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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.

The protein C receptor (PROCR) plays a crucial role in regulating coagulation and maintaining vascular integrity. Mutations in PROCR gene have been associated with thrombophilia and coagulation abnormalities. This study aimed to identify, characterize, and functionally analyze pathogenic nonsynonymous single nucleotide polymorphisms (nsSNPs) in PROCR via an integrative computational approach. Text mining revealed that PROCR is frequently associated with thrombophilia, highlighting its role in coagulation regulation. Protein‒protein interaction analysis revealed strong interactions between PROCR and key coagulation proteins, including F2, F5, THBD, and SERPINC1, suggesting its central role in the coagulation cascade. Functional predictions were generated using SIFT, PolyPhen-2, PROVEAN, and FATHMM, with further scoring by REVEL, ClinPred, and CADD. Structural and stability assessments were conducted using HOPE, I-Mutant2.0, MUpro, and AlphaFold substitution modeling. Evolutionary conservation was analyzed via PhyloP, PhastCons, GERP ++ , and ConSurf to assess residue functional importance. Posttranslational modifications (PTMs) were investigated using MusiteDeep, PhosphoSitePlus, and SwissPalm. Human Phenotype Ontology (HPO) mapping was used to explore clinical correlations. Six variants N64T, F93L, R113C, P145L, R173H, and R236W were consistently predicted to be deleterious. F93L showed the highest AlphaFold pathogenicity score (0.97), indicating significant structural disruption. N64T, P145L, and R173H were highly conserved residues, supporting functional relevance. PTM analysis revealed that N64T overlapped with a validated N-glycosylation site, whereas R113C, R173H, and R236W affected phosphorylation and ubiquitination. Structural modeling revealed disruptions in protein folding, secondary structure, aggregation potential, and chaperone-binding capacity. HPO mapping associated several variants with coagulation disorders, cardiovascular dysfunction, and systemic phenotypes. This analysis highlights PROCR variants with predicted structural and regulatory disruption. Variants F93L, N64T, and R236W may increase thrombophilia risk by impairing EPCR function and interactions with coagulation factors. These findings support using computational pipelines to prioritize functionally relevant nsSNPs and provide basis for future experimental validation and personalized risk assessment in thrombotic disorders.

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.

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.

Malaria pathology is driven by the accumulation of Plasmodium falciparum -infected erythrocytes in microvessels 1 . This process is mediated by the polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins of the parasite. A subset of PfEMP1 variants that bind to human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis 2 . A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here we describe two broadly reactive and inhibitory human monoclonal antibodies to CIDRα1. The antibodies isolated from two different individuals exhibited similar and consistent EPCR-binding inhibition of diverse CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins, as well as parasite sequestration in bioengineered 3D human brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with three different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies are likely to represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria. Two broadly reactive and inhibitory human monoclonal antibodies against the malaria parasite Plasmodium falciparum have been characterized, providing insights into immunity, prevention and treatment of severe malaria.

Pathological retinal neovascularization (RNV) is one of the leading causes of blindness worldwide; however, its underlying mechanism remains unclear. Here, we found that the expression of endothelial protein C receptor ( Epcr ) was increased during RNV, and its ligand was elevated in the serum or vitreous body of patients with proliferative diabetic retinopathy. Deleting endothelial Epcr or using an EPCR-neutralizing antibody ameliorated pathological retinal angiogenesis. EPCR promoted endothelial heme catabolism and carbon monoxide release through heme oxygenase 1 (HO-1). Inhibition of heme catabolism by deleting endothelial Ho-1 or using an HO-1 inhibitor suppressed pathological angiogenesis in retinopathy. Conversely, supplementation with carbon monoxide rescued the angiogenic defects after endothelial Epcr or Ho-1 deletion. Our results identified EPCR-dependent endothelial heme catabolism as an important contributor to pathological angiogenesis, which may serve as a potential target for treating vasoproliferative retinopathy. Pathological retinal neovascularization (RNV) is a major cause of blindness. Here, the authors show that endothelial protein C receptor (EPCR) promotes RNV through carbon monoxide derived from heme catabolism, and blocking EPCR offers therapeutic potential for the treating RNV.

Flow cytometric analysis of cell surface antigens is a powerful tool for the isolation and characterization of stem cells residing in adult tissues. In contrast to the collection of hematopoietic stem cells, the process of enzymatic digestion is usually necessary to prepare mesenchymal stem cells (MSCs) suspensions, which can influence the expression of cell surface markers. In this study, we examined the effects of various cell-detaching reagents and digestion times on the expression of stem cell-related surface antigens and MSC functions. Human MSCs were detached from dishes using four different reagents: trypsin, TrypLE, collagenase, and a non enzymatic cell dissociation reagent (C5789; Sigma-Aldrich). Following dissociation reagent incubations ranging from 5 to 120 min, cell surface markers were analyzed by flow cytometry. Trypsin and TrypLE quickly dissociated the cells within 5 min, while collagenase and C5789 required 60 min to obtain maximum cell yields. C5789 significantly decreased cell viability at 120 min. Trypsin treatment significantly reduced CD44+, CD55+, CD73+, CD105+, CD140a+, CD140b+, and CD201+ cell numbers within 30 min. Collagenase treatment reduced CD140a expression by 30 min. In contrast, TrypLE treatment did not affect the expression of any cell surface antigens tested by 30 min. Despite the significant loss of surface antigen expression after 60 min of treatment with trypsin, adverse effects of enzymatic digestion on multipotency of MSCs were limited. Overall, our data indicated that TrypLE is advantageous over other cell dissociation reagents tested for the rapid preparation of viable MSC suspensions.

Background Activated protein C/endothelial protein C receptor (APC/EPCR) axis is physiologically involved in anticoagulant and cytoprotective activities in endothelial cells. Emerging evidence indicates that EPCR also plays a role in breast stemness and human tumorigenesis. Yet, its contribution to breast cancer progression and metastasis has not been elucidated. Methods Transcriptomic status of EPCR was examined in a cohort of 286 breast cancer patients. Cell growth kinetics was evaluated in control and EPCR and SPARC/osteonectin, Cwcv, and kazal-like domains proteoglycan (SPOCK1/testican 1) silenced breast cancer cells in 2D, 3D, and in co-culture conditions. Orthotopic tumor growth and lung and osseous metastases were evaluated in several human and murine xenograft breast cancer models. Tumor-stroma interactions were further studied in vivo by immunohistochemistry and flow cytometry. An EPCR-induced gene signature was identified by microarray analysis. Results Analysis of a cohort of breast cancer patients revealed an association of high EPCR levels with adverse clinical outcome. Interestingly, EPCR knockdown did not affect cell growth kinetics in 2D but significantly reduced cell growth in 3D cultures. Using several human and murine xenograft breast cancer models, we showed that EPCR silencing reduced primary tumor growth and secondary outgrowths at metastatic sites, including the skeleton and the lungs. Interestingly, these effects were independent of APC ligand stimulation in vitro and in vivo. Transcriptomic analysis of EPCR-silenced tumors unveiled an effect mediated by matricellular secreted proteoglycan SPOCK1/testican 1. Interestingly, SPOCK1 silencing suppressed in vitro 3D growth. Moreover, SPOCK1 ablation severely decreased orthotopic tumor growth and reduced bone metastatic osteolytic tumors. High SPOCK1 levels were also associated with poor clinical outcome in a subset breast cancer patients. Our results suggest that EPCR through SPOCK1 confers a cell growth advantage in 3D promoting breast tumorigenesis and metastasis. Conclusions EPCR represents a clinically relevant factor associated with poor outcome and a novel vulnerability to develop combination therapies for breast cancer patients.

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.

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.