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Unlabelled: Essentials Epithelial cell apoptosis is critical in the pathogenesis of idiopathic pulmonary fibrosis. Protein S, a circulating anticoagulant, inhibited apoptosis of lung epithelial cells. Overexpression of protein S in lung cells reduced bleomycin-induced pulmonary fibrosis. Intranasal therapy with exogenous protein S ameliorated bleomycin-induced pulmonary fibrosis., Summary: Background Pulmonary fibrosis is the terminal stage of interstitial lung diseases, some of them being incurable and of unknown etiology. Apoptosis plays a critical role in lung fibrogenesis. Protein S is a plasma anticoagulant with potent antiapoptotic activity. The role of protein S in pulmonary fibrosis is unknown. Objectives To evaluate the clinical relevance of protein S and its protective role in pulmonary fibrosis. Methods and Results The circulating level of protein S was measured in patients with pulmonary fibrosis and controls by the use of enzyme immunoassays. Pulmonary fibrosis was induced with bleomycin in transgenic mice overexpressing human protein S and wild-type mice, and exogenous protein S or vehicle was administered to wild-type mice; fibrosis was then compared in both models. Patients with pulmonary fibrosis had reduced circulating levels of protein S as compared with controls. Inflammatory changes, the levels of profibrotic cytokines, fibrosis score, hydroxyproline content in the lungs and oxygen desaturation were significantly reduced in protein S-transgenic mice as compared with wild-type mice. Wild-type mice treated with exogenous protein S showed significant decreases in the levels of inflammatory and profibrotic markers and fibrosis in the lungs as compared with untreated control mice. After bleomycin infusion, mice overexpressing human protein S showed significantly low caspase-3 activity, enhanced expression of antiapoptotic molecules and enhanced Akt and Axl kinase phosphorylation as compared with wild-type counterparts. Protein S also inhibited apoptosis of alveolar epithelial cells in vitro. Conclusions These observations suggest clinical relevance and a protective role of protein S in pulmonary fibrosis., (© 2016 International Society on Thrombosis and Haemostasis.)

Background: Alcohol consumption is a major cause of liver injury but the mechanisms are not completely understood. Protein S (PS) is an anticoagulant glycoprotein with multiple functions. The role of PS in liver injury is unknown., Objectives: This study investigated the role of PS in acute alcoholic hepatitis., Methods: A mouse overexpressing human PS (hPS-TG) was generated in which acute hepatitis was induced by intraperitoneal injection of ethanol., Results: The levels of serum liver enzymes and liver tissue inflammatory cytokines and the degree of hepatic steatosis were significantly increased in hPS-TG mice treated with ethanol compared with ethanol-treated wild type (WT) mice. Cell expansion, activation and inhibition of apoptosis were significantly augmented in natural killer T (NKT) cells from hPS-TG mice compared with WT mice. Liver mononuclear cells from hPS-TG mice express higher levels of inflammatory cytokines than those from WT mice after stimulation with a specific stimulant of NKT cells in vitro. In a co-culture system of hepatocytes and NKT cells, the effects of PS on ethanol-mediated cell injury were suppressed by a CD1d neutralizing antibody. Alcoholic liver injury was significantly improved in mice pre-treated with PS siRNA and anti-protein S antibody compared with control mice. Patients with alcoholic hepatitis showed significantly increased plasma PS levels and enhanced liver expression of PS and CD1d compared with controls., Conclusions: The results of this study suggest that PS exacerbates acute alcoholic hepatitis by inhibiting apoptosis of activated NKT cells., (© 2014 International Society on Thrombosis and Haemostasis.)

Background: Apart from its role in the coagulation system, thrombin plays an important role in the inflammatory response through its protease-activated receptors (PARs). However, the role of thrombin in the immune response is not clear., Objective: To evaluate whether thrombin has a modulatory role in allergic bronchial asthma., Methods: Bronchial asthma was induced in mice by intraperitoneal sensitization and inhalation challenge with ovalbumin. Thrombin or its inhibitors were administered by inhalation before each allergen challenge., Results: Mice with low but sustained coagulation activation had reduced allergic inflammation, and allergic asthma was inhibited by low doses of thrombin but worsened by high doses. Allergic asthma was worsened by antithrombin, argatroban, hirudin, and anti-thrombomodulin antibody. Mice with a higher level of an inhibitor of both thrombin and activated protein C had worse disease. Heterozygous PAR-1 mice had less allergic inflammation, but PAR-1 agonist worsened it. Allergic bronchial inflammation was worsened in mice that received adoptive transfer of PAR-1 agonist-treated Th2 cells as compared with controls. Low levels of thrombin suppressed the maturation and secretion of cytokines in dendritic cells, but high levels enhanced this., Conclusions: The effects of thrombin on allergic asthma are dose-dependent, with detrimental effects at high doses and protective effects at low doses. These data demonstrate that thrombin modulates the outcome in allergic bronchial asthma., (© 2013 International Society on Thrombosis and Haemostasis.)

Background: Activated protein C (APC) can regulate immune and inflammatory responses and apoptosis. Protein C transgenic mice develop less diabetic nephropathy but whether exogenous administration of APC suppresses established diabetic nephropathy is unknown., Objectives: We investigated the therapeutic potential of APC in mice with streptozotocin-induced diabetic nephropathy., Methods: Diabetes was induced in unilaterally nephrectomized C57/Bl6 mice using intraperitoneal (i.p.) injection of streptozotocin. Four weeks later, the mice were treated with i.p. exogenous APC every other day for 1 month., Results: APC-treated mice had a significantly improved blood nitrogen urea-to-creatinine ratio, urine total protein to creatinine ratio and proteinuria, and had significantly less renal fibrosis as measured by the levels of collagen and hydroxyproline. The renal tissue concentration of monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF) and the RNA expression of platelet-derived growth factor (PDGF), transforming growth factor-β1 and connective tissue growth factor (CTGF) were significantly lower in APC-treated mice than in untreated animals. The percentage of apoptotic cells was reduced and the expression of podocin, nephrin and WT-1 in the glomeruli was significantly improved in mice treated with APC compared with untreated mice. The levels of coagulation markers were not affected by APC treatment., Conclusion: Exogenous APC improves renal function and mitigates pathological changes in mice with diabetic nephropathy by suppressing the expression of fibrogenic cytokines, growth factors and apoptosis, suggesting its potential usefulness for the therapy of this disease., (© 2012 International Society on Thrombosis and Haemostasis.)

Background: Activation of the complement system has been implicated in tumor growth. The antifibrinolytic protein, activated thrombin-activatable fibrinolysis inhibitor (TAFIa), can modulate the activation of the complement system by inactivating the anaphylatoxins C3a and C5a. The apolipoprotein-E (ApoE) genotype has been associated with carcinogenesis., Objective: The aim of this study was to evaluate whether TAFIa can affect the development of cancer in the ApoE-deficient mouse model., Methods: TAFI and ApoE double-knockout mice were generated. A group of mice was treated with the diabetogenic and carcinogenic compound streptozotocin (stz). Mice treated with saline, single knockout mice and wild-type (wt) mice served as controls., Results: Six months after treatment with stz, mice were sacrificed. Hepatic tumors were found in male double-knockout mice treated with stz but none was found in control animals that were not treated with stz or in single knockout of ApoE or wt animals. There was no significant difference in coagulation system activation between the groups of mice. The plasma concentrations of C5a, factor D and transforming growth factor-β1 were increased in TAFI/ApoE double-deficient mice treated with stz compared with the mice of the same genotype treated with saline., Conclusion: Apo-E deficiency alone was not associated with tumors but the lack of TAFI appears to make the mice permissive for tumor formation in ApoE mice., (© 2010 International Society on Thrombosis and Haemostasis.)

Summary: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a circulating zymogen that is activated physiologically by the thrombin/thrombomodulin complex to activated TAFI (TAFIa) which is a basic carboxypeptidase. Substrates include fibrin, leading to a reduction in rate of plasmin generation, and several proinflammatory mediators such as bradykinin, thrombin-cleaved osteopontin and complement factor C5a. TAFI-deficient mice have no phenotype without being challenged and TAFIa appears to play a limited role in physiological fibrinolysis in vivo. In several disease models, the TAFI-deficient mice have different outcomes from the wild type (WT), but whether the difference is beneficial or an exacerbation of the disease depends on the model. The consequences of TAFI deficiency include increased plasmin as a result of enhanced incorporation of plasminogen and tissue plasminogen activator into the fibrin clot, but also loss of its ability to degrade other substrates, with the resultant up-regulation of several proinflammatory mediators, including C5a. Criteria are recommended to demonstrate that a substrate is a physiological substrate of TAFIa.

Background: The fibrinolytic system has been implicated in the pathogenesis of pulmonary hypertension (PH). Thrombin-activatable fibrinolysis inhibitor (TAFI) inhibits fibrinolysis and therefore its absence would be expected to increase fibrinolysis and ameliorate PH., Objective: The objective of the present study was to evaluate the effect of TAFI deficiency on pulmonary hypertension in the mouse., Methods and Results: PH was induced in C57/Bl6 wild-type (WT) or TAFI-deficient (KO) mice by weekly subcutaneous treatment with 600 mg kg(-1) monocrotaline (MCT) for 8 weeks. PH was inferred from right heart hypertrophy measured using the ratio of right ventricle-to-left ventricle-plus-septum weight [RV/(LV+S)]. Pulmonary vascular remodeling was analyzed by morphometry. TAFI-deficient MCT-treated and wild-type MCT-treated mice suffered similar weight loss. TAFI-deficient MCT-treated mice had reduced levels of total protein and tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), transforming growth factor-beta (TGF-beta) and monocyte chemoattractant protein-1 (MCP-1) in bronchial alveolar lavage compared with wild-type MCT-treated mice. The ratio of RV to (LV+S) weight was significantly higher in WT/MCT than in KO/MCT mice. The pulmonary artery wall area and vascular stenosis were both greater in MCT-treated WT mice compared with MCT-treated TAFI-deficient mice., Conclusions: TAFI-deficient MCT-treated mice had less pulmonary hypertension, vascular remodeling and reduced levels of cytokines compared with MCT-treated WT animals, possibly as a result of reduced coagulation activation.

Objective: Protein S may exert an anticoagulant activity by enhancing the anticoagulant activity of activated protein C and/or by directly inhibiting the prothrombinase complex. Protein S itself may also directly regulate inflammatory responses and apoptosis. The role of protein S in acute lung injury (ALI) was unknown. This study evaluated the effect of protein S on ALI in the mouse., Methods: Animal ALI was induced in C57/BL6 mice by intratracheal instillation of lipopolysaccharide (LPS). Mice were treated with protein S or saline by intraperitoneal injection 1 h before LPS instillation., Results: Activated protein or protein S alone and combined activated protein C + protein S therapy decreased inflammatory markers and cytokines in mice with acute lung injury. In LPS-treated mice compared with controls ALI was induced as shown by significantly increased levels of total protein, tumor necrosis factor-alpha, interleukin-6 and monocyte chemoattractant protein-1 in the bronchoalveolar lavage fluid. Mice with ALI treated with protein S had significantly decreased concentrations of tumor necrosis factor-alpha and interleukin-6 in the lung compared with untreated animals. Thrombin-antithrombin III, a marker of the activity of the coagulation cascade, was unchanged. Protein S inhibited the expression of cytokines in vitro and increased activation of the Axl tyrosine kinase pathway in A549 epithelial cells., Conclusion: Protein S protects against LPS-induced ALI, possibly by directly inhibiting the local expression of inflammatory cytokines without affecting coagulation.

Background: C4b-binding protein (C4BP), a multimeric protein structurally composed of alpha chains (C4BPalpha) and a beta chain (C4BPbeta), regulates the anticoagulant activity of protein S (PS). Patients with sepsis have increased levels of plasma C4BP, which appears to be induced by interleukin (IL)-6. However, it is not fully understood how lipopolysaccharide (LPS) and IL-6 affect the plasma C4BP antigen level and C4BPalpha and C4BPbeta expression in hepatocytes., Objectives: To assess the effect of LPS and IL-6 on plasma C4BP, PS-C4BP complex levels, PS activity, and C4BP expression by rat liver in vivo and on C4BP expression by isolated rat hepatocytes in vitro., Results: Plasma C4BP antigen level transiently decreased from 2 to 12 h after LPS (2 mg kg(-1)) injection, and then it abruptly increased up to 24 h after LPS injection. Plasma C4BP antigen level increased until 8 h after IL-6 (10 microg kg(-1)) injection, and then gradually decreased up to 24 h after IL-6 injection. LPS significantly decreased the protein and mRNA expression of both C4BPalpha and C4BPbeta in rat hepatocytes, and this effect was inhibited by NFkappaB and MEK/ERK inhibitors. IL-6 mediated increase in C4BPbeta expression in rat hepatocytes, which leads to increased plasma PS-C4BP complex level and to decreased plasma PS activity, was inhibited by inhibition of STAT-3., Conclusion: LPS decreases both C4BPalpha and C4BPbeta expression via the NFkappaB and MEK/ERK pathways, whereas IL-6 specifically increases C4BPbeta expression via the STAT-3 pathway, causing an increase in plasma PS-C4BP complex, and thus decreasing the anticoagulant activity of PS.

Background: Thrombin-activatable fibrinolysis inhibitor (TAFI) has been reported to affect wound healing and fibrotic processes, but its role in renal tubulointerstitial fibrosis remains unknown., Objective: To study its potential role, we compared TAFI-deficient and wild-type mice for the degree of renal fibrosis caused by unilateral ureteral obstruction (UUO)., Methods: The grade of tubulointerstitial fibrosis, the activity of plasmin, MMP-2 and MMP-9 were evaluated on days 4 and 9 after UUO., Results: The renal content of hydroxyproline and the activity of plasmin, MMP-2 and MMP-9 were significantly increased in kidneys with UUO from TAFI-deficient mice compared with those from wild-type mice. These differences disappeared when animals with UUO from both groups were treated with the plasmin inhibitor tranexamic acid. The renal concentrations of fibrogenic cytokines were also significantly elevated in kidneys with UUO from TAFI-deficient mice compared with those from wild-type mice., Conclusion: The results of this study suggest that increased renal activity of plasmin in TAFI-deficient mice causes increased renal interstitial fibrosis in obstructive nephropathy.

Background: Hepatocyte growth factor (HGF) plays an important role in tissue repair and regeneration. HGF activator (HGFA), a factor XIIa-like serine protease, activates HGF precursor to HGF. The precursor of HGFA, proHGFA, is activated by thrombin generated at sites of tissue injury. It is known that protein C inhibitor (PCI), an inhibitor of activated protein C (APC), also inhibits thrombin-thrombomodulin (TM) complex., Objectives: In the present study we evaluated the effect of PCI on thrombin-catalyzed proHGFA activation in the presence of TM, and on HGFA activity., Results: PCI did not inhibit thrombin-TM-mediated proHGFA activation, but it directly inhibited activated HGFA by forming an enzyme inhibitor complex. The second-order rate constants (m(-1) min(-1)) of the reaction between HGFA and PCI in the presence or absence of heparin (10 U mL(-1)) were 4.3 x 10(6) and 4.0 x 10(6), respectively. The inhibition of HGFA by PCI resulted in a significant decrease of HGFA-catalyzed activation of HGF precursor. Exogenous HGFA added to normal human plasma formed a complex with plasma PCI, and this complex formation was competitively inhibited by APC in the presence of heparin, but very weakly in the absence of heparin. We also demonstrated using recombinant R362A-PCI that Arg362 residue of PCI is important for HGFA inhibition by PCI as judged from the three-dimensional structures constructed using docking models of PCI and HGFA or APC., Conclusion: These observations indicate that PCI is a potent inhibitor of activated HGFA, suggesting a novel function for PCI in the regulation of tissue repair and regeneration.

Background: Liver dysfunction caused by intrasinusoidal microthrombi is frequently observed in patients with cirrhosis after hepatectomy, but the mechanistic pathway remains unknown., Objective: In the present study, we evaluated the expression of protein S (PS) in hepatocytes and sinusoidal endothelial cells (SECs) from rats with dimethylnitrosoamine-induced cirrhosis before and after hepatectomy., Results: The plasma level of PS antigen was significantly decreased in cirrhotic rats as compared to control rats treated with vehicle. PS expression was significantly decreased in hepatocytes isolated from cirrhotic rats as compared to controls. In contrast, PS expression was significantly increased in SECs isolated from rats with cirrhosis as compared to controls. Interleukin-6 (IL-6) upregulated the expression of PS in hepatocytes, and tumor necrosis factor-alpha (TNF-alpha) decreased its expression in SECs from both cirrhotic and normal rats. The production of IL-6 and TNF-alpha by Kupffer cells and SECs was decreased in rats with cirrhosis as compared to controls. After hepatectomy, microthrombus formation was markedly enhanced in sinusoids from rats with cirrhosis, and the plasma levels of IL-6 and TNF-alpha were significantly increased in rats with cirrhosis as compared to controls. Furthermore, PS production in SECs was decreased, whereas that in hepatocytes was significantly increased in cirrhotic rats as compared to controls., Conclusions: These findings suggest that PS expression is differently regulated in hepatocytes and SECs of rats with cirrhosis before and after hepatectomy, that the expression of PS is regulated by locally released inflammatory cytokines, and that decreased expression of PS in SECs may cause liver microthrombus formation, which is frequently observed in patients with cirrhosis after hepatectomy.

Background: Protein C inhibitor (PCI) plays a role in multiple biological processes including fertilization, coagulation, fibrinolysis and kinin systems., Objectives: We hypothesized that PCI participates in the pathogenesis of pulmonary hypertension. To demonstrate this, we compared the development of pulmonary hypertension in mice overexpressing PCI in the lung with wild-type (WT) mice. Pulmonary hypertension was induced by s.c. injection of 600 mg kg-1 of monocrotaline weekly for 8 weeks., Results: Right ventricular arterial pressure was significantly increased in monocrotaline-treated WT mice compared with that in monocrotaline-treated transgenic mice. Bronchoalveolar lavage fluid (BALF) levels of thrombin-antithrombin complex, monocyte chemoattractant protein-1 and platelet-derived growth factor, and the plasma level of tumor necrosis factor-alpha were significantly increased in monocrotaline-treated WT mice as compared with monocrotaline-treated PCI transgenic mice. Increased level of PCI-thrombin complex was detected in BALF from monocrotaline-treated PCI transgenic mice as compared with saline-treated PCI transgenic mice., Conclusions: This study showed that increased expression of PCI in the lung is protective against monocrotaline-induced pulmonary hypertension, suggesting a potential beneficial effect of PCI for the therapy of this disease.

Background: The vitamin K-dependent protein S (PS), mainly synthesized in hepatocytes and endothelial cells, plays a critical role in the anticoagulant activity of plasma. The decreased plasma level of PS in sepsis is associated with thrombotic tendency, but the mechanism is unclear., Objectives: In the present study, we examined the effect of lipopolysaccharide (LPS) on PS expression in vivo in rat liver, and in vitro in isolated hepatocytes and sinusoidal endothelial cells (SECs) from normal rats., Results: LPS induced a progressive decrease of plasma PS antigen level up to 12 h with a slight recovery at 24 h, and a transient decrease of liver PS mRNA level at 4-8 h with a complete recovery at 24 h. In the in vitro studies, LPS decreased PS antigen and mRNA levels in both hepatocytes and SECs. After LPS treatment, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) transiently increased in plasma. IL-6 increased the protein expression of PS from hepatocytes, while TNF-alpha decreased it from SECs. LPS increased CD14 in hepatocytes and decreased it in SECs, but did not affect toll-like receptor-4 (TLR-4) expression in both cells. Antirat CD14 and antirat TLR-4 antibodies inhibited LPS-induced NFkappaB activation, and a NFkappaB inhibitor suppressed LPS-induced decreased PS expression in both cells. Furthermore, MEK inhibitor blocked LPS-induced decreased PS expression in both cells., Conclusions: These findings suggest that LPS-induced decreased PS expression in hepatocytes and SECs is mediated by MEK/ERK signaling and NFkappaB activation and that membrane-bound CD14 and TLR-4 are involved in this mechanism. These findings may explain in part the decreased level of plasma PS and thrombotic tendency in sepsis.

It has been previously demonstrated that activated protein C (APC) plays an important role in the inhibition of inflammation in the gastric mucosa from patients with Helicobacter pylori infection. However, the role of gastric epithelial cells in the anti-inflammatory activity of APC remains unknown. In the present study, we evaluated the anti-inflammatory activity of APC and the expression of thrombomodulin (TM) and endothelial protein C receptor (EPCR) in gastric epithelial cells. The gastric epithelial cell lines, MKN-1 and AGS, and gastric biopsy samples from patients with and without H. pylori infection were used in the experiments. Polymerase chain reaction showed that gastric epithelial cell lines express EPCR and TM. Flow cytometry analysis also showed EPCR expression in both cells. H. pylori infection significantly increased EPCR expression compared with non-infected cells. Similar results were observed in vivo when samples from patients with and without H. pylori infection were analyzed for EPCR protein expression. Significant TM activity was found on AGS and MKN-1 cells stimulated with LPS from Escherichia coli and VacA antigen. APC was able to significantly inhibit the secretion of MCP-1 and IL-1beta induced by H. pylori homogenate in AGS cells. APC also remarkably suppressed the mRNA expression and secretion of MCP-1 from AGS cells infected with H. pylori. These results demonstrated the expression of components of the protein C pathway on gastric epithelial cells and that APC may play a critical role in the protection against gastric mucosal inflammation.

In humans, protein C inhibitor (PCI) is expressed in various tissues and present in many body fluids including plasma and seminal fluid. In rodents, PCI is expressed in reproductive organs only and is absent in plasma. In this study, we characterized the tissue expression and physiological role of PCI in novel human PCI gene transgenic (TG) mice. Northern blot and immunohistochemical analyses demonstrated that human PCI is expressed in liver hepatocytes, renal epithelial cells as well as heart, brain and reproductive organs of the TG mice. This PCI tissue distribution is similar to that found in humans. PCI in plasma of TG mice showed the same immunological and functional properties as human plasma PCI. Next, we evaluated the effect of PCI on coagulation, inflammation and tissue damage in lipopolysaccharide-treated TG mice. The results suggested that PCI efficiently inhibits not only the anticoagulant and anti-inflammatory activities of exogenously injected human activated protein C (APC) but also that of endogenously produced APC in mice with endotoxemia. These findings suggest that PCI exerts a procoagulant and proinflammatory effect by inhibiting APC. We believe our results also show how useful these TG mice may be for assessing the therapeutic effect of human APC in vivo and for evaluating the role of PCI in human physiological and pathological conditions.