PAR-3 Activation by Activated Protein C: a Novel Podocyte Protective Signalling Pathway

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 329-329
Author(s):  
Thati Madhusudhan ◽  
Hongjie Wang ◽  
Sandra Müller-Krebs ◽  
Vedat Schwenger ◽  
Martin G. Zeier ◽  
...  
Keyword(s):  
Protein C ◽  
Limited Proteolysis ◽  
Activated Protein C ◽  
Signalling Pathway ◽  
Diabetic Patients ◽  
Cytoprotective Effect ◽  
Filtration Barrier ◽  
Foot Process Effacement ◽  
Foot Process

Abstract Abstract 329 Activated protein C (APC) has anti-coagulant and cytoprotective effects. Recently we have shown that APC protects against diabetic nephropathy by inhibiting endothelial and podocyte apoptosis. The cytoprotecive effects of APC in endothelial cells require endothelial protein C receptor (EPCR) and protease activated recetor-1 (PAR-1). However, the signalling mechanism through which APC exerts its cytoprotective effects in podocytes is not known. Here we have used a mouse model of LPS-mediated podocyte injury and show that APC protects against LPS induced podocyte apoptosis and proteinuria. These cytoprotective effects of APC require activation of PAR-3. Supplementary in vitro experiments were performed using immortalized differentiated human and mouse podocytes to delineate the receptor mechanism. LPS administration (200 μg/mice, i.p.) induced podocyte apoptosis, foot process effacement and albuminuria by 24h. Administration of APC (20 μg/ mice, i.v, 6 h after LPS injection) protected against podocyte apoptosis, foot process effacement and albuminuria. Pre-incubation of APC with HAAPC antibody which blocks the anticoagulant, but not the cytoprotective properties of APC did not abolish the cytoprotective effect, establishing that the protective effect of APC is independent of its anticoagulant property. To demonstrate the direct cytoprotective effect of APC on podocytes, FITC labelled APC was administered i.v. Within 3 min FITC-APC accumulated in the pericapillary space of mouse glomeruli. In addition significantly elevated levels of PC were detected in urine samples of LPS treated mice as well as of diabetic patients or mice. These results suggest that APC transverses the glomerular filtration barrier, thus being able to directly modulate podocyte signalling in vivo. Furthermore experiments with PAR-3-/- mice, EPCR deficient (EPCRd/d) mice or PAR-1 antagonist (pepducin PI pal12S) showed that the cytoprotective effect of APC in podocytes is independent of EPCR but requires PAR-3 and PAR-1. In vitro APC (2 nM) inhibited puromycin aminonucleoside (PAN) induced podocyte apoptosis. These anti-apoptotic effect of APC require limited proteolysis of PAR-3 and cross-activation of either PAR-1 (mouse, rat podocytes) or PAR-2 (human podocytes). In addition, ectopic expression of PAR-3 in mesangial cells which lack PAR-3 is sufficient to render these cells APC sensitive and inhibit staurosporine induced apoptosis. In conclusion, we demonstrate a novel cyto-protective mechamism of APC in an acute model of nephropathy, which depends on APC mediated limited proteolysis of PAR-3. This novel podocyte protective signalling pathway may lay ground to the delineation of new pathophysiological concepts and therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

Activated Protein C Controls p66shc: A Novel Defense Mechanism in Diabetic Nephropathy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2204-2204
Author(s):  
Fabian M Bock ◽  
Khurrum Shahzad ◽  
Thati Madhusudhan ◽  
Hongjie Wang ◽  
Peter P Nawroth ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Diabetic Nephropathy ◽  
Protein C ◽  
Defense Mechanism ◽  
Conflicts Of Interest ◽  
Mitochondrial Protein ◽  
Activated Protein C ◽  
Cytoprotective Effect ◽  
Mitochondrial Translocation

Abstract Abstract 2204 Introduction: Previously it has been shown that activated protein C (APC) protects against diabetic nephropathy (DN). APC's cytoprotective effect in DN may be related to its in vitro ability to downregulate reactive oxygen species (ROS) and maintain mitochondrial membrane potential in glucose stressed cells. Cellular targets, through which APC, an extracellular serine protease, regulates mitochondrial function, are unknown. Since mice lacking the ROS-inducing mitochondrial protein p66shc are protected from DN we hypothesized that APC's cytoprotective effect may be mechanistically linked to p66shc. Material & methods: Immortalized mouse podocytes and murine endothelial cells were stressed in vitro with glucose (30 mM). In mice persistent hyperglycaemia was induced by streptozotocin injections. Markers of DN were analysed in wild-type mice, mice expressing a thrombomodulin (TM) mutant lacking cofactor activity towards protein C activation (TMPro/Pro), mice expressing high levels of APC (hPChigh), and p66−/− x TMPro/Pro double mutant mice. Immunoblotting, RT-PCR, cellular subfractionation, immunohistochemistry and immunofluorescensce were employed for analyses. Results: 8-OH-Desoxyguanosine, a marker for ROS, is induced in diabetic mice. Nitrotyrosine, a marker for ROS-dependent damage, localizes mainly to podocytes. This is paralleled by p66shc-upregulation in glomerular podocytes. These effects are aggravated in diabetic TMPro/Pro, ameliorated in aPChigh, and reversed in p66shc x TMPro/Pro mice. In vitro APC counteracts glucose dependent upregulation of p66shc and inhibits mitochondrial translocation of p66shc in mouse podocytes, but not in endothelial cells in vitro. Conclusion: This study identifies a novel mechanism through which APC mediates cytoprotection. In glucose stressed podocytes, but not endothelial cells, APC prevents mitochondrial translocation of the ROS-inducing redox-protein p66shc, which is required for protection against DN in vivo. Disclosures: No relevant conflicts of interest to declare.

Dermatan Sulfate and LMW Heparin Enhance the Anticoagulant Action of Activated Protein C

1999 ◽  
Vol 82 (11) ◽  
pp. 1462-1468 ◽  
Author(s):  
José Fernández ◽  
Jari Petäjä ◽  
John Griffin
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Protein C ◽  
Dermatan Sulfate ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor V ◽  
Factor Va ◽  
Anticoagulant Action

SummaryUnfractionated heparin potentiates the anticoagulant action of activated protein C (APC) through several mechanisms, including the recently described enhancement of proteolytic inactivation of factor V. Possible anticoagulant synergism between APC and physiologic glycosaminoglycans, pharmacologic low molecular weight heparins (LMWHs), and other heparin derivatives was studied. Dermatan sulfate showed potent APC-enhancing effect. Commercial LMWHs showed differing abilities to promote APC activity, and the molecular weight of LMWHs correlated with enhancement of APC activity. Degree of sulfation of the glycosaminoglycans influenced APC enhancement. However, because dextran sulfates did not potentiate APC action, the presence of sulfate groups per se on a polysaccharide is not sufficient for APC enhancement. As previously for unfractionated heparin, APC anticoagulant activity was enhanced by glycosaminoglycans when factor V but not factor Va was the substrate. Thus, dermatan sulfate and LMWHs exhibit APC enhancing activity in vitro that could be of physiologic and pharmacologic significance.

A Sensitive and Facile Assay for the Measurement of Activated Protein C Activity Levels in Vivo

1993 ◽  
Vol 69 (05) ◽  
pp. 441-447 ◽  
Author(s):  
Carolyn L Orthner ◽  
Billy Kolen ◽  
William N Drohan
Keyword(s):  
Protein C ◽  
Plasma Concentrations ◽  
Limited Proteolysis ◽  
Activated Protein C ◽  
Microtiter Plate ◽  
Reversible Inhibitor ◽  
Activity Levels ◽  
Standard Curve ◽  
Agarose Beads

SummaryActivated protein C (APC) is a serine protease which plays an important role as a naturally occurring antithrombotic enzyme. APC, which is formed by thrombin-catalyzed limited proteolysis of the zymogen protein C, functions as an anticoagulant by proteolytic inactivation of the coagulation cofactors VIIIa and Va. APC is inhibited by several members of the serpin family as well a by α2-macroglobulin. APC is being developed as a therapeutic for the prevention and treatment of thrombosis. We have developed an assay to quantify circulating levels of enzymatically active APC during its administration to patients, in healthy individuals, and in various disease states. This assay utilizes an EDTA-dependent anti-protein C monoclonal antibody (Mab) 7D7B10 to capture both APC and protein C from plasma, prepared from blood collected in an anticoagulant supplemented with the reversible inhibitor p-aminobenzamidine. Mab 7D7B10-derivatized agarose beads are added to the wells of a 96-well filtration plate, equilibrated with Tris-buffered saline, and incubated for 10 min with 200 μl of plasma. After washing, APC and protein C are eluted from the immunosorbent beads with a calcium-containing buffer into the wells of a 96-well microtiter plate containing antithrombin III (ATIII) and heparin. The amidolytic activity of APC is then measured on a kinetic plate reader following the addition of L-pyroglutamyl-L-prolyl-L-arginine-p-nitroanilide (S-2366) substrate.The rate of substrate hydrolysis was proportional to APC concentration over a 200-fold concentration range (5.0 to 1,000 ng/ml) when measured continuously over a 15 to 30 min time period. The coefficient of variation was 5.9% at 35 ng/ml and 8.8% at 350 ng/ml APC. The sensitivity of the assay could be increased by measuring the amount of color produced after longer incubation times in the endpoint mode. The measured APC activity levels were little affected by varying protein C or prothrombin over the extremes of 0 to 150% of normal plasma concentrations. By constructing the standard curve in protein C-deficient plasma, the concentration of APC activity in normal pooled plasma was determined to be 2.8 ng/ml (45 pM), which represents 0.08% of the protein C concentration. The assay was approximately 50-fold more sensitive than the identical assay, but using Mab-coated microtiter wells rather than immunosorbent beads as the capture step.

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

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.

Exosite-dependent regulation of factor VIIIa by activated protein C

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 4802-4807 ◽  
Author(s):  
Chandrashekhara Manithody ◽  
Philip J. Fay ◽  
Alireza R. Rezaie
Keyword(s):  
Protein C ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Activated Protein C ◽  
Coagulation Cascade ◽  
Factor Va ◽  
Factor Viiia

AbstractActivated protein C (APC) is a natural anticoagulant serine protease in plasma that down-regulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. Recent results have indicated that basic residues of 2 surface loops known as the 39-loop (Lys37-Lys39) and the Ca2+-binding 70-80–loop (Arg74 and Arg75) are critical for the anticoagulant function of APC. Kinetics of factor Va degradation by APC mutants in purified systems have demonstrated that basic residues of these loops are involved in determination of the cleavage specificity of the Arg506 scissile bond on the A2 domain of factor Va. In this study, we characterized the properties of the same exosite mutants of APC with respect to their ability to interact with factor VIIIa. Time course of the factor VIIIa degradation by APC mutants suggested that the same basic residues of APC are also critical for recognition and degradation of factor VIIIa. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of the factor VIIIa cleavage reactions revealed that these residues are involved in determination of the specificity of both A1 and A2 subunits in factor VIIIa, thus facilitating the cleavages of both Arg336 and Arg562 scissile bonds in the cofactor.

Platelet-mediated proteolytic down regulation of the anticoagulant activity of protein S in individuals with haematological malignancies

2012 ◽  
Vol 107 (03) ◽  
pp. 468-476 ◽  
Author(s):  
Ilze Dienava-Verdoold ◽  
Marina R. Marchetti ◽  
Liane C. J. te Boome ◽  
Laura Russo ◽  
Anna Falanga ◽  
...  
Keyword(s):  
Protein C ◽  
Normal Values ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Intact Protein ◽  
The Impact ◽  
Independent Protein

SummaryThe natural anticoagulant protein S contains a so-called thrombin-sensitive region (TSR), which is susceptible to proteolytic cleavage. We have previously shown that a platelet-associated protease is able to cleave protein S under physiological plasma conditions in vitro. The aim of the present study was to investigate the relation between platelet-associated protein S cleaving activity and in vivo protein S cleavage, and to evaluate the impact of in vivo protein S cleavage on its anticoagulant activity. Protein S cleavage in healthy subjects and in thrombocytopenic and thrombocythaemic patients was evaluated by immunological techniques. Concentration of cleaved and intact protein S was correlated to levels of activated protein C (APC)-dependent and APC-independent protein S anticoagulant activity. In plasma from healthy volunteers 25% of protein S is cleaved in the TSR. While in plasma there was a clear positive correlation between levels of intact protein S and both APC-dependent and APC-independent protein S anticoagulant activities, these correlations were absent for cleaved protein S. Protein S cleavage was significantly increased in patients with essential thrombocythaemia (ET) and significantly reduced in patients with chemotherapy-induced thrombocytopenia. In ET patients on cytoreductive therapy, both platelet count and protein S cleavage returned to normal values. Accordingly, platelet transfusion restored cleavage of protein S to normal values in patients with chemotherapy-induced thrombocytopenia. In conclusion, proteases from platelets seem to contribute to the presence of cleaved protein S in the circulation and may enhance the coagulation response in vivo by down regulating the anticoagulant activity of protein S.

scholarly journals Turnover of *I-protein C inhibitor and *I-alpha 1-antitrypsin and their complexes with activated protein C

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2290-2295 ◽  
Author(s):  
M Laurell ◽  
J Stenflo ◽  
TH Carlson
Keyword(s):  
Complex Formation ◽  
Protein C ◽  
Activated Protein C ◽  
Human Protein ◽  
Relative Contribution ◽  
Protein C Inhibitor ◽  
The Difference ◽  
Alpha 1 Antitrypsin

Abstract The rates of clearance and catabolism of human protein C inhibitor (PCI) and human alpha 1-antitrypsin (alpha 1-AT) and their complexes with human activated protein C (APC) were studied in the rabbit. The radioiodinated-free inhibitors had biologic half-lives of 23.4 and 62.1 hours, respectively, while the corresponding *I-labeled activated- protein C complexes were cleared with half-lives of 19.6 +/- 3.1 and 72.2 +/- 6.1 minutes. Complex clearances were linked to their catabolism as shown by a correlation between clearance and the appearance of free radioiodine in the plasma. Thus, the difference in the rates of catabolism would result in a fivefold greater amount of alpha 1-AT-APC complex than PCI-APC complex 1 hour after the formation of equal amounts of these in vivo. These results lead to the conclusion that the relative contribution of PCI and alpha 1-AT to the physiologic inhibition of APC cannot be determined only from the rates of the formation of these complexes in vitro, or from measurement of their levels in plasma. The APC-PCI complex is unstable as compared with the APC-alpha 1-AT complex, compounding the problem of estimating rates of complex formation from their levels in plasma.

scholarly journals Activation of Notch1 signaling in podocytes by glucose-derived AGEs contributes to proteinuria

2020 ◽  
Vol 8 (1) ◽  
pp. e001203
Author(s):  
Rajkishor Nishad ◽  
Prajakta Meshram ◽  
Ashish Kumar Singh ◽  
G Bhanuprakash Reddy ◽  
Anil Kumar Pasupulati
Keyword(s):  
Notch Signaling ◽  
Epithelial To Mesenchymal Transition ◽  
Mesenchymal Transition ◽  
Notch1 Signaling ◽  
Reverse Transcription Pcr ◽  
Filtration Barrier ◽  
Foot Process ◽  
Design And Methods

IntroductionAdvanced glycation end-products (AGEs) are implicated in the pathogenesis of diabetic nephropathy (DN). Previous studies have shown that AGEs contribute to glomerulosclerosis and proteinuria. Podocytes, terminally differentiated epithelial cells of the glomerulus and the critical component of the glomerular filtration barrier, express the receptor for AGEs (RAGE). Podocytes are susceptible to severe injury during DN. In this study, we investigated the mechanism by which AGEs contribute to podocyte injury.Research design and methodsGlucose-derived AGEs were prepared in vitro. Reactivation of Notch signaling was examined in AGE-treated human podocytes (in vitro) and glomeruli from AGE-injected mice (in vivo) by quantitative reverse transcription-PCR, western blot analysis, ELISA and immunohistochemical staining. Further, the effects of AGEs on epithelial to mesenchymal transition (EMT) of podocytes and expression of fibrotic markers were evaluated.ResultsUsing human podocytes and a mouse model, we demonstrated that AGEs activate Notch1 signaling in podocytes and provoke EMT. Inhibition of RAGE and Notch1 by FPS-ZM1 (N-Benzyl-4-chloro-N-cyclohexylbenzamide) and DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenyl glycine t-butylester), respectively, abrogates AGE-induced Notch activation and EMT. Inhibition of RAGE and Notch1 prevents AGE-induced glomerular fibrosis, thickening of the glomerular basement membrane, foot process effacement, and proteinuria. Furthermore, kidney biopsy sections from people with DN revealed the accumulation of AGEs in the glomerulus with elevated RAGE expression and activated Notch signaling.ConclusionThe data suggest that AGEs activate Notch signaling in the glomerular podocytes. Pharmacological inhibition of Notch signaling by DAPT ameliorates AGE-induced podocytopathy and fibrosis. Our observations suggest that AGE-induced Notch reactivation in mature podocytes could be a novel mechanism in glomerular disease and thus could represent a novel therapeutic target.

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

Rheumatology ◽  
2019 ◽  
Vol 58 (10) ◽  
pp. 1850-1860 ◽  
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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