53% in C. maxima highly polymorphic SSRs. Out of these, 129 primers were validated on 24 citrus genotypes through wet-lab experiment. We found 127 (98.45%) polymorphic HvSSRs on 24 genotypes. The utility of the developed HvSSRs was demonstrated by analysing genetic diversity of 181 citrus genotypes using 17 HvSSRs spanning nine citrus chromosomes and were divided into 11 main groups through 17 HvSSRs. These chromosome-specific SSRs will serve as a powerful genomic tool used for future QTL mapping, molecular breeding, investigation of population genetic diversity, comparative mapping, and evolutionary studies among citrus and other relative genera/species., (© 2023. The Author(s).)

Background/aim: The Philadelphia chromosome-negative (Ph-) myeloproliferative neoplasms (MPNs) are a group of blood cancers that arise from abnormal growth of blood cells in the bone marrow. Patients with MPNs are at increased risk for life-threatening thromboembolic complications. The detection of JAK2V617F in endothelial cells (ECs) brought a new perspective to the research of thromboembolic events. However, the mechanisms by which the mutation contributes to risk have yet to be entirely understood. Consequently, the objective of this study was to investigate how JAK2V617F impacts endothelial cells by considering thermoregulation., Materials and Methods: We applied our previously created model for EC that was genetically modified with JAK2 wild type (WT)-GFP and JAK2V617F-GFP lentiviruses; the cells were cultured for 48 h at 37°C for normothermia and 32°C for mild hypothermia. We examined the effect of thermoregulation on infection efficiency and the expression of cell surface markers, including endothelial protein C receptor (EPCR), thrombomodulin (TM), and tissue factor (TF), which are related to the coagulation pathways. Furthermore, the microparticle production from the genetically modified EC (EMPs) was analyzed., Results: We found suppression of the expression of coagulation factors, including EPCR, TM, and TF in JAK2V617F positive ECs under mild hypothermia. JAK2V617F-positive ECs showed slightly higher EMP production under mild hypothermia., Conclusion: Although the molecular mechanisms of the thermal effects on the tumor microenvironment with JAK2V617F and its effect on EMP production and coagulation are not known yet, the therapy-oriented effect of thermoregulation might be considered in future studies., (Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Plasmodium parasites responsible for malaria follow a complex life cycle of which half takes place inside the human host. Parasites present diverse antigens at different stages of their life cycle and interact with many surface molecules to attach to and enter host cells. The CIDRα1 domain of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) in infected erythrocytes adheres to one such vascular receptor endothelial protein C receptor (EPCR). EPCR is implicated in the pathogenesis of severe malaria as preferential binding of CIDRα1 to endothelium results in widespread sequestration of infected erythrocytes leading to endothelium inflammation and severe disease. A single EPCR variant S219G is clinically reported to provide protection from severe malaria. In this work, we have collated all single nucleotide polymorphisms (SNPs) in EPCR from dbSNP. We structurally mapped the SNPs on the three-dimensional complex of EPCR and PfEMP1 CIDRα1. Analysis shows that most EPCR mutations lie on the receptor surface and are non-conservative. Of the 11 mutations in the CIDRα1-interaction region of EPCR, S88P, L96V/I, and R98L/H/P/C are seen with comparably higher occurrences in diverse populations. Our structural analysis details a framework of the interactions between the parasite ligand and host factor EPCR. These structural glimpses provide a blueprint for designing both field-based variant sequencing studies and vaccine development., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

Endothelial protein C receptor (EPCR) has emerged as one of the most conserved and reliable surface markers for the prospective identification and isolation of hematopoietic stem cells (HSCs). Prior studies have consistently demonstrated that EPCR expression enriches HSCs capable of long-term multilineage repopulation in both mouse and human across different hematopoietic tissues, including bone marrow (BM), fetal liver and ex vivo HSC expansion cultures. However, little is known about the expression profiles of EPCR in multipotent progenitor (MPP) populations located immediately downstream of HSCs in the hematopoietic hierarchy and which play a major role in sustaining lifelong blood cell production. Here, we incorporate EPCR antibody detection into a multi-parameter flow cytometric panel, which allows accurate identification of HSCs and five MPP subsets (MPP1-5) in mouse BM. Our data reveal that all MPP populations contain EPCR-expressing cells. Multipotent MPP1 and MPP5 contain higher proportion of EPCR + cells compared to the more lineage-biased MPP2-4. Notably, high expression of EPCR enriches phenotypic HSC and MPP5, but not MPP1. Comparison of EPCR expression profiles between young and old BM reveals ageing mediated expansion of EPCR-expressing cells only in HSCs, but not in any of the MPP populations. Collectively, our study provides a comprehensive characterization of the surface expression pattern of EPCR in mouse HSC and MPP1-5 cells during normal and aged hematopoiesis., (Copyright © 2023. Published by Elsevier B.V.)

Background: Due to their chronic hypercoagulable status, thalassemic individuals are at an elevated risk of developing thromboembolic sequence consequences. The goal of the current study is to assesses the EPCR gene polymorphism and soluble EPCR in Egyptian thalassemic children and its role in hypercoagulable state., Research Design and Methods: Eighty children diagnosed as thalassemia major and 80 healthy youngsters as a control group. The EPCR gene was identified using a restriction fragment length polymerase chain reaction (RFLP PCR). Additionally, we assessed the soluble EPCR levels using an enzyme-linked immunosorbent assay (ELISA)., Results: Frequency of 1651C-G EPCR, the GC genotype was strongly related with an increased risk of coagulation (OR = 1.83 (0.64-5.26), P = 0.0.016). In addition, soluble EPCR was considerably higher in patients with thalassemia than in controls, P value <0.001. Our study revealed significance difference between soluble EPCR and different genotypes., Conclusion: Polymorphisms in the EPCR gene and an elevated soluble EPCR level in patients with β-thalassemia major may contribute to these patients' hemostatic derangement in thalassemic Egyptian children.

The endothelial protein C receptor (EPCR)-rs867186 G allele has been linked to high plasma levels of soluble EPCR (sEPCR) and controversially associated with either susceptibility or resistance to severe and cerebral malaria. In this study, quantitative enzyme-linked immunosorbent assay and sequencing were used to assess sEPCR levels and EPCR-rs867186 polymorphism in blood samples from Beninese children with different clinical presentations of malaria. Our findings show that sEPCR levels were higher at hospital admission than during convalescence and that EPCR-rs867186 G allele was associated with increased sEPCR plasma levels, malaria severity, and mortality rate (P < .001, P = .03, and P = .04, respectively), suggesting a role of sEPCR in the pathogenesis of severe malaria., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The development of cardiovascular disease shows increase after contracting coronavirus 2019 (COVID-19) disease and myocardial damage is observed in patients who have had the disease severely. The relationship between genetic cardiovascular risk factors with COVID-19 infection was investigated in our study. One hundred thirty-five patients, 27 of whom were COVID-19 (-) and 108 were COVID-19 (+) patients, were included in the study. Patients were divided into three groups ([COVID-19 [-], COVID-19 [+] asymptomatic, and COVID-19 [+] symptomatic + patients with pulmonary involvement]). Genetic cardiovascular risk factors were examined in blood samples taken from the patients with new generation sequencing analysis. In the clinical classification, there were no significant differences between the three groups in fibrinogen beta chain-455G>A, human platelet antigen 1 (HPA1b)/platelet receptor GPIIIa/(ITGB3) (HPA1a/b; GpIIIa; integrin beta 3 L33P), ACE I/D, AGT (M268T), AGTR1 (1166A>C), Apo E (E2/E3/E4) (rs7412, rs429358), eNOS (786T>C), eNOS (894G>T) genes (p > 0.05). However, significant differences were observed in PROCR H3 haplotype/G (endothelial protein C receptor gene [EPCR] 4600A>G [A3 haplotype]), PROCR H1 haplotype/C (EPCR 4678G>C [A1 haplotype]) genes (p < 0.05). When COVID-19 (+) and COVID-19 (-) groups were compared, it was observed that the infection was more common in people with PROCR H1 haplotype/C and PROCR H3 haplotype/G genotypes (p < 0.05). PROCR H1 and PROCR H3 haplotypes may be an important factor in contracting COVID-19 disease. In people with COVID-19 disease, revealing PROCR genetic differences and measuring sEPCR levels will be beneficial in the follow-up of the disease., (© 2022 Wiley Periodicals LLC.)

Background:  The protein C (PC) anticoagulant system has a key role in maintaining hemostatic balance. One missense (Ser219Gly) variant in the PC receptor ( PROCR ) was associated with venous thromboembolism (VTE) in genome-wide association studies., Objectives:  This study aimed to determine the thrombotic risk of rare and common PROCR variants in a large population-based cohort of middle-aged and older adults., Methods:  The exonic sequence of PROCR was analyzed for the Ser219Gly variant and other qualifying variants in 28,794 subjects (born 1923-1950, 60% women) without previous VTE, who participated in the Malmö Diet and Cancer study (1991-1996). Incidence of VTE was followed up until 2018. Qualifying variants were defined as loss-of-function or nonbenign (PolyPhen-2) missense variants with minor allele frequencies (MAFs) <0.1%., Results:  Re-sequencing identified 36 PROCR variants in the study population (26,210 non-VTE exomes and 2,584 VTE exomes), 11 synonymous, 22 missense, and three loss-of-function variants. Kaplan-Meier analysis of the known Ser219Gly variant (rs867186) showed that homozygosity for this variant increased the risk of disease, whereas heterozygosity showed no effect. Cox multivariate regression analysis revealed an adjusted hazard ratio (HR) of 1.5 (95% confidence interval [CI]: 1.1-2.0). Fifteen rare variants were classified as qualifying and were included in collapsing analysis (burden test and SKAT-O). They did not contribute to risk. However, a Arg113Cys missense variant (rs146420040; MAF = 0.004) showed an increased VTE risk (HR = 1.3; 95% CI: 1.0-1.9)., Conclusion:  Homozygosity for the Ser219Gly variant and a previously identified functional PROCR variant (Arg113Cys) was associated with VTE. Other variants did not contribute to VTE., Competing Interests: None declared., (Thieme. All rights reserved.)

Vascular establishment is one of the early events in embryogenesis. It is believed that vessel-initiating endothelial progenitors cluster to form the first primitive vessel. Understanding the molecular identity of these progenitors is crucial in order to elucidate lineage hierarchy. In this study, we identify protein C receptor (Procr) as an endothelial progenitor marker and investigate the role of Procr+ progenitors during embryonic vascular development. Using a ProcrmGFP-2A-lacZ reporter, we reveal a much earlier Procr expression (embryonic day 7.5) than previously acknowledged (embryonic day 13.5). Genetic fate-mapping experiments using ProcrCre and ProcrCreER demonstrate that Procr+ cells give rise to blood vessels throughout the entire embryo proper. Single-cell RNA-sequencing analyses place Procr+ cells at the start of endothelial commitment and maturation. Furthermore, targeted ablation of Procr+ cells results in failure of vessel formation and early embryonic lethality. Notably, genetic fate mapping and scRNA-seq pseudotime analysis support the view that Procr+ progenitors can give rise to hemogenic endothelium. In this study, we establish a Procr expression timeline and identify Procr+ vessel-initiating progenitors, and demonstrate their indispensable role in establishment of the vasculature during embryo development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The protein C receptor (Procr) has been implicated as a stem cell surface marker in several tissues. It is unknown whether Procr acts as a functional signaling receptor in stem cells. Here, by conditional knockout in mammary stem cells (MaSCs), we demonstrate that Procr is essential for mammary gland development and homeostasis. Through proteomics profiling, we identify that, upon stimulation by the ligand protein C, Procr interacts with heat shock protein 90 (HSP90AA1) via its short cytoplasmic tail, recruiting Src and IGF1R to the complex at the plasma membrane. We show that Procr acts as a signaling receptor of protein C in regulation of MaSCs through HSP90, Src, and IGF1R in vitro. In vivo, IGF1R deletion in MaSCs displays similar phenotypes to Procr deletion. These findings illustrate the essential role of Procr signaling in MaSC maintenance, shedding light onto the molecular regulation by Procr in tissue stem cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Inc.)

Ovarian surface epithelium (OSE) undergoes recurring ovulatory rupture and OSE stem cells rapidly generate new cells for the repair. How the stem cell activation is triggered by the rupture and promptly turns on proliferation is unclear. Our previous study has identified that Protein C Receptor (Procr) marks OSE progenitors. In this study, we observed decreased adherent junction and selective activation of YAP signaling in Procr progenitors at OSE rupture site. OSE repair is impeded upon deletion of Yap1 in these progenitors. Interestingly, Procr+ progenitors show lower expression of Vgll4, an antagonist of YAP signaling. Overexpression of Vgll4 in Procr+ cells hampers OSE repair and progenitor proliferation, indicating that selective low Vgll4 expression in Procr+ progenitors is critical for OSE repair. In addition, YAP activation promotes transcription of the OSE stemness gene Procr . The combination of increased cell division and Procr expression leads to expansion of Procr+ progenitors surrounding the rupture site. These results illustrate a YAP-dependent mechanism by which the stem/progenitor cells recognize the murine ovulatory rupture, and rapidly multiply their numbers, highlighting a YAP-induced stem cell expansion strategy., Competing Interests: JW, CL, LH, ZX, LB, WY, ZW, YL, DG, JF, LZ No competing interests declared, YZ Reviewing editor, eLife, (© 2022, Wang et al.)

Many individual genetic risk loci have been associated with multiple common human diseases. However, the molecular basis of this pleiotropy often remains unclear. We present an integrative approach to reveal the molecular mechanism underlying the PROCR locus, associated with lower coronary artery disease (CAD) risk but higher venous thromboembolism (VTE) risk. We identify PROCR-p.Ser219Gly as the likely causal variant at the locus and protein C as a causal factor. Using genetic analyses, human recall-by-genotype and in vitro experimentation, we demonstrate that PROCR-219Gly increases plasma levels of (activated) protein C through endothelial protein C receptor (EPCR) ectodomain shedding in endothelial cells, attenuating leukocyte-endothelial cell adhesion and vascular inflammation. We also associate PROCR-219Gly with an increased pro-thrombotic state via coagulation factor VII, a ligand of EPCR. Our study, which links PROCR-219Gly to CAD through anti-inflammatory mechanisms and to VTE through pro-thrombotic mechanisms, provides a framework to reveal the mechanisms underlying similar cross-phenotype associations., (© 2022. The Author(s).)

Background: Protein C receptor (Procr) has recently been shown to mark resident adult stem cells in the mammary gland, vascular system, and pancreatic islets. More so, high Procr expression was also detected and used as indicator for subsets of triple-negative breast cancers (TNBCs). Previous study has revealed Procr as a target of Wnt/β-catenin signaling; however, direct upstream regulatory mechanism of Procr remains unknown. To comprehend the molecular role of Procr during physiology and pathology, elucidating the upstream effectors of Procr is necessary. Here, we provide a system for screening negative regulators of Procr, which could be adapted for broad molecular analysis on membrane proteins., Results: We established a screening system which combines CRISPR-Cas9 guided gene disruption with fluorescence activated cell sorting technique (FACS). CommaDβ (murine epithelial cells line) was used for the initial Procr upstream effector screening using lentiviral CRISPR-gRNA library. Shortlisted genes were further validated through individual lentiviral gRNA infection followed by Procr expression evaluation. Adam17 was identified as a specific negative inhibitor of Procr expression. In addition, MDA-MB-231 cells and Hs578T cells (human breast cancer cell lines) were used to verify the conserved regulation of ADAM17 over PROCR expression., Conclusion: We established an efficient CRISPR-Cas9/FACS screening system, which identifies the regulators of membrane proteins. Through this system, we identified Adam17 as the negative regulator of Procr membrane expression both in mammary epithelial cells and breast cancer cells., (© 2021. The Author(s).)

Sequestration of Plasmodium falciparum ( P. falciparum )-infected erythrocytes to host endothelium through the parasite-derived P. falciparum erythrocyte membrane protein 1 ( Pf EMP1) adhesion proteins is central to the development of malaria pathogenesis. Pf EMP1 proteins have diversified and expanded to encompass many sequence variants, conferring each parasite a similar array of human endothelial receptor-binding phenotypes. Here, we analyzed RNA-seq profiles of parasites isolated from 32 P. falciparum- infected adult travellers returning to Germany. Patients were categorized into either malaria naive (n = 15) or pre-exposed (n = 17), and into severe (n = 8) or non-severe (n = 24) cases. For differential expression analysis, Pf EMP1-encoding var gene transcripts were de novo assembled from RNA-seq data and, in parallel, var- expressed sequence tags were analyzed and used to predict the encoded domain composition of the transcripts. Both approaches showed in concordance that severe malaria was associated with Pf EMP1 containing the endothelial protein C receptor (EPCR)-binding CIDRα1 domain, whereas CD36-binding Pf EMP1 was linked to non-severe malaria outcomes. First-time infected adults were more likely to develop severe symptoms and tended to be infected for a longer period. Thus, parasites with more pathogenic Pf EMP1 variants are more common in patients with a naive immune status, and/or adverse inflammatory host responses to first infections favor the growth of EPCR-binding parasites., Competing Interests: JW, GT, TT, RK, BK, JS, Hv, JS, HS, RW, LT, FL, AS, IB, ET, RF, TO, TL, TG, MD, AB No competing interests declared, (© 2021, Wichers et al.)

Streptococcus pneumoniae is the leading cause of hospital community-acquired pneumonia. Patients with pneumococcal pneumonia may develop complicated parapneumonic effusions or empyema that can lead to pleural organization and subsequent fibrosis. The pathogenesis of pleural organization and scarification involves complex interactions between the components of the immune system, coagulation, and fibrinolysis. EPCR (endothelial protein C receptor) is a critical component of the protein C anticoagulant pathway. The present study was performed to evaluate the role of EPCR in the pathogenesis of S. pneumoniae infection-induced pleural thickening and fibrosis. Our studies show that the pleural mesothelium expresses EPCR. Intrapleural instillation of S. pneumoniae impairs lung compliance and lung volume in wild-type and EPCR-overexpressing mice but not in EPCR-deficient mice. Intrapleural S. pneumoniae infection induces pleural thickening in wild-type mice. Pleural thickening is more pronounced in EPCR-overexpressing mice, whereas it is reduced in EPCR-deficient mice. Markers of mesomesenchymal transition are increased in the visceral pleura of S. pneumoniae- infected wild-type and EPCR-overexpressing mice but not in EPCR-deficient mice. The lungs of wild-type and EPCR-overexpressing mice administered intrapleural S. pneumoniae showed increased infiltration of macrophages and neutrophils, which was significantly reduced in EPCR-deficient mice. An analysis of bacterial burden in the pleural lavage, the lungs, and blood revealed a significantly lower bacterial burden in EPCR-deficient mice compared with wild-type and EPCR-overexpressing mice. Overall, our data provide strong evidence that EPCR deficiency protects against S. pneumoniae infection-induced impairment of lung function and pleural remodeling.

Antiphospholipid antibodies (aPLs) cause severe autoimmune disease characterized by vascular pathologies and pregnancy complications. Here, we identify endosomal lysobisphosphatidic acid (LBPA) presented by the CD1d-like endothelial protein C receptor (EPCR) as a pathogenic cell surface antigen recognized by aPLs for induction of thrombosis and endosomal inflammatory signaling. The engagement of aPLs with EPCR-LBPA expressed on innate immune cells sustains interferon- and toll-like receptor 7-dependent B1a cell expansion and autoantibody production. Specific pharmacological interruption of EPCR-LBPA signaling attenuates major aPL-elicited pathologies and the development of autoimmunity in a mouse model of systemic lupus erythematosus. Thus, aPLs recognize a single cell surface lipid-protein receptor complex to perpetuate a self-amplifying autoimmune signaling loop dependent on the cooperation with the innate immune complement and coagulation pathways., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Extensive fibrin deposition in the lungs and altered levels of circulating blood coagulation proteins in COVID-19 patients imply local derangement of pathways that limit fibrin formation and/or promote its clearance. We examined transcriptional profiles of bronchoalveolar lavage fluid (BALF) samples to identify molecular mechanisms underlying these coagulopathies. mRNA levels for regulators of the kallikrein-kinin (C1-inhibitor), coagulation (thrombomodulin, endothelial protein C receptor), and fibrinolytic (urokinase and urokinase receptor) pathways were significantly reduced in COVID-19 patients. While transcripts for several coagulation proteins were increased, those encoding tissue factor, the protein that initiates coagulation and whose expression is frequently increased in inflammatory disorders, were not increased in BALF from COVID-19 patients. Our analysis implicates enhanced propagation of coagulation and decreased fibrinolysis as drivers of the coagulopathy in the lungs of COVID-19 patients., Competing Interests: AM receives research funding from Novo Nordisk and has received honoraria for serving on Novo Nordisk advisory boards. AW receives research funding from Takeda and Bristol Myers Squibb, DG receives research funding from Bayer and has received honoraria for serving on Anthos, Bristol-Myers Squibb, Ionis and Janssen advisory boards. MG, CA, JM, BA, DJ No competing interests declared, (© 2021, Mast et al.)

The Plasmodium falciparum erythrocyte-membrane-protein-1 (PF3D7&#95;1150400/PF11&#95;0521) contains both domain cassette DC13 and DBLβ3 domain binding to EPCR and ICAM-1 receptors, respectively. This type of PfEMP1 proteins with dual binding specificity mediate specific interactions with brain micro-vessels endothelium leading to the development of cerebral malaria (CM). Using plasma collected from children at time of hospital admission and after 30 days, we study an acquisition of IgG response to PF3D7&#95;1150400/PF11&#95;0521 DC13 and DBLβ3&#95;D4 recombinant constructs, and five peptides located within these constructs, specifically in DBLα1.7&#95;D2 and DBLβ3&#95;D4 domains. We found significant IgG responses against the entire DC13, PF11&#95;0521&#95;DBLβ3&#95;D4 domain, and peptides. The responses varied against different peptides and depended on the clinical status of children. The response was stronger at day 30, and mostly did not differ between CM and uncomplicated malaria (UM) groups. Specifically, the DBLβ3 B3-34 peptide that contains essential residues involved in the interaction between PF11&#95;0521 DBLβ3&#95;D4 domain and ICAM-1 receptor demonstrated significant increase in reactivity to IgG1 and IgG3 antibodies at convalescence. Further, IgG reactivity in CM group at time of admission against functionally active (ICAM-1-binding) PF11&#95;0521 DBLβ3&#95;D4 domain was associated with protection against severe anemia. These results support development of vaccine based on the PF3D7&#95;1150400/PF11&#95;0521 structures to prevent CM.

Objective: Thrombophilia is known to be associated with poor pregnancy outcomes. In this study, three thrombophilic gene polymorphisms, including EPCR (Ser219Gly), F11 (rs4253417) and F7 (323 Ins10) were investigated in an Iranian population of women in order to determine the correlation between thrombophilia and recurrent pregnancy loss (RPL)., Methods: Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) were used to evaluate the frequency of three candidate thrombophilic risk factors for recurrent pregnancy loss. The frequencies of the polymorphisms were compared between the case (144 patients with a history of at least two miscarriages) and the control (150 healthy women with no abortion) group., Results: Our results show that EPCR and FVII polymorphisms of the patient and control group have the same genotype frequency, and the difference is not statistically significant ( p -value > .05). Regarding FXI polymorphism, TT genotype frequency was higher in the patient group than the control group ( p -value < .05); however, CT heterozygote form was higher in the control group compared to the patient group ( p -value < .05)., Conclusion: In FXI polymorphism, T allele is possibly an RPL risk factor and C allele has a protective role. Thus, wild type FXI could be related to RPL, but EPCR and FVII polymorphism have no such correlation.

PfEMP1 is the major antigen involved in Plasmodium falciparum -infected erythrocyte sequestration in cerebrovascular endothelium. While some PfEMP1 domains have been associated with clinical phenotypes of malaria, formal associations between the expression of a specific domain and the adhesion properties of clinical isolates are limited. In this context, 73 cerebral malaria (CM) and 98 uncomplicated malaria (UM) Beninese children were recruited. We attempted to correlate the cytoadherence phenotype of Plasmodium falciparum isolates with the clinical presentation and the expression of specific PfEMP1 domains. Cytoadherence level on Hbec-5i and CHO-ICAM-1 cell lines and var genes expression were measured. We also investigated the prevalence of the ICAM-1-binding amino acid motif and dual receptor-binding domains, described as a potential determinant of cerebral malaria pathophysiology. We finally evaluated IgG levels against PfEMP1 recombinant domains (CIDRα1.4, DBLβ3, and CIDRα1.4-DBLβ3). CM isolates displayed higher cytoadherence levels on both cell lines, and we found a correlation between CIDRα1.4-DBLβ1/3 domain expression and CHO-ICAM-1 cytoadherence level. Endothelial protein C receptor (EPCR)-binding domains were overexpressed in CM isolates compared to UM whereas no difference was found in ICAM-1-binding DBLβ1/3 domain expression. Surprisingly, both CM and UM isolates expressed ICAM-1-binding motif and dual receptor-binding domains. There was no difference in IgG response against DBLβ3 between CM and UM isolates expressing ICAM-1-binding DBLβ1/3 domain. It raises questions about the role of this motif in CM pathophysiology, and further studies are needed, especially on the role of DBLβ1/3 without the ICAM-1-binding motif. IMPORTANCE Cerebral malaria pathophysiology remains unknown despite extensive research. PfEMP1 proteins have been identified as the main Plasmodium antigen involved in cerebrovascular endothelium sequestration, but it is unclear which var gene domain is involved in Plasmodium cytoadhesion. EPCR binding is a major determinant of cerebral malaria whereas the ICAM-1-binding role is still questioned. Our study confirmed the EPCR-binding role in CM pathophysiology with a major overexpression of EPCR-binding domains in CM isolates. In contrast, ICAM-1-binding involvement appears less obvious with A-type ICAM-1-binding and dual receptor-binding domain expression in both CM and UM isolates. We did not find any variations in ICAM-1-binding motif sequences in CM compared to UM isolates. UM and CM patients infected with isolates expressing the ICAM-1-binding motif displayed similar IgG levels against DBLβ3 recombinant protein. Our study raises interrogations about the role of these domains in CM physiopathology and questions their use in vaccine strategies against cerebral malaria., (Copyright © 2020 Joste et al.)

Background: Endothelial protein C receptor (EPCR) is a membranous protein that can be combined with a variety of ligands and plays important roles in anticoagulant and anti-inflammation. Recent reports have shown that surface EPCR expression on T cells is negatively associated with Th17 differentiation and is co-expressed with other immunosuppressive molecules, such as The programmed cell death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4). Hence, we hypothesized that EPCR may play a critical role in rheumatoid arthritis (RA) disease progression that is mediated by Th17 differentiation. In order to explore the role of EPCR on RA disease pathogenesis, we detected membranous EPCR (mEPCR) expression in CD4 + T cells and soluble EPCR (sEPCR) expression in the sera of RA patients., Methods: The proportion of CD4 + /EPCR + T cells in the peripheral blood of RA patients was detected by flow cytometry, and the expression of sEPCR in the sera of RA patients was detected by enzyme-linked immunosorbent assay (ELISA). For in vitro experiments, protein C (PC) and EPCR recombinant proteins were used to block peripheral blood mononuclear cell (PBMC) activation and to detect Th17 differentiation. For in vivo experiments in DBA/1 mice with collagen-induced arthritis (CIA), we administered PC and EPCR recombinant proteins, monitored disease progression, and evaluated the role of EPCR in disease progression., Results: The proportion of CD4 + /EPCR + T cells in the peripheral blood of RA patients was lower than that of osteoarthritis (OA) patients, while the expression level of sEPCR in the sera of RA patients was concomitantly higher than that in OA patients. Subsequent analysis revealed that sEPCR expression was positively correlated with rheumatoid factors (RF) and other inflammatory indicators in RA patients. Further studies confirmed that sEPCR administration alleviated the progression of collagen-induced arthritis and partially blocked the therapeutic effect of PC in CIA mice., Conclusion: Soluble EPCR is associated with RA disease progression and induces disease remission in CIA mice by inhibiting Th17 differentiation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Endothelial protein C receptor (EPCR) plays an anticoagulant and anti-inflammatory role by promoting the activation of protein C by thrombin bound to thrombomodulin (TBM). Incompatibility between pig TBM and human/primate thrombin is thought to contribute to dysregulated coagulation in pig-to-primate organ xenografts, and expression of human TBM (hTBM) in pigs has shown benefit in preclinical models. However, it is not known whether there are incompatibilities-or molecular barriers-between endogenous pig EPCR (pEPCR) and transgenically expressed human TBM., Aim: To clone and express pEPCR, and determine its function in the human protein C pathway in vitro., Methods: Pig endothelial protein C receptor cDNA was generated from pig lung RNA by RT-PCR. Primate COS-7 transfectants expressing various combinations of human and pig TBM and EPCR were incubated with human thrombin and human protein C, and tested for TBM cofactor activity., Results: The predicted protein sequence of pEPCR shared 72.3% amino acid sequence identity with hEPCR, and residues critical for protein C binding were conserved. COS-7 cells transfected with hEPCR, pEPCR or vector showed minimal TBM cofactor activity (0.13 ± 0.04, 0.13 ± 0.02 and 0.14 ± 0.06 U, respectively). The cofactor activity of hTBM-transfected cells (1.18 ± 0.29 U) was 8-fold higher than vector-transfected cells (P = .004) and further increased 4-fold and 3-fold by co-transfection with hEPCR (5.01 ± 1.12 U, P = .004) or pEPCR (3.73 ± 0.65 U, P = .003), respectively., Conclusions: Our data show that pEPCR is largely compatible with the human TBM/thrombin complex, when expressed on COS-7 cells in vitro, promoting the activation of human protein C. These findings suggest that endogenous pEPCR will enhance the activity of transgenic hTBM in the xenograft setting., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Preeclampsia is a severe pregnancy-related disease that is found in 3%-5% of pregnancies worldwide and is primarily related to the decreased proliferation and invasion of trophoblast cells and abnormal uterine spiral artery remodelling. However, studies on the pathogenesis of placental trophoblasts are insufficient, and the aetiology of PE remains unclear. Here, we report that endothelial protein C receptor (EPCR), a transmembrane glycoprotein, was down-regulated in placentas from preeclamptic patients. Moreover, lack of EPCR significantly reduced the trophoblast cell proliferation, invasion and tube formation capabilities. Microscale thermophoresis analysis showed that EPCR directly bound to protease-activated receptor 1 (PAR-1), a G protein-coupled receptor. This change resulted in a substantial reduction in active Rac1 and caused excessive actin rearrangement. Our findings reveal a previously unidentified role of EPCR in the regulation of trophoblast proliferation, invasion and tube formation through promotion of actin polymerization, which is required for normal placental development., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background and Purpose: The involvement of protein C (PC) pathway components in multiple sclerosis (MS) has scarcely been explored. The aim was to investigate their levels in relation to clinical and neurodegenerative magnetic resonance imaging (MRI) outcomes in patients., Methods: In all, 138 MS patients and 42 healthy individuals were studied. PC, protein S (PS) and soluble endothelial protein C receptor (sEPCR) were evaluated by multiplex assays and enzyme-linked immunosorbent assay. Regression analyses between 3 T MRI outcomes and PC pathway components were performed. ancova was used to compare MRI volumes based on protein level quartiles. Partial correlation was assessed amongst levels of PC pathway components and hemostasis protein levels, including soluble thrombomodulin (sTM), heparin cofactor II (HCII), plasminogen activator inhibitor 1 (PAI-1) and factor XII (FXII). The variation of PC concentration across four time points was evaluated in 32 additional MS patients., Results: There was an association between PC concentration, mainly reflecting the zymogen PC, and MRI measures for volumes of total gray matter (GM) (P = 0.003), thalamus (P = 0.007), cortex (P = 0.008), deep GM (P = 0.009) and whole brain (P = 0.026). Patients in the highest PC level quartile were characterized by the lowest GM volumes. Correlations of PC-HCII, PC-FXII and sEPCR-sTM values were detectable in MS patients, whilst PC-PS and PS-PAI-1 correlations were present in healthy individuals only., Conclusions: Protein C plasma concentrations might be associated with neurodegenerative MRI outcomes in MS. Several differences in correlation amongst protein plasma levels suggest dysregulation of PC pathway components in MS patients. The stability of PC concentration over time supports a PC investigation in relation to GM atrophy in MS., (© 2019 European Academy of Neurology.)

Hematopoiesis is dynamically regulated to maintain blood system function under nonhomeostatic conditions such as inflammation and injury. However, common surface marker and hematopoietic stem cell (HSC) reporter systems used for prospective enrichment of HSCs have been less rigorously tested in these contexts. Here, we use two surface markers, EPCR/CD201 and CD34, to re-analyze dynamic changes in the HSC-enriched phenotypic SLAM compartment in a mouse model of chronic interleukin (IL)-1 exposure. EPCR and CD34 coordinately identify four functionally and molecularly distinct compartments within the SLAM fraction, including an EPCR + /CD34 - fraction whose long-term serial repopulating activity is only modestly impacted by chronic IL-1 exposure, relative to unfractionated SLAM cells. Notably, the other three fractions expand in frequency following IL-1 treatment and represent actively proliferating, lineage-primed cell states with limited long-term repopulating potential. Importantly, we find that the Fgd5-ZSGreen HSC reporter mouse enriches for molecularly and functionally intact HSCs regardless of IL-1 exposure. Together, our findings provide further evidence of dynamic heterogeneity within a commonly used HSC-enriched phenotypic compartment under stress conditions. Importantly, they also indicate that stringency of prospective isolation approaches can enhance interpretation of findings related to HSC function when studying models of hematopoietic stress., Competing Interests: Conflict of interest disclosure The authors declare no competing interests., (Copyright © 2019 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Sweet taste receptor (STR) is a C GPCR family member and a suggested drug target for metabolic disorders such as diabetes. Detailed characteristics of the molecule as well as its ligand interactions mode are yet considerably unclear due to experimental study limitations of transmembrane proteins. An in silico study was designed to find the putative carbohydrate binding sites on STR. To this end, α-D-glucose and its α-1,4-oligomers (degree of polymerization up to 14) were chosen as probes and docked into an ensemble of different conformations of the extracellular region of STR monomers (T1R2 and T1R3), using AutoDock Vina. Ensembles had been sampled from an MD simulation experiment. Best poses were further energy-minimized in the presence of water molecules with Amber14 forcefield. For each monomer, four distinct binding regions consisting of one or two binding pockets could be distinguished. These regions were further investigated with regard to hydrophobicity and hydrophilicity of the residues, as well as residue compositions and non-covalent interactions with ligands. Popular binding regions showed similar characteristics to carbohydrate binding modules (CBM). Observation of several conserved or semi-conserved residues in these binding regions suggests a possibility to extrapolate the results to other C GPCR family members. In conclusion, presence of CBM in STR and, by extrapolation, in other C GPCR family members is suggested, similar to previously proposed sites in gut fungal C GPCRs, through transcriptome analyses. STR modes of interaction with carbohydrates are also discussed and characteristics of non-covalent interactions in C GPCR family are highlighted.

Ovarian surface epithelium (OSE) undergoes recurring ovulatory rupture and repair. The OSE replenishing mechanism post ovulation remains unclear. Here we report that the expression of Protein C Receptor (Procr) marks a progenitor population in adult mice that is responsible for OSE repair post ovulation. Procr+  cells are the major cell source for OSE repair. The mechanism facilitating the rapid re-epithelialization is through the immediate expansion of Procr+  cells upon OSE rupture. Targeted ablation of Procr+  cells impedes the repairing process. Moreover, Procr+  cells displayed robust colony-formation capacity in culture, which we harnessed and established a long-term culture and expansion system of OSE cells. Finally, we show that Procr+  cells and previously reported Lgr5+ cells have distinct lineage tracing behavior in OSE homeostasis. Our study suggests that Procr marks progenitor cells that are critical for OSE ovulatory rupture and homeostasis, providing insight into how adult stem cells respond upon injury.

The functional heterogeneity of hematopoietic stem cells (HSCs) has been comprehensively investigated by single-cell transplantation assay. However, the heterogeneity regarding their physiological contribution remains an open question, especially for those with life-long hematopoietic fate of rigorous self-renewing and balanced differentiation capacities. In this study, we revealed that Procr expression was detected principally in phenotypical vascular endothelium co-expressing Dll4 and CD44 in the mid-gestation mouse embryos, and could enrich all the HSCs of the embryonic day 11.5 (E11.5) aorta-gonad-mesonephros (AGM) region. We then used a temporally restricted genetic tracing strategy to irreversibly label the Procr-expressing cells at E9.5. Interestingly, most labeled mature HSCs in multiple sites (such as AGM) around E11.5 were functionally categorized as lymphomyeloid-balanced HSCs assessed by direct transplantation. Furthermore, the labeled cells contributed to an average of 7.8% of immunophenotypically defined HSCs in E14.5 fetal liver (FL) and 6.9% of leukocytes in peripheral blood (PB) during one-year follow-up. Surprisingly, in aged mice of 24 months, the embryonically tagged cells displayed constant contribution to leukocytes with no bias to myeloid or lymphoid lineages. Altogether, we demonstrated, for the first time, the existence of a subtype of physiologically long-lived balanced HSCs as hypothesized, whose precise embryonic origin and molecular identity await further characterization., (Copyright © 2019 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

Breast cancer is a heterogeneous disease. In particular, triple-negative breast cancer (TNBC) comprises various molecular subgroups with unclear identities and currently has few targeted treatment options. Our previous study identified protein C receptor (Procr) as a surface marker on mammary stem cells (MaSCs) located in the basal layer of the normal mammary gland. Given the possible connection of TNBC with basal layer stem cells, we conducted comparative analyses of Procr in breast cancers of mouse and human origin. In mouse mammary tumors, we showed that Procr + cells are enriched for cancer stem cells (CSCs) in Wnt1 basal-like tumors, but not in Brca1 basal-like tumors or PyVT luminal tumors. In human cancers, PROCR was robustly expressed in half of TNBC cases. Experiments with patient-derived xenografts (PDXs) revealed that PROCR marks CSCs in this discrete subgroup (referred to as PROCR + TNBC). Interfering with the function of PROCR using an inhibitory nanobody reduced the CSC numbers, arrested tumor growth and prevented rapid tumor recurrence. Our data suggest a key role of MaSC in breast tumorigenesis. Moreover, our work indicates that PROCR can be used as a biomarker to stratify TNBC into clinically relevant subgroups and may provide a novel targeted treatment strategy for this clinically important tumor subtype.

Objective: To investigate association between the single nucleotide polymorphisms of endothelial protein C receptor (EPCR) gene and the risk of Kawasaki disease (KD) in a Chinese children.
 Methods: A total of 103 KD patients including 23 patients with coronary artery lesions (CAL) and 158 controls were recruited. Seven tagging SNPs (rs6088738, rs2069940, rs2069945, rs2069952, rs867186, rs9574, and rs1415774) of EPCR gene were selected for TaqMan allelic discrimination assay. The plasma soluble EPCR (sEPCR) levels of 53 KD and 52 healthy children were detected by ELISA.
 Results: We found a significant association between rs2069952, rs9574 or rs1415774 and higher probability for the occurrence of KD but not CAL formation. Interestingly, males with these 3 SNPs and rs2069945 SNPs bore a much greater risk of KD than females. The level of plasma sEPCR in children with KD didnot predict the formation of CAL. However, the allele G of rs867186 in EPCR was associated with the increased level of plasma sEPCR in KD patients.
 Conclusion: The SNPs of EPCR are associated with KD susceptibility in a Chinese Han children.

Xenotransplantation research has made considerable progress in recent years, largely through the increasing availability of pigs with multiple genetic modifications. We suggest that a pig with nine genetic modifications (ie, currently available) will provide organs (initially kidneys and hearts) that would function for a clinically valuable period of time, for example, >12 months, after transplantation into patients with end-stage organ failure. The national regulatory authorities, however, will likely require evidence, based on in vitro and/or in vivo experimental data, to justify the inclusion of each individual genetic modification in the pig. We provide data both from our own experience and that of others on the advantages of pigs in which (a) all three known carbohydrate xenoantigens have been deleted (triple-knockout pigs), (b) two human complement-regulatory proteins (CD46, CD55) and two human coagulation-regulatory proteins (thrombomodulin, endothelial cell protein C receptor) are expressed, (c) the anti-apoptotic and "anti-inflammatory" molecule, human hemeoxygenase-1 is expressed, and (d) human CD47 is expressed to suppress elements of the macrophage and T-cell responses. Although many alternative genetic modifications could be made to an organ-source pig, we suggest that the genetic manipulations we identify above will all contribute to the success of the initial clinical pig kidney or heart transplants, and that the beneficial contribution of each individual manipulation is supported by considerable experimental evidence., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Cerebral malaria is a severe neurological complication associated with sequestration of Plasmodium falciparum -infected erythrocytes (IE) in the brain microvasculature, but the specific binding interactions remain under debate. Here, we have generated an engineered three-dimensional (3D) human brain endothelial microvessel model and studied P. falciparum binding under the large range of physiological flow velocities that occur in both health and disease. Perfusion assays on 3D microvessels reveal previously unappreciated phenotypic heterogeneity in parasite binding to tumor necrosis factor alpha (TNF-α)-activated brain endothelial cells. While clonal parasite lines expressing a group B P. falciparum erythrocyte membrane protein 1 (PfEMP1) present an increase in binding to activated 3D microvessels, P. falciparum - IE expressing DC8-PfEMP1 present a decrease in binding. The differential response to endothelium activation is mediated by surface expression changes of endothelial protein C receptor (EPCR) and intercellular adhesion molecule 1 (ICAM-1). These findings demonstrate heterogeneity in parasite binding and provide evidence for a parasite strategy to adapt to a changing microvascular environment during infection. The engineered 3D human brain microvessel model provides new mechanistic insight into parasite binding and opens opportunities for further studies on malaria pathogenesis and parasite-vessel interactions. IMPORTANCE Cerebral malaria research has been hindered by the inaccessibility of the brain. Here, we have developed an engineered 3D human brain microvessel model that mimics the blood flow rates and architecture of small blood vessels to study how P. falciparum - infected human erythrocytes attach to brain endothelial cells. By studying parasite lines with different adhesive properties, we show that the malaria parasite binding rate is heterogeneous and strongly influenced by physiological differences in flow and whether the endothelium has been previously activated by TNF-α, a proinflammatory cytokine that is linked to malaria disease severity. We also show the importance of human EPCR and ICAM-1 in parasite binding. Our model sheds new light on how P. falciparum binds within brain microvessels and provides a powerful method for future investigations of recruitment of human brain pathogens to the blood vessel lining of the brain., (Copyright © 2019 Bernabeu et al.)

Mast cells (MCs) are immune sentinels, but whether they also function as antigen-presenting cells (APCs) remains elusive. Using mouse models of MC deficiency, we report on MC-dependent recruitment and activation of multiple T cell subsets to the skin and draining lymph nodes (DLNs) during dengue virus (DENV) infection. Newly recruited and locally proliferating γδ T cells were the first T cell subset to respond to MC-driven inflammation, and their production of IFN-γ was MC dependent. MC-γδ T cell conjugates were observed consistently in infected peripheral tissues, suggesting a new role for MCs as nonconventional APCs for γδ T cells. MC-dependent γδ T cell activation and proliferation during DENV infection required T cell receptor (TCR) signaling and the nonconventional antigen presentation molecule endothelial cell protein C receptor (EPCR) on MCs. γδ T cells, not previously implicated in DENV host defense, killed infected targeted DCs and contributed to the clearance of DENV in vivo. We believe immune synapse formation between MCs and γδ T cells is a novel mechanism to induce specific and protective immunity at sites of viral infection.

Background and Purpose: Polymorphisms in coagulation genes have been associated with early-onset ischemic stroke. Here we pursue an a priori hypothesis that genetic variation in the endothelial-based receptors of the thrombomodulin-protein C system (THBD and PROCR) may similarly be associated with early-onset ischemic stroke. We explored this hypothesis utilizing a multi-stage design of discovery and replication., Methods: Discovery was performed in the Genetics-of-Early-Onset Stroke (GEOS) Study, a biracial population-based case-control study of ischemic stroke among men and women aged 15-49 including 829 cases of first ischemic stroke (42.2% African-American) and 850 age-comparable stroke-free controls (38.1% African-American). Twenty-four single-nucleotide-polymorphisms (SNPs) in THBD and 22 SNPs in PROCR were evaluated. Following LD pruning (r2≥0.8), we advanced uncorrelated SNPs forward for association analyses. Associated SNPs were evaluated for replication in an early-onset ischemic stroke population (onset-age<60 years) consisting of 3676 cases and 21118 non-stroke controls from 6 case-control studies. Lastly, we determined if the replicated SNPs also associated with older-onset ischemic stroke in the METASTROKE data-base., Results: Among GEOS Caucasians, PROCR rs9574, which was in strong LD with 8 other SNPs, and one additional independent SNP rs2069951, were significantly associated with ischemic stroke (rs9574, OR = 1.33, p = 0.003; rs2069951, OR = 1.80, p = 0.006) using an additive-model adjusting for age, gender and population-structure. Adjusting for risk factors did not change the associations; however, associations were strengthened among those without risk factors. PROCR rs9574 also associated with early-onset ischemic stroke in the replication sample (OR = 1.08, p = 0.015), but not older-onset stroke. There were no PROCR associations in African-Americans, nor were there any THBD associations in either ethnicity., Conclusion: PROCR polymorphisms are associated with early-onset ischemic stroke in Caucasians., Competing Interests: Drs. Cole, Mitchell, Kittner, Longstreth, and Worrall are supported by research grants from National Institutes of Health (NIH). Dr Cole is supported by a research grants from the American Heart Association and Bayer Pharmaceuticals. Dr. Worrall is Deputy Editor for AAN/Neurology. Dr. Boncoraglio is supported by a research grant from the Fondazione IRCCS Istituto Neurologico Carlo Besta. Dr. Metso is supported by grants from the Finnish Medical Foundation, the Orion Farmos Research Foundation, the Maud Kuistila Memorial Foundation, and the Emil Aaltonen Foundation. Dr. Danesh serves on advisory boards for Novartis, Merck Sharp & Dohme UK, Sanofi, the Medical Research Council, and Wellcome Trust and is a consultant for Takeda. These do not alter the authors adherence to PLOS ONE policies on sharing data and materials. The other authors report no disclosures.

Introduction: Genetic variants in the endothelial protein C receptor gene (PROCR) may contribute to the thrombosis risk by regulating levels of the soluble form of this protein (sEPCR). We evaluated whether PROCR polymorphisms and sEPCR levels play a role in the thrombotic manifestations of antiphospholipid syndrome., Materials and Methods: One hundred and seventy-five patients (62 with primary antiphospholipid syndrome, 30 with antiphospholipid syndrome associated with systemic lupus erythematosus, 40 with systemic lupus erythematosus without antiphospholipid antibodies and 43 with systemic lupus erythematosus and antiphospholipid antibodies) and 66 healthy controls were included. PROCR H1 and H3 haplotypes were determined by genotyping 7014G/C and 1651C/G tag-polymorphisms, respectively. sEPCR levels were determined by enzyme-linked immunosorbent assay., Results: PROCR haplotype distribution was similar among groups of patients and controls. PROCR H1 and H3 haplotypes were less prevalent in antiphospholipid syndrome patients with arterial thrombosis than those without arterial thrombosis, but statistical significance was only reached for the H1 haplotype (58.0% vs. 85.7%, p = 0.003; odds ratio: 0.23 [95% CI 0.08-0.65]). No relationship between the PROCR H1 and H3 haplotypes and venous thrombosis was found. sEPCR levels were higher in H3 than in H1 carriers (175.5 [95% CI 60.9-290.1] ng/ml vs. 69.1 [95% CI 61.5-76.9] ng/ml, p < 0.01). No relationship of sEPCR with arterial or venous thrombosis was found., Conclusion: The PROCR H1 haplotype was less frequently found in APS patients with arterial thrombosis, suggesting a protective effect of PROCR H1 against arterial thrombosis in these patients. No relationship between sEPCR and thrombosis was found., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cell-type origin is one of the factors that determine molecular features of tumors, but resources to validate this concept are scarce because of technical difficulties in propagating major cell types of adult organs. Previous attempts to generate such resources to study breast cancer have yielded predominantly basal-type cell lines. We have created a panel of immortalized cell lines from core breast biopsies of ancestry-mapped healthy women that form ductal structures similar to normal breast in 3D cultures and expressed markers of major cell types, including the luminal-differentiated cell-enriched ERα-FOXA1-GATA3 transcription factor network. We have also created cell lines from PROCR (CD201) + /EpCAM - cells that are likely the "normal" counterpart of the claudin-low subtype of breast cancers. RNA-seq and PAM50-intrinsic subtype clustering identified these cell lines as the "normal" counterparts of luminal A, basal, and normal-like subtypes and validated via immunostaining with basal-enriched KRT14 and luminal-enriched KRT19. We further characterized these cell lines by flow cytometry for distribution patterns of stem/basal, luminal-progenitor, mature/differentiated, multipotent PROCR + cells, and organogenesis-enriched epithelial/mesenchymal hybrid cells using CD44/CD24, CD49f/EpCAM, CD271/EpCAM, CD201/EpCAM, and ALDEFLUOR assays and E-cadherin/vimentin double staining. These cell lines showed interindividual heterogeneity in stemness/differentiation capabilities and baseline activity of signaling molecules such as NF-κB, AKT2, pERK, and BRD4. These resources can be used to test the emerging concept that genetic variations in regulatory regions contribute to widespread differences in gene expression in "normal" conditions among the general population and can delineate the impact of cell-type origin on tumor progression. Significance: In addition to providing a valuable resource for the breast cancer research community to investigate cell-type origin of different subtypes of breast cancer, this study highlights interindividual differences in normal breast, emphasizing the need to use "normal" cells from multiple sources as controls to decipher the effects of cancer-specific genomic aberrations. Cancer Res; 78(17); 5107-23. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Humans have a number of nonclassical major histocompatibility complex (MHC) class I molecules that are quite divergent from the classical ones, and that may have separated from the classical lineage in pre-mammalian times. To estimate when in evolution the respective nonclassical lineages separated from the classical lineage, we first identified "phylogenetic marker motifs" within the evolution of classical MHC class I; the selected motifs are rather specific for and rather stably inherited within clades of species. Distribution of these motifs in nonclassical MHC class I molecules indicates that the lineage including the nonclassical MHC class I molecules CD1 and PROCR separated from the classical lineage before the emergence of tetrapod species, and that the human nonclassical MHC class I molecules FCGRT, MIC/ULBP/RAET, HFE, MR1, and ZAG show similarity with classical MHC class I at the avian/reptilian level. An MR1-like α1 exon sequence was identified in turtle. Our system furthermore indicates that the lineage UT, hitherto only found in non-eutherian mammals, predates tetrapod existence, and we identified UT genes in reptiles. If only accepting wide distribution of a lineage among extant species as true evidence for ancientness, the oldest identified nonclassical MHC class I lineage remains the fish-specific lineage Z, which was corroborated in the present study by finding both Z and classical-type MHC class I sequences in a primitive fish, the bichir. In short, we gained important new insights into the evolution of classical MHC class I motifs and the probable time of origin of nonclassical MHC class I lineages.

There has recently been considerable progress in the results of pig organ transplantation in nonhuman primates, largely associated with the availability of (i) pigs genetically engineered to overcome coagulation dysregulation, and (ii) novel immunosuppressive agents. The barriers of thrombotic microangiopathy and/or consumptive coagulation were believed to be associated with (i) activation of the graft vascular endothelial cells by a low level of antipig antibody binding and/or complement deposition and/or innate immune cell activity, and (ii) molecular incompatibilities between the nonhuman primate and pig coagulation-anticoagulation systems. The introduction of a human coagulation-regulatory transgene, for example, thrombomodulin, endothelial protein C receptor, into the pig vascular endothelial cells has contributed to preventing a procoagulant state from developing, resulting in a considerable increase in graft survival. In the heterotopic (non-life-supporting) heart transplant model, graft survival has increased from a maximum of 179 days in 2005 to 945 days. After life-supporting kidney transplantation, survival has been extended from 90 days in 2004 to 499 days. In view of the more complex coagulation dysfunction seen after pig liver and, particularly, lung transplantation, progress has been less dramatic, but the maximum survival of a pig liver has been increased from 7 days in 2010 to 29 days, and of a pig lung from 4 days in 2007 to 9 days. There is a realistic prospect that the transplantation of a kidney or heart, in combination with a conventional immunosuppressive regimen, will enable long-term recipient survival.

Recent studies show that endothelial cell protein C receptor (EPCR) interacts with diverse ligands, in addition to its known ligands protein C and activated protein C (APC). We showed in earlier studies that procoagulant clotting factor VIIa (FVIIa) binds EPCR and downregulates EPCR-mediated anticoagulation and induces an endothelial barrier protective effect. Here, we investigated the effect of FVIIa's interaction with EPCR on endothelial cell inflammation and lipopolysaccharide (LPS)-induced inflammatory responses in vivo. Treatment of endothelial cells with FVIIa suppressed tumor necrosis factor α (TNF-α)- and LPS-induced expression of cellular adhesion molecules and adherence of monocytes to endothelial cells. Inhibition of EPCR or protease-activated receptor 1 (PAR1) by either specific antibodies or small interfering RNA abolished the FVIIa-induced suppression of TNF-α- and LPS-induced expression of cellular adhesion molecules and interleukin-6. β-Arrestin-1 silencing blocked the FVIIa-induced anti-inflammatory effect in endothelial cells. In vivo studies showed that FVIIa treatment markedly suppressed LPS-induced inflammatory cytokines and infiltration of innate immune cells into the lung in wild-type and EPCR-overexpressing mice, but not in EPCR-deficient mice. Mechanistic studies revealed that FVIIa treatment inhibited TNF-α-induced ERK1/2, p38 MAPK, JNK, NF-κB, and C-Jun activation indicating that FVIIa-mediated signaling blocks an upstream signaling event in TNFα-induced signaling cascade. FVIIa treatment impaired the recruitment of TNF-receptor-associated factor 2 into the TNF receptor 1 signaling complex. Overall, our present data provide convincing evidence that FVIIa binding to EPCR elicits anti-inflammatory signaling via a PAR1- and β-arrestin-1 dependent pathway. The present study suggests new therapeutic potentials for FVIIa, which is currently in clinical use for treating bleeding disorders., (© 2018 by The American Society of Hematology.)

Purpura fulminans is a deadly complication of Neisseria meningitidis infections due to extensive thrombosis of microvessels. Although a Disseminated Intra-vascular Coagulation syndrome (DIC) is frequently observed during Gram negative sepsis, it is rarely associated with extensive thrombosis like those observed during meningococcemia, suggesting that the meningococcus induces a specific dysregulation of coagulation. Another specific feature of N. meningitidis pathogenesis is its ability to colonize microvessels endothelial cells via type IV pili. Importantly, endothelial cells are key in controlling the coagulation cascade through the activation of the potent anticoagulant Protein C (PC) thanks to two endothelial cell receptors among which the Endothelial Protein C Receptor (EPCR). Considering that congenital or acquired deficiencies of PC are associated with purpura fulminans, we hypothesized that a defect in the activation of PC following meningococcal adhesion to microvessels is responsible for the thrombotic events observed during meningococcemia. Here we showed that the adhesion of N. meningitidis on endothelial cells results in a rapid and intense decrease of EPCR expression by inducing its cleavage in a process know as shedding. Using siRNA experiments and CRISPR/Cas9 genome edition we identified ADAM10 (A Disintegrin And Metalloproteinase-10) as the protease responsible for this shedding. Surprisingly, ADAM17, the only EPCR sheddase described so far, was not involved in this process. Finally, we showed that this ADAM10-mediated shedding of EPCR induced by the meningococcal interaction with endothelial cells was responsible for an impaired activation of Protein C. This work unveils for the first time a direct link between meningococcal adhesion to endothelial cells and a severe dysregulation of coagulation, and potentially identifies new therapeutic targets for meningococcal purpura fulminans.

Endothelial protein C receptor (EPCR) has been implicated in the carcinogenesis of diverse tumor types. This tumor-promoting effect of EPCR is associated with the upregulation of activated protein C and the activation of protease-activated receptor 1 (PAR-1). However, the exact role of EPCR in gastric cancer (GC) and the mechanisms underlying the regulation of EPCR remain elusive. In the present study, we investigated the effects of EPCR on human GC cells, as well as the underlying mechanisms. An siRNA inference system was used to knock down the expression of EPCR in GC cells, and CCK-8, colony formation and Transwell assays were performed to determine the effects of EPCR knockdown on the proliferation and migration of the tumor cells. Additionally, cell cycle distribution and apoptosis were assessed by flow cytometry, and activated PAR-1 levels were determined by cell ELISA. The results indicated that the proliferation, clonogenicity and migration were significantly reduced and that the cell cycle was arrested in the Gap 1 phase by EPCR knockdown in SGC7901 and AGS cells. Meanwhile, apoptosis was promoted by EPCR knockdown in the two cell lines. The activation of PAR-1 on the cell surface of SGC7901 and AGS cells was significantly reduced after the knockdown of EPCR. By contrast, blockade of PAR-1 reduced the proliferation and migration of gastric cells in vitro. Additionally, after the knockdown of EPCR or treatment with PAR-1 antibody, the expression of pERK1/2 was significantly downregulated in the SGC7901 and AGS cells, while the expression levels of p-AKT (S473) and p-AKT (T308) were unchanged. The findings of the present study demonstrated that EPCR exerts pro-carcinogenic effects in GC cells in a PAR-1-dependent manner via the ERK1/2-MAPK pathway. Thus, EPCR may be a potential molecular diagnostic or therapeutic target for GC.

The protein C receptor (PROCR) has emerged as a stem cell marker in several normal tissues and has also been implicated in tumor progression. However, the functional role of PROCR and the signaling mechanisms downstream of PROCR remain poorly understood. Here, we dissected the PROCR signaling pathways in breast cancer cells. Combining protein array, knockdown, and overexpression methods, we found that PROCR concomitantly activates multiple pathways. We also noted that PROCR-dependent ERK and PI3k-Akt-mTOR signaling pathways proceed through Src kinase and transactivation of insulin-like growth factor 1 receptor (IGF-1R). These pathway activities led to the accumulation of c-Myc and cyclin D1. On the other hand, PROCR-dependent RhoA-ROCK-p38 signaling relied on coagulation factor II thrombin receptor (F2R). We confirmed these findings in primary cells isolated from triple-negative breast cancer-derived xenografts (PDX) that have high expression of PROCR. To the best our knowledge, this is the first comprehensive study of PROCR signaling in breast cancer cells, and its findings also shed light on the molecular mechanisms of PROCR in stem cells in normal tissue., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Essentials: Essentials A fraction of coagulation factor VII circulates in blood as an activated protease (FVIIa). We evaluated FVIIa and FVIIa-antithrombin (FVIIa-AT) levels in the Cardiovascular Health Study. Polymorphisms in the F7 and PROCR loci were associated with FVIIa and FVIIa-AT levels. FVIIa may be an ischemic stroke risk factor in older adults and FVIIa-AT may assess mortality risk., Summary: Background A fraction of coagulation factor (F) VII circulates as an active protease (FVIIa). FVIIa also circulates as an inactivated complex with antithrombin (FVIIa-AT). Objective Evaluate associations of FVIIa and FVIIa-AT with genome-wide single nucleotide polymorphisms (SNPs) and incident coronary heart disease, ischemic stroke and mortality. Patients/Methods We measured FVIIa and FVIIa-AT in 3486 Cardiovascular Health Study (CHS) participants. We performed a genome-wide association scan for FVIIa and FVIIa-AT in European-Americans (n = 2410) and examined associations of FVII phenotypes with incident cardiovascular disease. Results In European-Americans, the most significant SNP for FVIIa and FVIIa-AT was rs1755685 in the F7 promoter region on chromosome 13 (FVIIa, β = -25.9 mU mL -1 per minor allele; FVIIa-AT, β = -26.6 pm per minor allele). Phenotypes were also associated with rs867186 located in PROCR on chromosome 20 (FVIIa, β = 7.8 mU mL -1 per minor allele; FVIIa-AT, β = 9.9 per minor allele). Adjusted for risk factors, a one standard deviation higher FVIIa was associated with increased risk of ischemic stroke (hazard ratio [HR], 1.12; 95% confidence interval [CI], 1.01, 1.23). Higher FVIIa-AT was associated with mortality from all causes (HR, 1.08; 95% CI, 1.03, 1.12). Among European-American CHS participants the rs1755685 minor allele was associated with lower ischemic stroke (HR, 0.69; 95% CI, 0.54, 0.88), but this association was not replicated in a larger multi-cohort analysis. Conclusions The results support the importance of the F7 and PROCR loci in variation in circulating FVIIa and FVIIa-AT. The findings suggest FVIIa is a risk factor for ischemic stroke in older adults, whereas higher FVIIa-AT may reflect mortality risk., (© 2017 International Society on Thrombosis and Haemostasis.)

The present study investigated the influence of cigarette smoke extract (CSE) and nicotine on the expression of thrombomodulin (TM) and endothelial protein C receptor (EPCR) in human umbilical vein endothelial cells (HUVECs). Smoking is associated with intravascular thrombosis. As a vital anticoagulation cofactor, TM is located on the endothelial cell surface and regulates intravascular coagulation by binding to thrombin, hence activating protein C. Activated protein C is a natural anticoagulant that interacts with EPCR to enhance the function of anticoagulation system. The effects of CSE (0.5‑5%) and nicotine (10‑3‑10‑9 mol/l) on the expression of TM and EPCR in HUVECs were observed. Reverse transcription‑quantitative polymerase chain reaction and flow cytometric analysis techniques were used for detecting TM and EPCR mRNA and protein expression levels, respectively. After 6‑h exposure, TM protein and mRNA expression levels decreased in a dose‑dependent manner. Stimulation with 5% CSE for 0, 6, 10, 12 and 24 h led to a decrease in the levels of TM mRNA and protein over time, which reached a peak at 12 h. The levels were significantly reduced compared with the control group (P<0.001). However, CSE had no effect on EPCR. Furthermore, nicotine had no influence on TM and EPCR. In conclusion, the present study supports a novel molecular mechanism of cigarette smoking‑associated thrombosis by the decreased expression of TM. Further studies are required to identify specific components in CSE responsible for decreasing TM expression and its associated consequences.

Endothelial protein C receptor (EPCR) is a specific receptor for anticoagulant protein C and expressed by human epidermis and cultured keratinocytes. Here we investigated whether: (a) the level of EPCR in keratinocytes is associated with their growth potential; and (b) EPCR is a potential marker for human epidermal stem cells. Human keratinocytes isolated from foreskins or adult skin tissues were transfected with EPCR siRNA or EPCR overexpressing plasmids. Cell proliferation, long term proliferation potential, colony forming efficiency (CFE), and in vitro epidermal regeneration ability of EPCR high and EPCR l ° w cells were assessed. The expression and colocalization of EPCR with stem cell markers p63, integrin β1, and activation of MAP kinases were detected by flow cytometry, immunofluorescence staining, or Western blot. Results showed that EPCR was highly expressed by the basal layer of skin epidermis. EPCR high cells were associated with the highest levels of p63 and integrin β1. Most EPCR high cells were smaller in size, formed larger colonies and had a greater long term growth potential, CFE, holoclone formation, and in vitro epidermal regeneration ability when compared to EPCR l ° w cells. Blocking EPCR resulted in keratinocyte apoptosis, particularly in nondifferentiated conditions. Cell proliferation and p63 expression were reduced by blocking EPCR and enhanced by overexpressing this receptor. These data indicate that EPCR can regulate p63, is associated with highly proliferative keratinocytes, and is a potential human epidermal stem cell marker. Stem Cells 2017;35:1786-1798., (© 2017 AlphaMed Press.)

In addition to its procoagulant and proinflammatory functions mediated by cleavage of fibrinogen and PAR1, the trypsin-like protease thrombin activates the anticoagulant protein C in a reaction that requires the cofactor thrombomodulin and the endothelial protein C receptor. Once in the circulation, activated protein C functions as an anticoagulant, anti-inflammatory and regenerative factor. Hence, availability of a protein C activator would afford a therapeutic for patients suffering from thrombotic disorders and a diagnostic tool for monitoring the level of protein C in plasma. Here, we present a fusion protein where thrombin and the EGF456 domain of thrombomodulin are connected through a peptide linker. The fusion protein recapitulates the functional and structural properties of the thrombin-thrombomodulin complex, prolongs the clotting time by generating pharmacological quantities of activated protein C and effectively diagnoses protein C deficiency in human plasma. Notably, these functions do not require exogenous thrombomodulin, unlike other anticoagulant thrombin derivatives engineered to date. These features make the fusion protein an innovative step toward the development of protein C activators of clinical and diagnostic relevance.

Protein C is an environmentally modifiable anticoagulant, which protects against venous thrombosis, whether it also protects against ischaemic heart disease is unclear, based on observational studies and relatively small genetic studies. It was our study aim to clarify the role of protein C in ischaemic heart disease. The risk of coronary artery disease/myocardial infarction (CAD/MI) was assessed according to genetically predicted protein C in very large studies. Associations with lipids and diabetes were similarly assessed to rule out effects via traditional cardiovascular disease risk factors. Separate sample instrumental variable analysis with genetic instruments (Mendelian randomisation) was used to obtain an unconfounded estimate of the association of protein C (based on (rs867186 (PROCR), rs3746429 (EDEM2), rs7580658 (inter/PROC)) with CAD/MI in an extensively genotyped case (n=64374)-control (n=130681) study, CARDIoGRAMplusC4D. Associations with lipids and diabetes were similarly assessed using the Global Lipids Genetics Consortium Results (n=196,475) and the DIAbetes Genetics Replication And Meta-analysis case (n=34,380)-control (n=114,981) study. Genetically predicted protein C was negatively associated with CAD/MI, odds ratio (OR) 0.85 µg/ml, 95 % confidence interval 0.80 to 0.90, but had no such negative association with lipids or diabetes. Results were similar for the SNP rs867186 functionally relevant to protein C, and including additional potentially pleiotropic SNPs (rs1260326 (GCKR), rs17145713 (BAZ1B) and rs4321325 (CYP27C1)). In conclusion, protein C may protect against CAD/MI. Whether environmental or dietary items that raise protein C protect against ischaemic cardiovascular disease by that mechanism should be investigated.