scholarly journals Activated protein C and PAR1‐derived and PAR3‐derived peptides are anti‐inflammatory by suppressing macrophage NLRP3 inflammasomes

2020 ◽  
Vol 19 (1) ◽  
pp. 269-280
Author(s):  
Laura D. Healy ◽  
José A. Fernández ◽  
Laurent O. Mosnier ◽  
John H. Griffin
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Anti Inflammatory ◽  
Nlrp3 Inflammasomes

Signaling by Activated Protein C in Endothelial Cells Is PAR1 Dependent but Distinct from Thrombin Signaling.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 221-221 ◽  
Author(s):  
Matthias Riewald ◽  
Clemens Feistritzer ◽  
Wolfram Ruf
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Protein C ◽  
Biological Effects ◽  
Activated Protein C ◽  
Cell Protein ◽  
Protein Levels ◽  
Thrombospondin 1 ◽  
Anti Inflammatory

Abstract Thrombin initiates fibrin formation and platelet activation, and activates protein C, generating activated protein C (APC) that inhibits blood coagulation by a negative feedback loop. Thrombin has also proinflammatory effects through activation of cellular protease activated receptor-1 (PAR1). Endothelial cell protein C receptor (EPCR) can bind both protein C and APC and activation of EPCR-bound protein C is enhanced. Results from animal models and clinical trials indicate that APC has potent protective effects in systemic inflammation that are independent from its well established anticoagulant function and recombinant APC was recently approved to treat patients with severe sepsis. The molecular basis for APC’s anti-inflammatory effects is incompletely understood. We have identified PAR1 and EPCR as part of a novel APC signaling pathway in quiescent endothelial cells, raising the question how the same receptor PAR1 can mediate both pro- and anti-inflammatory effects. In an overexpression system in PAR-deficient fibroblasts, wildtype PAR2 but not a PAR2 variant with an Arg36 to Ala substitution at the P1 position was activated by APC, indicating that APC can activate PAR2 in addition to PAR1 through a canonical cleavage mechanism. Therefore, we tested whether endothelial cell PAR2 can be activated by APC under conditions where endogenous PAR2 expression is upregulated. Even when PAR2 expression was highly upregulated in inflammatory cytokine-stimulated human umbilical endothelial cells (HUVECs), signaling by APC was strictly dependent on PAR1 cleavage and signaling. Consistent with these results in HUVECs, intravenous injection of APC in wildtype, PAR1−/−, and PAR2−/− mice demonstrated that PAR1 is the major murine receptor that mediates induction of the transcript for monocyte chemoattractant protein-1 in the lung in response to APC. This indicates that indeed the same receptor PAR1 mediates signaling by APC and thrombin both in vitro and in vivo. To test the possibility that APC diminishes proinflammatory thrombin-PAR1 signaling by downregulating cellular levels of functional PAR1, we tested whether preincubation with APC can desensitize Erk1/2 phosphorylation by thrombin. Phospho-Erk1/2 was induced by APC dependent upon PAR1 cleavage, but APC-pretreated cells still responded to PAR1-dependent thrombin signaling, suggesting that only a fraction of the cellular PAR1 is subject to cleavage by APC. These results indicate that APC does not block thrombin signaling by desensitation at the receptor level. Large-scale gene expression profiling demonstrated that APC and thrombin had specific effects on gene expression in tumor necrosis factor α (TNFα )-perturbed endothelial cells that were not detected in quiescent cells. Transcripts for several proapoptotic genes including p53 and thrombospondin-1 were downregulated by APC but not by thrombin or PAR1 agonist peptides in TNFα-stimulated HUVECs. Western blotting confirmed that in TNFα-perturbed HUVECs pretreatment with APC significantly reduced the increase in cellular p53 protein levels in response to the cytotoxic doxorubicin. This APC effect was dependent on EPCR binding and PAR1 cleavage. Thrombospondin-1 protein levels were similarly downregulated by APC but upregulated by thrombin. Both down- and upregulation by APC and thrombin, respectively, were PAR1 dependent. These findings demonstrate that the same receptor on the same cell type can mediate opposite biological effects and they suggest that EPCR cosignaling may modify PAR1-dependent APC signaling in endothelial cells.

Engineering a Disulfide Bond To Stabilize the Calcium Binding Loop of Activated Protein C Eliminates Its Anticoagulant but Not Protective Signaling Properties.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 62-62
Author(s):  
Jong-Sup Bae ◽  
Likui Yang ◽  
Chandrashekhara Manithody ◽  
Alireza R. Rezaie
Keyword(s):  
Severe Sepsis ◽  
Endothelial Cell ◽  
Disulfide Bond ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Cell Permeability ◽  
Endothelial Protein C Receptor ◽  
Anti Inflammatory ◽  
Protective Signaling

Abstract In addition to its potent anticoagulant activity, activated protein C (APC) also exhibits anti-inflammatory and cytoprotective properties. The latter properties of APC are thought to be responsible for its beneficial effect in treating severe sepsis patients. A higher incidence of bleeding due to its anticoagulant function has been found to be a major drawback of APC as an effective anti-inflammatory drug. Thus, there is intense interest in developing APC variants that have no anticoagulant activity, but have normal protective properties. In this study, we have prepared a protein C variant in which an engineered disulfide bond between two β-sheets has stabilized the functionally critical Ca2+-binding 70–80 loop of the molecule. The 70–80 loop of this mutant no longer binds Ca2+ and the activation of the mutant by thrombin has been enhanced 60–80-fold independent of thrombomodulin. The anticoagulant activity of the APC variant has been essentially eliminated as determined by both factor Va degradation and plasma-based clotting assays. However, the endothelial protein C receptor- and protease-activated receptor-1-dependent protective signaling properties of the APC variant have remained intact as determined by staurosporine-induced endothelial cell apoptosis, thrombin-induced endothelial cell permeability and TNF-α-mediated neutrophil adhesion and migration assays. These results suggest that modulation of the structure and/or activity of APC by Ca2+ is required for its anticoagulant, but not for its anti-inflammatory activity, thus the protein C variant may potentially be a safer drug for treating severe sepsis patients in both activated and zymogenic forms.

Recombinant human activated protein C (rhAPC; drotrecogin alfa [activated]) has minimal effect on markers of coagulation, fibrinolysis, and inflammation in acute human endotoxemia

Blood ◽  
2003 ◽  
Vol 102 (6) ◽  
pp. 2093-2098 ◽  
Author(s):  
Ulla Derhaschnig ◽  
Rosemarie Reiter ◽  
Paul Knöbl ◽  
Magdalena Baumgartner ◽  
Priska Keen ◽  
...  
Keyword(s):  
Protein C ◽  
Drotrecogin Alfa ◽  
Activated Protein C ◽  
Host Responses ◽  
Low Grade ◽  
Human Endotoxemia ◽  
Drotrecogin Alfa Activated ◽  
Anti Inflammatory ◽  
To Receive ◽  
Thrombin Formation

Abstract Inflammatory and procoagulant host responses are closely related in sepsis. The protein C pathway serves as a regulatory pathway with anti-inflammatory and anticoagulant properties. Recently, recombinant human activated protein C (rhAPC) was shown to reduce mortality in severe sepsis. Nevertheless, the effects of rhAPC in humans are still ill defined. The infusion of low endotoxin doses into humans provides a standardized model to study inflammatory and hemostatic mechanisms. Thus, we investigated whether rhAPC acts as an anticoagulant or anti-inflammatory drug in human endotoxemia. There were 24 volunteers randomized to receive either 24 μg/kg per hour rhAPC or placebo intravenously for 8 hours. Lipopolysaccharide (LPS, 2 ng/kg) was administered 2 hours after starting the infusions. rhAPC decreased basal tissue factor (TF)–mRNA expression, and thrombin formation and action. In contrast, rhAPC did not significantly blunt LPS-induced thrombin generation. Consistently, rhAPC did not reduce LPS-induced levels of TF-mRNA or D-dimer and had no effect on fibrinolytic activity or inflammation. Finally, endogenous APC formation was enhanced during endotoxemia and appeared to be associated with inflammation rather than thrombin formation. In conclusion, even low-grade endotoxemia induces significant protein C activation. Infusion of rhAPC decreases “spontaneous” activation of coagulation but does not blunt LPS-induced, TF-mediated coagulation in healthy volunteers, which is in contrast to a number of anticoagulants.

Recombinant activated protein C attenuates coagulopathy and inflammation when administered early in murine pneumococcal pneumonia

2011 ◽  
Vol 106 (12) ◽  
pp. 1189-1196 ◽  
Author(s):  
Cornelis van ’t Veer ◽  
Joris Roelofs ◽  
Bruce Gerlitz ◽  
Brian Grinnell ◽  
Marcel Levi ◽  
...  
Keyword(s):  
Protein C ◽  
Pneumococcal Pneumonia ◽  
Therapeutic Response ◽  
Activated Protein C ◽  
Coagulation Activation ◽  
Cytokines And Chemokines ◽  
Anti Inflammatory ◽  
D Dimer ◽  
Bacterial Outgrowth

SummaryRecombinant human activated protein C (APC), which has both anticoagulant and anti-inflammatory properties, improves survival of patients with severe sepsis. This beneficial effect is especially apparent in patients with pneumococcal pneumonia. Earlier treatment with APC in sepsis has been associated with a better therapeutic response as compared to later treatment. In a mouse model it was recently confirmed that recombinant murine (rm-)APC decreases coagulation activation and improves survival in pneumococcal pneumonia; however, APC did not impact on the inflammatory response. The aim of this study was to determine the effect of APC treatment instigated early in infection on activation of coagulation and inflammation after induction of pneumococcal pneumonia. Mice were infected intranasally with viable S. pneumoniae. Mice were treated with rm-APC (125 μg) or vehicle intraperitoneally 12 hours after infection and were sacrificed after 20 hours, after which blood and organs were harvested for determination of bacterial outgrowth, coagulation activation and inflammatory markers. In this early treatment model, rm-APC treatment inhibited pulmonary and systemic activation of coagulation as reflected by lower levels of throm-bin-antithrombin complexes and D-dimer. Moreover, rm-APC reduced the levels of a large number of cytokines and chemokines in the lung. When administered early in pneumococcal pneumonia, rm-APC inhibits systemic and pulmonary activation of coagulation and moreover exerts various anti-inflammatory effects in the lung.

scholarly journals A thrombomodulin mutation that impairs activated protein C generation results in uncontrolled lung inflammation during murine tuberculosis

Blood ◽  
2005 ◽  
Vol 106 (8) ◽  
pp. 2761-2768 ◽  
Author(s):  
Sebastiaan Weijer ◽  
Catharina W. Wieland ◽  
Sandrine Florquin ◽  
Tom van der Poll
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Inflammatory Response ◽  
Lung Inflammation ◽  
Protein C ◽  
Essential Role ◽  
Activated Protein C ◽  
Wild Type ◽  
Anti Inflammatory

AbstractThrombomodulin (TM) plays an essential role in the generation of activated protein C (APC), a mediator with both anticoagulant and anti-inflammatory properties, and is preferentially expressed in lungs. To investigate the role of TM in the coagulant and inflammatory response in the lung during tuberculosis, mice with a mutation in the TM gene (Thbd), which results in a minimal capacity for APC generation (TMpro/pro mice), were intranasally infected with live virulent Mycobacterium tuberculosis. Whereas pulmonary tuberculosis was not associated with activation of coagulation in either wild-type or TMpro/pro mice, 5 weeks after infection TMpro/pro mice displayed an uncontrolled inflammatory response in their lungs, as reflected by higher lung weights, a diminished ability to form well-shaped granulomas, elevated levels of proinflammatory cytokines, and concurrently reduced concentrations of anti-inflammatory cytokines. During a 36-week follow-up after infection with a lower dose of M tuberculosis, 35% of TMpro/pro mice died from week 28 onward versus none of the wild-type mice, and the surviving TMpro/pro mice displayed increased lung inflammation accompanied by higher mycobacterial loads in liver and spleen. These data suggest that a TM mutation that impairs APC generation results in uncontrolled lung inflammation during tuberculosis.

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