Activated Protein C Cellular Pathways Regulating Thrombosis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-44-SCI-44
Author(s):  
John H. Griffin ◽  
Laurent O. Mosnier
Keyword(s):  
Endothelial Cells ◽  
Cell Signaling ◽  
Protein C ◽  
Activated Protein C ◽  
Caveolin 1 ◽  
Multiple Receptors ◽  
Injury Models ◽  
Research Institute

Abstract SCI-44 Plasma protein C is known for its mild deficiency linked to venous thrombosis risk and severe deficiency linked to neonatal purpura fulminans. Activated protein C (APC) exerts both anticoagulant activity via proteolytic inactivation of factors Va and VIIIa and cellular cytoprotective actions via direct initiation of cell signaling. Based on studies of engineered APC mutants and the use of genetically modified mice, APC’s cell signaling actions are thought to drive murine APC’s mortality reduction in sepsis models, neuroprotective actions in brain injury models, and nephroprotective effects in kidney injury models. These actions in vivo are generally suggested to involve multiple receptors (PAR1, endothelial protein C receptor [EPCR], PAR3, and CD11b), while in vitro studies implicate these receptors and potentially also other receptors (apoER2, β1 and β3 integrins, S1P1, and the angiopoietin/Tie-2 axis) for APC’s cellular effects. Crosstalk among these receptors may permit a timely integration of APC-induced signaling, which ultimately determines APC’s effects on a specific cell and organ. Central to many in vivo and in vitro published studies is the implication that APC provides beneficial effects via EPCR-dependent PAR1-dependent cell signaling. This central role for PAR1 poses the dilemma of how thrombin and APC can often seem to have opposing effects when activating PAR1. Microdomain-specific PAR1 signaling by APC versus thrombin may help explain some observations. Binding of protein C or APC to EPCR on endothelial cells appears to determine whether these proteins and PAR1 are or are not colocalized in microdomains with caveolin-1. APC’s activation of Rac1 via PAR1 requires EPCR and caveolin-1 whereas thrombin’s activation of RhoA via PAR1 is independent of EPCR and caveolin-1. We hypothesized that APC might cleave PAR1 not only at Arg41 but also at Arg46 with distinct consequences and that this could distinguish APC’s from thrombin’s signaling. We found that APC cleaved the PAR1 N-terminal synthetic TR33-66 peptide at Arg41 and also at another site distal from Arg41. Isolation of the novel proteolytic fragments and their MALDI-TOF analysis identified Arg46 as that cleavage site. When cells containing EPCR were transfected with secretable alkaline phosphatase (SEAP)-PAR1 wild type and mutant constructs, both thrombin and APC cleaved wt-PAR1 but not R41Q/R46Q-PAR1. As expected, thrombin also did not cleave R41A-PAR1 or R41Q-PAR1. But APC very efficiently cleaved both the R41A-PAR1 and the R41Q-PAR1 mutants. We tested a synthetic PAR1 analog peptide (Asn47-residue 66) to see if it could promote signaling. This PAR1 (47–66)-peptide increased Akt phosphorylation at Ser473 in endothelial cells over 30 minutes whereas neither a control scrambled sequence (47–66)-peptide nor a TRAP peptide had a similar effect. The PAR1 (47–66)-peptide, but the control scrambled sequence-peptide or TRAP, inhibited staurosporine-induced endothelial cell apoptosis. Thus, it appears that the new N-terminus generated by APC’s cleavage at Arg46 in PAR1 generates a novel tethered ligand, which could induce selective APC-like protective signaling. Hence, APC is capable of a unique, functionally significant cleavage of PAR1. Further in vitro and in vivo studies are needed to address a number of obvious questions. In summary, explanations for APC’s beneficial cellular cytoprotective effects may be found in its ability to signal via multiple receptors selectively located in different cell membrane microdomains and potentially also in its ability to activate PARs by cleavages at unique sites, which initiate unique signaling events on different cells in different organs. Disclosures: Griffin: ZZBiotech LLC: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Scripps Research Institute: Employment, Patents & Royalties. Mosnier:Scripps Research institute: Employment, Patents & Royalties.

Platelet factor 4 enhances generation of activated protein C in vitro and in vivo

Blood ◽  
2003 ◽  
Vol 102 (1) ◽  
pp. 146-151 ◽  
Author(s):  
Arne Slungaard ◽  
Jose A. Fernandez ◽  
John H. Griffin ◽  
Nigel S. Key ◽  
Janel R. Long ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Platelet Factor 4 ◽  
In Vivo Model ◽  
Platelet Factor ◽  
Umbilical Vein Endothelial Cells

Abstract Platelet factor 4 (PF4), an abundant platelet α-granule protein, accelerates in vitro generation of activated protein C (APC) by soluble thrombin/thrombomodulin (TM) complexes up to 25-fold. To test the hypothesis that PF4 similarly stimulates endothelium-associated TM, we assessed the influence of human PF4 on thrombin-dependent APC generation by cultured endothelial monolayers. APC generated in the presence of 1 to 100 μg PF4 was up to 5-fold higher than baseline for human umbilical vein endothelial cells, 10-fold higher for microvascular endothelial cells, and unaltered for blood outgrowth endothelial cells. In an in vivo model, cynomolgus monkeys (n = 6, each serving as its own control) were infused with either PF4 (7.5 mg/kg) or vehicle buffer, then with human thrombin (1.0 μg/kg/min) for 10 minutes. Circulating APC levels (baseline 3 ng/mL) peaked at 10 minutes, when PF4-treated and vehicle-treated animals had APC levels of 67 ± 5 ng/mL and 39 ± 2 ng/mL, respectively (P < .001). The activated partial thromboplastin time (APTT; baseline, 28 seconds) increased maximally by 27 ± 6 seconds in PF4-treated animals and by 9 ± 1 seconds in control animals at 30 minutes (P < .001). PF4-dependent increases in circulating APC and APTT persisted more than 2-fold greater than that of control's from 10 through 120 minutes (P ≤ .04). All APTT prolongations were essentially reversed by monoclonal antibody C3, which blocks APC activity. Thus, physiologically relevant concentrations of PF4 stimulate thrombin-dependent APC generation both in vitro by cultured endothelial cells and in vivo in a primate thrombin infusion model. These findings suggest that PF4 may play a previously unsuspected physiologic role in enhancing APC generation. (Blood. 2003;102:146-151)

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

Blood ◽  
2012 ◽  
Vol 120 (26) ◽  
pp. 5237-5246 ◽  
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.

scholarly journals Novel R41Q- and R46Q-PAR1-Modified Mice Enable Proof-of-Concept Studies for In Vivo Protective Mechanisms of Action for Activated Protein C (APC) in Sepsis and Stroke

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 13-13
Author(s):  
Ranjeet Kumar Sinha ◽  
Yaoming Wang ◽  
Zhen Zhao ◽  
Jose A. Fernandez ◽  
Naveen Gupta ◽  
...  
Keyword(s):  
Cell Signaling ◽  
Brain Damage ◽  
Mechanism Of Action ◽  
Protein C ◽  
Activated Protein C ◽  
Proof Of Concept ◽  
E Coli ◽  
Apc Mutations

Abstract Introduction: Although much is known about in vitro mechanisms for Activated Protein C (APC)'s beneficial actions on endothelial, neuronal, and epithelial cells, much less is known about its in vivo mechanism(s) of action. EPCR-bound APC can cleave protease activated receptor (PAR) 1 at Arg41 or Arg46 to initiate cell signaling. Notably, cleavage at Arg46 can initiate arrestin-dependent biased signaling whereas thrombin's canonical cleavage occurs at Arg41. Which PAR1 cleavage may mediate APC's in vivo benefits? Inferences often come from using PAR1 knockout mice. But knocking out PAR1 disrupts PAR1 interactomes, thereby potentially disrupting many protein-protein interactions, e.g., PAR heterodimers, etc. To elucidate PAR1-dependent aspects of APC's in vivo mechanism of action, we generated C57BL/6 strains carrying either Arg41Gln (R41Q) or Arg46Gln (R46Q) PAR1 point mutations. Using these strains, we determined whether or not recombinant murine signaling-selective APC mutants would reduce septic death or provide neuroprotection against stroke when mice carried PAR1 mutations that prevent PAR1 cleavages at either Arg41 or Arg46. Methods: Standard homologous recombination methods and C57BL/6-ES cells were used to make C57BL/6 strains carrying PAR1 mutations. Brain microvascular endothelial cells (BECs) were isolated from mice by published protocols. Bioassays using BECs and APC, thrombin, or a thrombin receptor activating peptide (TRAP) included: (1) endothelial barrier stabilization or disruption that was monitored using Trans-Endothelial Resistance (TER) (iCelligence, Acea, San Diego) and (2) induction of cell signaling that was quantified using in-cell Western blotting. The ability of 5A-APC (mutations: KKK191-193AAA+RR229-230AA) (0.2 mg/kg, i.v. at 0h and 6 h) to reduce death due to live E. coli-induced pneumonia was determined using standard protocols. The ability of 3K3A-APC (mutations: KKK191-193AAA) (0.04 mg/kg, i.v. at 4 h) to reduce brain damage caused by transient distal middle cerebral artery occlusion (tdMCAO) (1 h) was determined at 24 h, as described. Results : R46Q mice were normal in breeding whereas R41Q mice, like PAR1 KO, gave less than half the expected QQ or null homozygotes. PAR1 knockout-derived BECs showed no TER decrease for either TRAP or thrombin, proving PAR1 was needed. TER assays showed that TRAP disrupted endothelial barriers of BECs from QQ41-PAR1 and QQ46-PAR1 mice similar to BECs from WT-mice, proving the expression of normally functional PAR1 in each PAR1-mutated strain. As expected, thrombin decreased TER for BECs from WT mice and QQ46-PAR1 mice but not for BECs from QQ41-PAR1 mice. APC inhibited thrombin-induced TER decreases for BECs from WT mice but not for BECs from QQ46-PAR1 mice, showing R46 is required for APC's barrier stabilization. The QQ46-PAR1 BECs showed significantly reduced APC-induced phosphorylation of Akt whereas thrombin-induced phosphorylation of ERK was not significantly affected. Thus, in vitro studies showed the predicted retention or loss of responses to TRAP, thrombin or APC for each PAR1 mutation. In sepsis studies, 5A-APC reduced mortality from 50 % to 10 % in E. coli-induced pneumonia for WT-PAR1 and 41QQ-PAR1 mice (p < 0.01) but had no benefit for 46QQ-PAR1 mice. In tdMCAO stroke studies, 3K3A-APC significantly reduced infarct size, edema and neuronal apoptosis for WT mice and QQ41-PAR1 mice but had no detectable benefits for mice carrying QQ46-PAR1. In functional studies of forelimb asymmetry and foot fault tests at 24 h after tdMCAO, 3K3A-APC was beneficial for mice with QQ41-PAR1 but not for QQ46-PAR1. Conclusions : Genetically altered mice carrying 41QQ-PAR1 and 46QQ-PAR1 provide unique, powerful tools to explore in vivo requirements for Arg41 or Arg46 cleavages by APC or other proteases that initiate PAR1-dependent signaling. The failures of 5A-APC to reduce death following intratracheal E. coli in 46QQ-PAR1 mice and of 3K3A-APC to reduce brain damage following ischemia in 46QQ-PAR1 mice provide very clear in vivo proof-of-concept data for the hypothesis that APC's cleavage of PAR1 at Arg46 is central to its in vivo mechanism of action in these two very different pathologies. These results strongly support the concept that APC's biased PAR1-dependent signaling following Arg46 cleavage is central to APC's in vivo benefits. Figure. Figure. Disclosures Mosnier: The Scripps Research Institute: Patents & Royalties; Hematherix LLC: Membership on an entity's Board of Directors or advisory committees; Bayer: Honoraria, Speakers Bureau; Baxalta: Honoraria, Speakers Bureau.

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

2015 ◽  
Vol 114 (12) ◽  
pp. 1144-1155 ◽  
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.

In Vitro Protection of Human Endothelial Cells from Adenovirus Vector Toxicity by Activated Protein C.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3694-3694
Author(s):  
Jay Nelson Lozier ◽  
Felice D’Agnillo
Keyword(s):  
Endothelial Cells ◽  
Cell Death ◽  
Endothelial Cell ◽  
Protein C ◽  
First Generation ◽  
Activated Protein C ◽  
Adenovirus Vector ◽  
Dose Dependent

Abstract High dose adenovirus vector administration in vivo has been associated with toxicity toward many cell types, including endothelial cells. Some of the prominent pathological features of an adenovirus vector death in a gene therapy trial included capillary leak syndrome and disseminated intravascular coagulation (DIC). We investigated the hypothesis that activated protein C (APC) might have a protective effect on primary human microvascular endothelial cells exposed to a first-generation adenovirus vector. We exposed primary human endothelial cells to a first-generation (E1, E3 deleted) adenovirus vector, AVC3FIX5 at concentrations ranging from 103 to 105 vector particles per cell and showed dose-dependent cell death as early as 6 hours (40% cell death at the highest dose). Phase contrast and immunofluorescence microscopy revealed that some cells died rapidly by primary necrosis while others died by apoptosis over a longer time course. By 40 hours, only 40% of the cells were viable. We then tested the effect of pretreatment of endothelial cells with APC concentrations ranging from 1 nM to 100 nM. Dose-dependent protection was seen in which cell death was reduced to 9 and 2 % at APC concentrations of 50 and 100 nM, respectively. We also tested the effect of timing of the APC treatment and showed that 1 hour pre-treatment or concurrent APC treatment were protective, but APC administered one hour after the adenovirus exposure was substantially less protective. This suggested that APC exerts its protective actions on endothelial cells either by interfering with early steps in the interaction of the vector with the cells, (e.g., vector entry) or by modulating death signaling pathways. It has been proposed that APC protects against cell damage in sepsis by interaction with the endothelial cell protein C receptor (EPCR) and protease activated receptor 1 (PAR1) on the endothelial cell surface to induce MCP-1 and other immunomodulatory genes by proteolytic signaling (Riewald et al., Science296:1880–1882, 2002). Other investigators have shown protective effects of APC for endothelial cells subjected to hypoxia through normalization of levels of p53, Bax, and Bcl-2 gene expression (Cheng et al., Nat Med9:338–342, 2003). The APC concentrations in our experiments that were maximally protective (50–100 nM) were of the same order of magnitude as was shown to be protective in vitro by these investigators. If APC can be shown to have a protective effect against adenovirus-induced endothelial cell toxicity and DIC in vivo, this may be a useful therapeutic strategy to explore as treatement of gene therapy vector toxicity. Cell Viability vs. APC Concentration Cell Viability vs. APC Concentration

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.

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.

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.

Recombinant human activated protein C upregulates cyclooxygenase-2 expression in endothelial cells via binding to endothelial cell protein C receptor and activation of protease-activated receptor-1

2005 ◽  
Vol 93 (04) ◽  
pp. 743-750 ◽  
Author(s):  
Sarah Horn ◽  
Siegfried Lang ◽  
Kenji Fukudome ◽  
Adriane Nahrup ◽  
Ursula Hoffmann ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein C ◽  
Activated Protein C ◽  
Cell Protein ◽  
Mrna Levels ◽  
Endothelial Protein C Receptor ◽  
Drotrecogin Alfa Activated ◽  
Cox 2 ◽  
Vitro Effect

SummaryProstacyclin (PGI2) has beneficial cytoprotective properties, is a potent inhibitor of platelet aggregation and has been reported to improve microcirculatory blood flow during sepsis. The formation of PGI2 in response to proinflammatory cytokines is catalysed by the inducible cyclooxygenase (COX) isoform COX-2. Recombinant human activated protein C (rhAPC, drotrecogin alfa (activated)) was shown to have multiple biological activities in vitro and to promote resolution of organ dysfunction in septic patients. Whether rhAPC exerts its beneficial effects by modulating prostanoid generation is unknown up to now. It was therefore the aim of the study to examine the in vitro effect of rhAPC on COX-2-mRNA-expression and PGI2 release from human umbilical vein endothelial cells (HUVEC). We found that rhAPC, at supra-therapeutical concentrations (500ng/ml-20μg/ ml), upregulated the amount of COX-2-mRNA in HUVEC at t=3–9h and caused a time- and dose-dependent release of 6-keto PGF1α, the stable hydrolysis product of prostacyclin. RhAPC further increased the stimulating effect of tumor necrosis factor-α (TNF-α) and thrombin on COX-2-mRNA-levels. Transcript levels of cyclooxygenase-1 (COX-1) and prostagland-in I2 synthase, however, were unaffected by the stimulation with rhAPC or thrombin. The upregulatory effect on COX2-mRNA levels was specific for rhAPC since the zymogen protein C in equimolar concentrations had no effect on COX-2-mRNA-levels or 6keto PGF1α-release. Western Blot analysis revealed an increase of COX-2-protein content in HUVEC after treatment with rhAPC. As shown by experiments using monoclonal antibodies against the thrombin receptor PAR-1 (mAb=ATAP2) and against the endothelial protein C receptor (EPCR; mAb=RCR-252), the effect of rhAPC on COX-2-mRNA up-regulation was mediated by binding to the EPCR-receptor and signaling via PAR-1. These results demonstrate that induction of COX-2-expression is an important response of HUVEC to stimulation with rhAPC and may represent a new molecular mechanism, by which rhAPC promotes upregulation of prostanoid production in human endothelium.

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