Cytoprotective signaling by activated protein C requires protease-activated receptor-3 in podocytes

Abstract The cytoprotective effects of activated protein C (aPC) are well established. In contrast, the receptors and signaling mechanism through which aPC conveys cytoprotection in various cell types remain incompletely defined. Thus, within the renal glomeruli, aPC preserves endothelial cells via a protease-activated receptor-1 (PAR-1) and endothelial protein C receptor-dependent mechanism. Conversely, the signaling mechanism through which aPC protects podocytes remains unknown. While exploring the latter, we identified a novel aPC/PAR-dependent cytoprotective signaling mechanism. In podocytes, aPC inhibits apoptosis through proteolytic activation of PAR-3 independent of endothelial protein C receptor. PAR-3 is not signaling competent itself as it requires aPCinduced heterodimerization with PAR-2 (human podocytes) or PAR-1 (mouse podocytes). This cytoprotective signaling mechanism depends on caveolin-1 dephosphorylation. In vivo aPC protects against lipopolysaccharide-induced podocyte injury and proteinuria. Genetic deletion of PAR-3 impairs the nephroprotective effect of aPC, demonstrating the crucial role of PAR-3 for aPC-dependent podocyte protection. This novel, aPC-mediated interaction of PARs demonstrates the plasticity and cell-specificity of cytoprotective aPC signaling. The evidence of specific, dynamic signaling complexes underlying aPC-mediated cytoprotection may allow the design of cell type specific targeted therapies.

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Hyperantithrombotic, noncytoprotective Glu149Ala-activated protein C mutant

Blood ◽

10.1182/blood-2008-10-183327 ◽

2009 ◽

Vol 113 (23) ◽

pp. 5970-5978 ◽

Cited By ~ 50

Author(s):

Laurent O. Mosnier ◽

Antonella Zampolli ◽

Edward J. Kerschen ◽

Reto A. Schuepbach ◽

Yajnavalka Banerjee ◽

...

Keyword(s):

Protein C ◽

Anticoagulant Activity ◽

Activated Protein C ◽

Bleeding Risk ◽

Endothelial Protein C Receptor ◽

Antithrombotic Activity ◽

Cytoprotective Activity ◽

Protease Activated Receptor 1

Abstract Activated protein C (APC) reduces mortality in severe sepsis patients. APC exerts anticoagulant activities via inactivation of factors Va and VIIIa and cytoprotective activities via endothelial protein C receptor and protease-activated receptor-1. APC mutants with selectively altered and opposite activity profiles, that is, greatly reduced anticoagulant activity or greatly reduced cytoprotective activities, are compared here. Glu149Ala-APC exhibited enhanced in vitro anticoagulant and in vivo antithrombotic activity, but greatly diminished in vitro cytoprotective effects and in vivo reduction of endotoxin-induced murine mortality. Thus, residue Glu149 and the C-terminal region of APC's light chain are identified as functionally important for expression of multiple APC activities. In contrast to Glu149Ala-APC, 5A-APC (Lys191-193Ala + Arg229/230Ala) with protease domain mutations lacked in vivo antithrombotic activity, although it was potent in reducing endotoxin-induced mortality, as previously shown. These data imply that APC molecular species with potent antithrombotic activity, but without robust cytoprotective activity, are not sufficient to reduce mortality in endotoxemia, emphasizing the need for APC's cytoprotective actions, but not anticoagulant actions, to reduce endotoxin-induced mortality. Protein engineering can provide APC mutants that permit definitive mechanism of action studies for APC's multiple activities, and may also provide safer and more effective second-generation APC mutants with reduced bleeding risk.

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The ligand occupancy of endothelial protein C receptor switches the protease-activated receptor 1-dependent signaling specificity of thrombin from a permeability-enhancing to a barrier-protective response in endothelial cells

Blood ◽

10.1182/blood-2007-06-096651 ◽

2007 ◽

Vol 110 (12) ◽

pp. 3909-3916 ◽

Cited By ~ 152

Author(s):

Jong-Sup Bae ◽

Likui Yang ◽

Chandrashekhara Manithody ◽

Alireza R. Rezaie

Keyword(s):

Endothelial Cells ◽

Protein C ◽

Vascular Endothelial Cells ◽

Activated Protein C ◽

Catalytic Efficiency ◽

Endothelial Protein C Receptor ◽

New Paradigm ◽

Caveolin 1 ◽

Protease Activated Receptor 1 ◽

Protective Signaling

AbstractRecent studies have indicated that activated protein C (APC) may exert its cytoprotective and anti-inflammatory activities through the endothelial protein C receptor (EPCR)-dependent cleavage of protease-activated receptor 1 (PAR-1) on vascular endothelial cells. Noting that (1) the activation of protein C on endothelial cells requires thrombin, (2) relative to APC, thrombin cleaves PAR-1 with approximately 3 to 4 orders of magnitude higher catalytic efficiency, and (3) PAR-1 is a target for the proinflammatory activity of thrombin, it is not understood how APC can elicit a protective signaling response through the cleavage of PAR-1 when thrombin is present. In this study, we demonstrate that EPCR is associated with caveolin-1 in lipid rafts of endothelial cells and that its occupancy by the γ-carboxyglutamic acid (Gla) domain of protein C/APC leads to its dissociation from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway through coupling of PAR-1 to the pertussis toxin–sensitive Gi-protein. Thus, when EPCR is bound by protein C, the PAR-1 cleavage-dependent protective signaling responses in endothelial cells can be mediated by either thrombin or APC. These results provide a new paradigm for understanding how PAR-1 and EPCR participate in protective signaling events in endothelial cells.

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Cell painting with an engineered EPCR to augment the protein C system

Thrombosis and Haemostasis ◽

10.1160/th15-01-0079 ◽

2015 ◽

Vol 114 (12) ◽

pp. 1144-1155 ◽

Cited By ~ 6

Author(s):

Eveline A. M. Bouwens ◽

Fabian Stavenuiter ◽

Laurent O. Mosnier

Keyword(s):

Endothelial Cells ◽

Protein C ◽

Activated Protein C ◽

Wild Type ◽

Endothelial Protein C Receptor ◽

Gpi Anchor ◽

Akt Phosphorylation ◽

Caveolin 1 ◽

Dose Dependent

SummaryThe protein C (PC) system conveys beneficial anticoagulant and cytoprotective effects in numerous in vivo disease models. The endothelial protein C receptor (EPCR) plays a central role in these pathways as cofactor for PC activation and by enhancing activated protein C (APC)-mediated protease-activated receptor (PAR) activation. During inflammatory disease, expression of EPCR on cell membranes is often diminished thereby limiting PC activation and APC’s effects on cells. Here a caveolae-targeting glycosylphosphatidylinositol (GPI)-anchored EPCR (EPCR-GPI) was engineered to restore EPCR’s bioavailability via “cell painting.” The painting efficiency of EPCR-GPI on EPCR-depleted endothelial cells was time- and dose-dependent. The EPCR-GPI bioavailability after painting was long lasting since EPCR surface levels reached 400 % of wild-type cells after 2 hours and remained > 200 % for 24 hours. EPCR-GPI painting conveyed APC binding to EPCR-depleted endothelial cells where EPCR was lost due to shedding or shRNA. EPCR painting normalised PC activation on EPCR-depleted cells indicating that EPCR-GPI is functional active on painted cells. Caveolin-1 lipid rafts were enriched in EPCR after painting due to the GPI-anchor targeting caveolae. Accordingly, EPCR painting supported PAR1 and PAR3 cleavage by APC and augmented PAR1-dependent Akt phosphorylation by APC. Thus, EPCR-GPI painting achieved physiological relevant surface levels on endothelial cells, restored APC binding to EPCR-depleted cells, supported PC activation, and enhanced APC-mediated PAR cleavage and cytoprotective signalling. Therefore, EPCRGPI provides a novel tool to restore the bioavailability and functionality of EPCR on EPCR- depleted and -deficient cells.

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Abstract 34: Endogenous Superoxide Dismutase Protects From Impaired Generation of Activated Protein C and Enhanced Susceptibility to Experimental Thrombosis in Mice

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.34.suppl_1.34 ◽

2014 ◽

Vol 34 (suppl_1) ◽

Author(s):

Sanjana Dayal ◽

Sean X Gu ◽

Katinan M Wilson ◽

Ryan Hutchins ◽

Steven R Lentz

Keyword(s):

Superoxide Dismutase ◽

Protein C ◽

Activated Protein C ◽

Vena Cava ◽

Oxidative Modification ◽

Mrna Levels ◽

Endothelial Protein C Receptor ◽

Experimental Thrombosis

In vitro studies have suggested that reactive oxygen species such as superoxide can produce prothrombotic effects, including enhanced platelet activation, increased tissue factor (TF) expression, and an oxidative modification in thrombomodulin impairing its capacity to enhance the generation of activated protein C (APC) by thrombin. It is not known, however, if elevated levels of superoxide accelerate susceptibility to experimental thrombosis in vivo . We used mice genetically deficient in superoxide dismutase-1 (SOD1, an antioxidant enzyme that dismutates superoxide to hydrogen peroxide), to test the hypothesis that lack of SOD1 enhances susceptibility to thrombosis. Susceptibility to carotid artery thrombosis in a photochemical injury model demonstrated that Sod1-/- mice formed stable occlusions significantly faster than Sod1+/+ mice (P<0.05). In an inferior vena cava (IVC) stasis model Sod1- /- mice developed significantly larger thrombi 48 hours after IVC ligation (P<0.05 vs. Sod1+/+ mice). After activation with thrombin (0.5 U/ml) or convulxin (200 ng/ml), no differences in surface expression of P-selectin or binding of fibrinogen were observed between platelets from Sod1-/- and Sod1+/+ mice. The expression of TF mRNA in lung measured by real time qPCR showed similar levels in Sod1-/- and Sod1 +/+ mice. However, the activation of exogenous protein C by thrombin in lung homogenates was decreased in Sod1 -/- mice (P<0.05 vs. Sod1 +/+ mice). Further, in vivo generation of activated protein C in response to thrombin (40 U/Kg) infusion was significantly lower in Sod1-/- mice (P<0.05 vs. Sod1+/+ mice). No differences in mRNA levels for thrombomodulin or endothelial protein C receptor were detected in Sod1 -/- mice vs. Sod1 +/+ mice, suggesting that altered generation of activated protein C in Sod1-/- mice may be related to a direct oxidative effect on thrombomodulin. In accordance, thrombomodulin treated with xanthine/hypoxanthine showed 40% loss of ability to activate protein C that was overcome by addition of SOD and catalase (P<0.05). We conclude that endogenous SOD1 in mice protects from impaired generation of activated protein C and accelerated thrombosis.

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Activated protein C targets immune cells and rheumatoid synovial fibroblasts to prevent inflammatory arthritis in mice

Rheumatology ◽

10.1093/rheumatology/key429 ◽

2019 ◽

Vol 58 (10) ◽

pp. 1850-1860 ◽

Cited By ~ 4

Author(s):

Meilang Xue ◽

Suat Dervish ◽

Kelly J McKelvey ◽

Lyn March ◽

Fang Wang ◽

...

Keyword(s):

Protein C ◽

Activated Protein C ◽

Synovial Fibroblasts ◽

Endothelial Protein C Receptor ◽

Mouse Thymus ◽

Tnf Α ◽

Thymus Cells ◽

Proliferation And Invasion

Abstract Objectives To investigate whether activated protein C (APC), a physiological anticoagulant can inhibit the inflammatory/invasive properties of immune cells and rheumatoid arthritis synovial fibroblasts (RASFs) in vitro and prevent inflammatory arthritis in murine antigen-induced arthritis (AIA) and CIA models. Methods RASFs isolated from synovial tissues of patients with RA, human peripheral blood mononuclear cells (PBMCs) and mouse thymus cells were treated with APC or TNF-α/IL-17 and the following assays were performed: RASF proliferation and invasion by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell invasion assays, respectively; cytokines and signalling molecules using ELISA or western blot; Th1 and Th17 phenotypes in human PBMCs or mouse thymus cells by flow cytometry. The in vivo effect of APC was evaluated in AIA and CIA models. Results In vitro, APC inhibited IL-1β, IL-17 and TNF-α production, IL-17-stimulated cell proliferation and invasion and p21 and nuclear factor κB activation in RASFs. In mouse thymus cells and human PBMCs, APC suppressed Th1 and Th17 phenotypes. In vivo, APC inhibited pannus formation, cartilage destruction and arthritis incidence/severity in both CIA and AIA models. In CIA, serum levels of IL-1β, IL-6, IL-17, TNF-α and soluble endothelial protein C receptor were significantly reduced by APC treatment. Blocking endothelial protein C receptor, the specific receptor for APC, abolished the early or preventative effect of APC in AIA. Conclusion APC prevents the onset and development of arthritis in CIA and AIA models via suppressing inflammation, Th1/Th17 phenotypes and RASF invasion, which is likely mediated via endothelial protein C receptor.

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The Cardioprotective Signaling Activity of Activated Protein C in Heart Failure and Ischemic Heart Diseases

International Journal of Molecular Sciences ◽

10.3390/ijms20071762 ◽

2019 ◽

Vol 20 (7) ◽

pp. 1762 ◽

Cited By ~ 3

Author(s):

Di Ren ◽

Hemant Giri ◽

Ji Li ◽

Alireza R. Rezaie

Keyword(s):

Heart Failure ◽

Protein C ◽

Heart Diseases ◽

Ischemia Reperfusion ◽

Activated Protein C ◽

Ischemic Heart ◽

Signaling Networks ◽

Endothelial Protein C Receptor ◽

Signaling Mechanism ◽

Mtorc1 Signaling

Activated protein C (APC) is a vitamin-K dependent plasma serine protease, which functions as a natural anticoagulant to downregulate thrombin generation in the clotting cascade. APC also modulates cellular homeostasis by exhibiting potent cytoprotective and anti-inflammatory signaling activities. The beneficial cytoprotective effects of APC have been extensively studied and confirmed in a number of preclinical disease and injury models including sepsis, type-1 diabetes and various ischemia/reperfusion diseases. It is now well-known that APC modulates downstream cell signaling networks and transcriptome profiles when it binds to the endothelial protein C receptor (EPCR) to activate protease-activated receptor 1 (PAR1) on various cell types. However, despite much progress, details of the downstream signaling mechanism of APC and its crosstalk with other signaling networks are far from being fully understood. In this review, we focus on the cardioprotective properties of APC in ischemic heart disease and heart failure with a special emphasis on recent discoveries related to the modulatory effect of APC on AMP-activated protein kinase (AMPK), PI3K/AKT, and mTORC1 signaling pathways. The cytoprotective properties of APC might provide a novel strategy for future therapies in cardiac diseases.

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Effects of oral anticoagulant therapy and haplotype 1 of the endothelial protein C receptor gene on activated protein C levels

Thrombosis and Haemostasis ◽

10.1160/th11-07-0510 ◽

2012 ◽

Vol 107 (03) ◽

pp. 448-457 ◽

Cited By ~ 2

Author(s):

Pilar Medina ◽

Elena Bonet ◽

Silvia Navarro ◽

Laura Martos ◽

Amparo Estellés ◽

...

Keyword(s):

Anticoagulant Therapy ◽

Lupus Erythematosus ◽

Protein C ◽

Oral Anticoagulant Therapy ◽

Receptor Gene ◽

Oral Anticoagulants ◽

Activated Protein C ◽

Endothelial Protein C Receptor ◽

Haplotype 1

SummaryOral anticoagulants (OACs) reduce activated protein C (APC) plasma levels less than those of protein C (PC) in lupus erythematosus and cardiac patients. Carriers of the H1 haplotype of the endothelial PC receptor gene (PROCR) have higher APC levels than non-carriers. We aimed to confirm these results in a large group of patients treated with OACs because of venous thromboembolism (VTE) and to assess whether the effect is influenced by the PROCR H1 haplotype. We evaluated APC, PC, and factor (F)II levels in 502 VTE patients (158 with and 344 without OACs) and in 322 healthy individuals. Mean APC, PC and FII levels were significantly lower in OAC patients than in patients not taking OACs. During anticoagulant therapy, the FII/PC ratios were independent of the PC values, whereas APC/FII and APC/PC ratios significantly increased when FII and PC levels decreased. Of the 22 OAC patients carrying the H1H1genotype, 11 (50%) showed APC/PCag ≥2.0 and 10 (45%) APC/ FIIag ratios ≥2.0, whereas for the 49 OAC patients non-carrying the H1 haplotype these figures were 6 (12%) and 4 (8%), respectively (p<0.001). Barium citrate adsorption of plasma from OAC patients showed that most of the circulating free and complexed APC, but only part of PCag, is fully carboxylated. In conclusion, during anticoagulant therapy VT patients have APC levels disproportionately higher than the corresponding PC levels, mainly due to the presence of the PROCR H1 haplotype. Furthermore, a sufficiently carboxylated PC Gla-domain seems to be essential for PC activation in vivo.

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Endothelial Protein C Receptor (EPCR), Protease Activated Receptor-1 (PAR-1) and Their Interplay in Cancer Growth and Metastatic Dissemination

Cancers ◽

10.3390/cancers11010051 ◽

2019 ◽

Vol 11 (1) ◽

pp. 51 ◽

Cited By ~ 3

Author(s):

Marek Z. Wojtukiewicz ◽

Dominika Hempel ◽

Ewa Sierko ◽

Stephanie C. Tucker ◽

Kenneth V. Honn

Keyword(s):

Cell Surface ◽

Cancer Patients ◽

Cancer Progression ◽

Protein C ◽

Critical Role ◽

Activated Protein C ◽

Cancer Growth ◽

Endothelial Protein C Receptor ◽

Surface Receptors ◽

Protease Activated Receptor 1

Endothelial protein C receptor (EPCR) and protease activated receptor 1 (PAR-1) by themselves play important role in cancer growth and dissemination. Moreover, interactions between the two receptors are essential for tumor progression. EPCR is a cell surface transmembrane glycoprotein localized predominantly on endothelial cells (ECs). It is a vital component of the activated protein C (APC)—mediated anticoagulant and cytoprotective signaling cascade. PAR-1, which belongs to a family of G protein–coupled cell surface receptors, is also widely distributed on endothelial and blood cells, where it plays a critical role in hemostasis. Both EPCR and PAR-1, generally considered coagulation-related receptors, are implicated in carcinogenesis and dissemination of diverse tumor types, and their expression correlates with clinical outcome of cancer patients. Existing data explain some mechanisms by which EPCR/PAR-1 affects cancer growth and metastasis; however, the exact molecular basis of cancer invasion associated with the signaling is still obscure. Here, we discuss the role of EPCR and PAR-1 reciprocal interactions in cancer progression as well as potential therapeutic options targeted specifically to interact with EPCR/PAR-1-induced signaling in cancer patients.

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Biased agonism of protease-activated receptor 1 by activated protein C caused by noncanonical cleavage at Arg46

Blood ◽

10.1182/blood-2012-08-452169 ◽

2012 ◽

Vol 120 (26) ◽

pp. 5237-5246 ◽

Cited By ~ 139

Author(s):

Laurent O. Mosnier ◽

Ranjeet K. Sinha ◽

Laurent Burnier ◽

Eveline A. Bouwens ◽

John H. Griffin

Keyword(s):

Endothelial Cells ◽

Protein C ◽

Glycogen Synthase ◽

Activated Protein C ◽

The Novel ◽

Biased Agonism ◽

Protease Activated Receptor 1 ◽

Protective Signaling

Abstract Activated protein C (APC) exerts endothelial cytoprotective actions that require protease-activated receptor 1 (PAR1), whereas thrombin acting via PAR1 causes endothelial disruptive, proinflammatory actions. APC's activities, but not thrombin's, require PAR1 located in caveolae. PAR1 is a biased 7-transmembrane receptor because G proteins mediate thrombin's signaling, whereas β-arrestin 2 mediates APC's signaling. Here we elucidate novel mechanisms for APC's initiation of signaling. Biochemical studies of APC's protease specificity showed that APC cleaved PAR1 sequences at both Arg41 and Arg46. That PAR1 cleavage at Arg46 can occur on cells was supported by APC's cleavage of N-terminal-SEAP-tagged R41Q-PAR1 but not R41Q/R46Q-PAR1 mutants transfected into cells and by anti-PAR1 epitope mapping of APC-treated endothelial cells. A synthetic peptide composing PAR1 residues 47-66, TR47, stimulated protective signaling in endothelial cells as reflected in Akt and glycogen synthase kinase 3β phosphorylation, Ras-related C3 botulinum toxin substrate 1 activation, and barrier stabilization effects. In mice, the TR47 peptide reduced VEGF-induced vascular leakage. These in vitro and in vivo data imply that the novel PAR1 N-terminus beginning at residue Asn47, which is generated by APC cleavage at Arg46, mediates APC's cytoprotective signaling and that this unique APC-generated N-terminal peptide tail is a novel biased agonist for PAR1.

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