Protease Activated Receptor-1 Deficiency Research Articles

Tubulovascular protection from protease-activated receptor-1 depletion during AKI-to-CKD transition.

Thromboembolic events are prevalent in chronic kidney disease (CKD) patients due to increased thrombin generation leading to a hypercoagulable state. We previously demonstrated that inhibition of protease-activated receptor-1 (PAR-1) by vorapaxar reduces kidney fibrosis. We used an animal model of unilateral ischemia-reperfusion injury-induced CKD to explore the tubulovascular crosstalk mechanisms of PAR-1 in acute kidney injury (AKI)-to-CKD transition. During the early phase of AKI, PAR-1-deficient mice exhibited reduced kidney inflammation, vascular injury, and preserved endothelial integrity and capillary permeability. During the transition phase to CKD, PAR-1 deficiency preserved kidney function and diminished tubulointerstitial fibrosis via downregulated transforming growth factor-β/Smad signaling. Maladaptive repair in the microvasculature after AKI further exacerbated focal hypoxia with capillary rarefaction, which was rescued by stabilization of hypoxia-inducible factor and increased tubular vascular endothelial growth factor A in PAR-1-deficient mice. Chronic inflammation was also prevented with reduced kidney infiltration by both M1- and M2-polarized macrophages. In thrombin-induced human dermal microvascular endothelial cells (HDMECs), PAR-1 mediated vascular injury through activation of NF-κB and ERK MAPK pathways. Gene silencing of PAR-1 exerted microvascular protection via a tubulovascular crosstalk mechanism during hypoxia in HDMECs. Finally, pharmacologic blockade of PAR-1 with vorapaxar improved kidney morphology, promoted vascular regenerative capacity, and reduced inflammation and fibrosis depending on the time of initiation. Our findings elucidate a detrimental role of PAR-1 in vascular dysfunction and profibrotic responses upon tissue injury during AKI-to-CKD transition and provide an attractive therapeutic strategy for post-injury repair in AKI.

Protease-activated receptor 1 drives and maintains ductal cell fates in the premalignant pancreas and ductal adenocarcinoma.

Pancreatic acinar cells have high plasticity and can transdifferentiate into ductal‐like cells. This acinar‐to‐ductal metaplasia (ADM) contributes to tissue maintenance but may also contribute to the premalignant transformation that can eventually progress to pancreatic ductal adenocarcinoma (PDAC). Macrophages are key players in ADM, and macrophage‐secreted matrix metalloproteinase (MMP)‐9 induces ADM through yet unknown mechanisms. As we previously identified MMP9 as a novel agonist of protease‐activated receptor 1 (PAR1), a receptor that is known to orchestrate the cross‐talk between macrophages and tumor cells in PDAC, we here assessed the contribution of PAR1 to pancreatic cell fates. We found that genetic deficiency for PAR1 increases acinar gene expression programs in the healthy pancreas and that PAR1 deficiency limits ductal transdifferentiation in experimental systems for ADM. Moreover, PAR1 silencing in PDAC cells increases acinar marker expression. Changes in PDAC cell lines were associated with a downregulation of known Myc‐target genes, and Myc inhibition mimics PAR1 deficiency in enhancing acinar programs in healthy organoids and PDAC cells. Overall, we identify the PAR1‐Myc axis as a driver of ductal cell fates in premalignant pancreas and PDAC. Moreover, we show that cellular plasticity is not unique to acinar cells and that ductal regeneration into acinar‐like cells is possible even in the context of oncogenic KRAS activation.

Biased PAR-1 Signaling Regulates Thrombo-Inflammation in a Mouse Model of Sickle Cell Disease

Thrombin and activated protein C (APC) are serine proteases involved in coagulation and inflammatory responses. Protease activated receptor-1 (PAR-1) is a G-protein coupled receptor (GPCR) activated by proteolytic cleavage of the extracellular N-terminus. PAR-1 cleavage at R41 by thrombin or at R46 by APC creates unique tethered ligands that activate different signaling pathways. On endothelial cells, thrombin employs PAR-1 to promote endothelial barrier disruption, vascular leakage, and inflammation. In contrast, APC employs PAR-1 to stabilize endothelial barrier and provides anti-inflammatory and anti-apoptotic activities. These opposing effects of PAR-1 activation are a classic example of "GPCR biased signaling". We have previously demonstrated that PAR-1 deficiency in all hematopoietic cells reduces heme-induced vascular stasis in sickle mice. In contrast it does not affect the elevated plasma levels of IL-6 or thrombin-antithrombin (TAT) complexes observed in sickle cell disease (SCD) mice compared to non-SCD controls during steady state. In this study, we investigated the effects of thrombin- and APC-induced PAR-1 activation on thromboinflammation in the well-characterized Townes mouse model of SCD. Procoagulant and proinflammatory responses were analyzed in control (AA) and sickle (SS) mice at steady state (saline injection) and 5 hours after TNFα (2 µg/kg, i.p.) to mimic the pro-inflammatory milieu that occurs during painful crisis in SCD patients. As previously described, SS mice had elevated plasma levels of TAT compared to AA mice (8.6± 2.4 vs 3.5±0.7ng/mL; p<0.05), that were further enhanced in SS mice by TNFα injection (21.5 ± 4.2 ng/mL, p<0.001). A similar pattern was observed for plasma levels of IL-6 (AA: 2.4±2 vs SS: 18±7.8 ng/mL, p<0.05; SS+TNFα: 231 ± 32 ng/mL, p<0.0001 vs SS). Using these experimental conditions, we first investigated the role of endogenous APC in SCD. We used a rat anti-mouse protein C mAb (SPC-54), which inhibits the active site of APC, thereby blocking all its enzymatic activities, including PAR-1-dependent signaling and anticoagulant actions. SS mice were injected with anti-APC SPC54 (10 mg/kg) or control IgG antibodies. Eighteen hours later both groups of mice were injected with either saline or TNFα and plasma samples were collected 5 hours later. We TNFαSSdata suggest that endogenous APC has anticoagulant and anti-inflammatory roles for both the steady state and after TNFα challenge. To address the individual roles of thrombin and APC signaling on vascular inflammation in SCD, we used mice with point mutations in PAR-1 at R41 or R46. Mutation of R41 to Glu (R41Q) renders PAR-1 insensitive to thrombin while preserving APC-mediated cleavage at R46. Mutation of R46 to Glu (R46Q) ablates APC's cytoprotective effects. Bone marrow (BM) from SS mice was transplanted into PAR-1WT, PAR-1R41Q, or PAR-1R46Q mice to generate SSBM mice with either normal PAR-1 (SSBM/PAR-1WT) or the mutated form of this receptor on all non-hematopoietic cells (SSBM/PAR-1R41Q and SSBM/PAR-1R46Q). Four months after BM transplantation, plasma levels of TAT, IL-6, and HMGB1 were analyzed in all 3 groups at steady state or 5 hours after TNFα challenge. Neither the PAR-1R41Q nor PAR-1R46Q mutation on non-hematopoietic cells affected plasma levels of TAT, IL-6, or HMGB1 in SS mice at steady state. Interestingly, plasma levels of IL-6 were significantly elevated in SSBM/PAR-1R46Q mice and reduced in SSBM/PAR-1R41Q mice compared to SSBM/PAR-1WT mice after TNFα challenge, with a statistically significant difference between the two PAR-1 mutant groups. Furthermore plasma levels of TAT and HMGB1 were significantly reduced in SSBM/PAR-1R41Q but not changed in SSBM/PAR-1R46Q mice after TNFα challenge (Figure 2A-C). These data suggest that PAR-1 activation at R41 regulates TAT, IL-6, and HMGB1, whereas activation of R46 negatively regulates IL-6 in sickle mice during TNFα challenge. Our data imply that pharmacological approaches aiming to eliminate thrombin-dependent, while preserving APC-dependent PAR-1 activation, may be effective in attenuating thrombo-inflamatory complications associated with SCD. Studies evaluating the effects of R41Q and R46Q mutations on microvascular stasis in SS mice are currently ongoing. Disclosures No relevant conflicts of interest to declare.

Thrombin activation of PAR-1 contributes to microvascular stasis in mouse models of sickle cell disease

AbstractVaso-occlusive crisis (VOC) is the primary cause of morbidity and hospitalization in sickle cell disease (SCD); however, only 4 therapies (hydroxyurea, l-glutamine, crizanlizumab, and voxeletor) are currently approved in SCD. These agents limit the duration, severity, and frequency of crises. Activation of coagulation is a hallmark of SCD. Studies in animal models of SCD have shown that coagulation contributes to the chronic inflammation and end-organ damage associated with the disease; however, it is unknown whether coagulation directly contributes to the microvascular stasis that causes VOC. Herein, we demonstrate that inhibition of tissue factor (TF) and the downstream coagulation proteases factor Xa and thrombin significantly attenuates heme-induced microvascular stasis in mouse models of VOC. Pharmacologic inhibition of the principal thrombin receptor, protease activated receptor-1 (PAR-1), as well as deficiency of PAR-1 in all nonhematopoietic cells, also reduces stasis in sickle mice. PAR-1 deficiency was associated with reduced endothelial von Willebrand factor expression, which has been shown to mediate microvascular stasis. In addition, TF inhibition reduces lung vaso-occlusion in sickle mice mediated by arteriolar neutrophil-platelet microemboli. In sum, these results suggest that prophylactic anticoagulation might attenuate the incidence of VOC.

Recent Advances in the Understanding of Thrombosis.

Recent Advances in the Understanding of Thrombosis.

Inflammation and Tissue Damage Mediated By PAR-1 Expression Is Cell Type-Specific and Varies Depending on the Inflammatory Challenge

Protease activated receptor-1 (PAR-1) is a G protein-coupled receptor activated by cleavage of the extracellular domain, revealing an encrypted ligand. PAR-1 can be activated by several proteases, including thrombin and activated protein C, coupling hemostatic proteases to cell signaling. PAR-1 activation is a significant determinant of the inflammatory response in multiple settings. However, the fact that PAR-1 is expressed on many cell types makes a mechanistic understanding of the effector mechanisms challenging. We hypothesized that the effector cells expressing PAR-1 that drive inflammatory functions are contextually dependent on the type of inflammatory challenge. To test this hypothesis, we used recently-generated mice carrying a conditional (floxed) PAR-1 allele (PAR-1fl/fl) interbred with established transgenic mice expressing Cre recombinase in a cell type-specific manner in two experimental mouse models, an acetaminophen (APAP) hepatotoxicity model and a Citrobacter rodentium infectious colitis model. Cre recombinase was expressed under the control of the CMV promoter (global PAR-1 deletion), the Tie2 promoter (endothelial and hematopoietic cell PAR-1 deletion), and the LysM promoter (myeloid PAR-1 deletion). An established hepatocyte-tropic AAV Cre recombinase vector was used to delete hepatocyte-associated PAR-1.To define the cell type-specific role of PAR-1 in APAP induced liver damage, mice were challenged with APAP and euthanized 24 hours later for analyses. Control mice exposed to toxic APAP doses had a 10-100 fold increase in serum alanine transaminase (ALT) levels and ~50% hepatocyte necrosis based on morphometric analyses of liver tissue sections. Consistent with previous results, APAP-challenged mice with global PAR-1 deletion (CMV Cre/PAR-1fl/fl) demonstrated significantly lower serum ALT levels relative to control animals (PAR-1fl/fl) and significantly less hepatocyte necrosis. Neither AAV-Cre mediated deletion of PAR-1 in hepatocytes nor LysM Cre-mediated deletion in myeloid cells had any significant impact on APAP-induced liver injury. In contrast, deletion of PAR-1 in the endothelial and hematopoietic compartments (Tie2 Cre) phenocopied global PAR-1 deletion, resulting in a significant diminution in serum ALT and a 30% decrease in APAP-induced hepatocyte necrosis. Together, these results suggest that either a non-myeloid leukocyte and/or endothelial cells are the effector cells expressing PAR-1 that promote liver damage in this experimental context.To define the role of PAR-1 in the setting of infectious colitis, mice were challenged with C. rodentium by oral gavage and euthanized 14 days later for analyses. Global deletion of PAR-1 resulted in significantly less weight loss, longer colonic crypts, and a local diminution in multiple inflammatory cytokines (IFNγ, IL-1β, IL-17A, IL-22, and IL-6). In contrast to what was observed in APAP-induced liver injury, cell type-specific deletion of PAR-1 in myeloid cells (LysM Cre) resulted in a phenocopy of global PAR-1 deficiency, including blunted weight loss and longer colonic crypt lengths. The difference in colonic crypt length appeared to be due, at least in part, to genotype-dependent differences in colonic epithelial apoptosis, demonstrated by diminished staining for cleaved caspase 3 in crypts harvested from LysM Cre/PAR-1fl/fl mice relative to controls. Deletion of myeloid-associated PAR-1 also resulted in a local diminution in several cytokines (IFNγ, IL-1β, IL-17A, and IL-6) similar to global PAR-1 deficiency.Together, these results strengthen previous data showing the importance of PAR-1 in mediating multiple inflammatory pathologies. These data also highlight the finding that the specific effector cells expressing PAR-1 that promote inflammatory processes may differ depending on the type of inflammatory challenge. A detailed effector cell-specific understanding of the role of PAR-1 is critical to understanding the mechanisms coupling hemostatic proteases to inflammation and tissue injury and for defining the potential of PAR-1 as a target to mitigate these pathologies. DisclosuresPalumbo:Ionis Pharmaceuticals: Research Funding.

Protease‐activated receptor‐1 impedes prostate and intestinal tumor progression in mice

Essentials Protease activated receptor-1 (PAR-1) has been proposed to drive cancer progression. Surprisingly, PAR-1 deletion accelerated tumor progression in two distinct experimental settings. PAR-1 deletion was shown to limit the apoptosis of transformed epithelial cells. Thrombin- and activated protein C-mediated PAR-1 activation have unique effects on tumor cell biology. SUMMARY: Background Multiple studies have implicated protease-activated receptor-1 (PAR-1), a G-protein-coupled receptor activated by proteolytic cleavage of its N-terminus, as one target coupling thrombin-mediated proteolysis to tumor progression. Objective To analyze the role of PAR-1 in the setting of two distinct spontaneously developing tumor models in mice. Methods We interbred PAR-1-deficient mice with Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice, which spontaneously develop prostate tumors, and adenomatous polyposis coli Min (APCMin/+ ) mice, which spontaneously develop intestinal adenomas. Results Analyses of TRAMP mice with advanced disease (30 weeks) revealed that PAR-1 deficiency resulted in significantly larger and more aggressive prostate tumors. Prostates collected at an earlier time point (12 weeks of age) revealed that PAR-1 promotes apoptosis in transformed epithelia. In vitro analyses of TRAMP-derived cells revealed that activated protein C-mediated PAR-1 cleavage can induce tumor cell apoptosis, suggesting that tumor cell-intrinsic PAR-1 functions can limit tumor progression. Paralleling results in TRAMP mice, PAR-1-deficient APCMin/+ mice developed three-fold more adenomas than PAR-1-expressing mice, and the adenomas that formed were significantly larger. Moreover, loss of PAR-1 expression was shown to limit apoptosis in transformed intestinal epithelial cells. Conclusions Together, these results demonstrate a previously unrecognized role for PAR-1 in impeding tumor progression in vivo. These results also offer a cautionary note suggesting that long-term PAR-1 inhibition could increase malignancy risk in some contexts.

Protease activated receptor-1 signaling: its potential implication in HIV driven immune activation, inflammation/coagulation

Abstract Persistent immune activation remains the hallmark of HIV infection even in the context of successfully suppressed viremia. Its clinical significance is suggested by increased risk to develop non-AIDS defining illnesses such as cardiovascular disease. We previously have shown that human CD4 and CD8 T cells with effector memory and terminal effector memory phenotype expressed the protease activated receptor-1 (PAR1) and that expression was increased in CD8 T cells from HIV infected patients. Thrombin, in a PAR1 dependent fashion, enhanced CD8 T cell function measured by increased secretion of IFN-g and chemokinesis. PAR1 is the main receptor of thrombin and is also expressed on platelets and endothelial cells suggesting a cross-talk between the coagulation system and T cells at the site of vascular injury. To address the role of PAR1 in vivo, we used an infection model with the hepatotropic strain of lymphocytic choriomeningitis virus (LCMV-WE). To evaluate the role of PAR1 signaling in this model, we analyzed the T cells of littermate wild type control and PAR1−/−mice eight days following infection with LCMV-WE (2×105 pfu i.v.). In this model, we observed a similar expansion of total CD8 T cells in the spleens and livers of both WT and PAR1−/−mice at 8 days post infection. Despite similar expansion, PAR1 deficient mice showed impaired viral clearance at day 8 of infection. This lack of viral control was associated with a significant reduction in cytokine secretion by gp-33-specific CD8 T cells in the livers and spleens. These results suggest that PAR1 deficiency leads to an impair viral control that is in part mediated by a decrease ability to secrete cytokine by virus specific CD8 T cells.

Protease Activated Receptor-1 (PAR-1) Impedes Tumor Progression

Activation of cell signaling by thrombin through Protease Activated Receptor-1 (PAR-1) represents one important interface between blood coagulation and cell activation in response to injury and inflammation. In the context of cancer, PAR-1 has been suggested to promote tumor growth through mechanisms coupled to tumor cell proliferation, tumor cell migration, and the development of a supportive tumor stroma. Consistent with this view, both tumor cells and stromal cells express high levels of PAR-1, and elevated PAR-1 expression has been correlated with a poor prognosis across several tumor types. In the current studies, we tested the hypothesis that PAR-1 is a critical driver of tumorigenesis and tumor growth using murine models of genetically-induced prostate and intestinal tumor growth. To define the role of PAR-1 in prostate tumor progression, we interbred mice expressing the TRAMP transgene (transgenic adenocarcinoma of the mouse prostate; SV40 Large T antigen under the control of a probasin promoter) to PAR-1-deficient mice (PAR-1-/-) in order to generate male TRAMP mice with and without PAR-1 expression for detailed analyses of prostate tumor growth. Surprisingly, prostate tumors harvested from PAR-1-/- mice were significantly larger than those harvested from PAR-1+/+ mice. In order to begin to address the PAR-1 expressing cellular compartments responsible for prostate tumor inhibition, we subcutaneously inoculated immunocompetent C57Bl/6-derived PAR-1-/- and control mice with tumor cells derived from a C57Bl/6 TRAMP mouse. TRAMP-derived tumors grew indistinguishably in PAR-1-/- and control mice, suggesting that stromal-cell associated PAR-1 is dispensable for prostate tumor growth. We next tested the effect of tumor cell-intrinsic inhibition of PAR-1 in TRAMP tumor cells by viral transduction with a construct containing an shRNA against murine PAR-1 in parallel to a non-specific shRNA construct. Diminishing tumor cell-associated PAR-1 expression resulted in significantly more rapid tumor growth in vivo. In order to better define the role of tumor cell-intrinsic PAR-1 we harvested TRAMP tumor cells from a PAR-1 deficient mouse and grew these cells in vitro. We transduced these PAR-1-deficient prostate tumor cells with viral vectors conferring expression of WT murine PAR-1 (PAR-1+), a PAR-1 mutant lacking the thrombin cleavage (R41A mutant) or empty vector (PAR-1-). PAR-1- cells grew robustly and similarly to the parental cells in vitro with a doubling time of approximately 48 hours. Cells expressing the R41A mutant PAR-1 also grew robustly and similarly to PAR-1 deficient cells. However, PAR-1+ cells failed to show any signs of cell proliferation over the span of a 4 day observation period. Furthermore, PAR-1 expression dramatically altered the ability of TRAMP cells to demonstrate signs of cell spreading as measured by the frequency of pseudopodia per cell. As a means of determining the role of PAR-1 in tumorigenesis and tumor growth in another spontaneously occurring setting, we interbred PAR-1-/- mice with APCMin/+ mice genetically predisposed to intestinal adenoma formation due to loss of heterozygosity of the tumor suppressor adenomatous polyposis coli gene. Blinded quantitative histological analyses of the intestinal tracts of PAR-1-/- and PAR-1+/+ APCMin/+ mice revealed that PAR-1 deficiency resulted in a significant 2-fold increase in the number of adenomas observed. Furthermore, the adenomas observed in PAR-1-/- mice were significantly larger based on morphometric analyses of adenoma surface area in histological sections. In sum, these data demonstrate a surprising and unexpected role for PAR-1 in the inhibition of tumor growth in the context of two distinct tumor types. DisclosuresNo relevant conflicts of interest to declare.

Protease-Activated Receptor 1 Contributes to Angiotensin II-Induced Cardiovascular Remodeling and Inflammation

Background: Angiotensin II (Ang II) plays an important role in cardiovascular disease. It also leads to the activation of coagulation. The coagulation protease thrombin induces cellular responses by activating protease-activated receptor 1 (PAR-1). We investigated whether PAR-1 contributes to Ang II-induced cardiovascular remodeling and inflammation. Methods and Results: PAR-1^+/+ (wild-type; WT) and PAR-1^-/- mice were infused with Ang II (600 ng/kg/min) for up to 4 weeks. In WT mice, this dose of Ang II did not cause a significant increase in blood pressure but it did cause pathological changes in both the aorta and the heart. Ang II infusion resulted in vascular remodeling of the aorta, demonstrated by a significant increase in medial wall thickening and perivascular fibrosis. Importantly, both parameters were significantly attenuated by PAR-1 deficiency. Furthermore, perivascular fibrosis around coronary vessels was reduced in Ang II-treated PAR-1^-/- mice compared to WT mice. In addition, PAR-1 deficiency significantly attenuated Ang II induction of inflammatory cytokines and profibrotic genes in the aortas compared to WT mice. Finally, PAR-1 deficiency had no effect on Ang II-induced heart hypertrophy. However, the heart function measured by fractional shortening was less impaired in PAR-1^-/- mice than in WT mice. Conclusion: Our data indicate that PAR-1 plays a significant role in cardiovascular remodeling mediated by a blood pressure-independent action of Ang II.

Protease-activated receptor-1 inhibits proliferation but enhances leukemia stem cell activity in acute myeloid leukemia.

Eradication of leukemia stem cells (LSCs) is the ultimate goal of treating acute myeloid leukemia (AML). We recently showed that the combined loss of Runx1/Cbfb inhibited the development of MLL-AF9-induced AML. However, c-Kit+/Gr-1- cells remained viable in Runx1/Cbfb-deleted cells, indicating that suppressing RUNX activity may not eradicate the most immature LSCs. In this study, we found upregulation of several hemostasis-related genes, including the thrombin-activatable receptor PAR-1 (protease-activated receptor-1), in Runx1/Cbfb-deleted MLL-AF9 cells. Similar to the effect of Runx1/Cbfb deletion, PAR-1 overexpression induced CDKN1A/p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1 deficiency also prevented leukemia development induced by a small number of MLL-AF9 leukemia stem cells (LSCs) in vivo. PAR-1 deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose-dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there were a large number of LSCs, while a small number of LSCs required PAR-1 for their efficient growth. Mechanistically, PAR-1 increased the adherence properties of MLL-AF9 cells and promoted their engraftment to bone marrow. Taken together, these data revealed a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML.

From the Cover: Coagulation-Driven Hepatic Fibrosis Requires Protease Activated Receptor-1 (PAR-1) in a Mouse Model of TCDD-Elicited Steatohepatitis.

Emerging evidence supports a role for environmental chemical exposure in the pathology of non-alcoholic fatty liver disease (NAFLD), a disease process tightly linked to increased activity of the blood coagulation cascade. Exposure of C57BL/6 mice to the persistent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) recapitulates features of the NAFLD spectrum, including steatosis, hepatic injury, inflammation, and fibrosis. We assessed coagulation cascade activation, and determined the role of the thrombin receptor protease activated receptor-1 (PAR-1) in experimental TCDD-elicited NAFLD. Chronic exposure to TCDD (30 µg/kg every 4 days for 28 days) was associated with intrahepatic coagulation, indicated by increased plasma thrombin-antithrombin levels and hepatic fibrin(ogen) deposition. PAR-1 deficiency diminished TCDD-elicited body weight loss and relative liver weight was reduced in TCDD-exposed PAR-1-/- mice compared with TCDD-exposed wild-type mice. PAR-1 deficiency did not affect TCDD-induced hepatic steatosis or hepatocellular injury, as indicated by serum alanine aminotransferase activity. Despite a lack of effect on these 2 features of NAFLD pathology, PAR-1 deficiency was associated with a reduction in hepatic inflammation evident in liver histopathology, and reflected by a reduction in serum levels of the proinflammatory cytokine interleukin-6. Moreover, TCDD-driven hepatic collagen deposition was markedly reduced in PAR-1-deficient mice. These results indicate that experimental TCDD-elicited steatohepatitis is associated with coagulation cascade activation and PAR-1-driven hepatic inflammation and fibrosis.

The Tissue Factor/Thrombin/Protease-Activated Receptor 1 Pathway Enhances Double-Strand RNA Induced Immune Responses in Macrophages

Introduction: Co-regulation of the immune response and the coagulation cascade after infection is thought to be an ancient response to limit pathogen spread. Recently, we showed that activation of the thrombin receptor, protease-activated receptor 1 (PAR1), on fibroblasts enhanced the innate immune responses to RNA virus infection. Here, we investigated whether PAR1 activation by the extrinsic coagulation pathway contributes to dsRNA-induced innate immune responses in macrophages.Methods: Activation of the type-I interferon (IFN) pathway in the murine macrophage cell line RAW264.7 and bone-marrow derived macrophages (BMDM) from WT and PAR1-/- was analyzed after dsRNA (poly I:C) and/or PAR-1 stimulation. In addition, innate immune responses in the spleen were analyzed in vivo 4 hours after poly I:C (8mg/kg) injection in mice with reduced tissue factor expression (LowTF) or global PAR1 deletion (PAR1-/-) as well as in WT mice with a thrombin inhibitor (dabigatran etexilate, 10g/kg chow) or PAR-1 inhibitor (SCH79797, 25μg/kg). Lastly, we investigated the innate immune response in the spleen of WT and PAR1-/-mice after infection with the single-stranded RNA virus coxsackievirus B3.Results: RAW264.7 and BMDM exhibited a toll-like receptor 3 dependent induction of IFNβ and CXCL10 after poly I:C stimulation. Activation of PAR-1 with either thrombin or agonist peptide enhanced poly I:C induction of IFNβ and CXCL10. A deficiency of tissue factor levels, thrombin inhibition, PAR-1 inhibition or PAR1 deficiency resulted in reduced expression levels of type-I IFNs and IFN-response genes such as CXCL10 in the spleen and plasma in mice given poly I:C. Last, PAR1-/-mice exhibited impaired IFNβ immune response 4 days after coxsackievirus B3 infection compared to WT mice.Conclusion: Our study indicates that the coagulation dependent activation of PAR1 on macrophages is important for anti-viral responses to dsRNA. We speculate that PAR1 inhibition may interfere with anti-viral responses in humans. DisclosuresNo relevant conflicts of interest to declare.

PAR1 contributes to influenza A virus pathogenicity in mice

Influenza causes substantial morbidity and mortality, and highly pathogenic and drug-resistant strains are likely to emerge in the future. Protease-activated receptor 1 (PAR1) is a thrombin-activated receptor that contributes to inflammatory responses at mucosal surfaces. The role of PAR1 in pathogenesis of virus infections is unknown. Here, we demonstrate that PAR1 contributed to the deleterious inflammatory response after influenza virus infection in mice. Activating PAR1 by administering the agonist TFLLR-NH2 decreased survival and increased lung inflammation after influenza infection. Importantly, both administration of a PAR1 antagonist and PAR1 deficiency protected mice from infection with influenza A viruses (IAVs). Treatment with the PAR1 agonist did not alter survival of mice deficient in plasminogen (PLG), which suggests that PLG permits and/or interacts with a PAR1 function in this model. PAR1 antagonists are in human trials for other indications. Our findings suggest that PAR1 antagonism might be explored as a treatment for influenza, including that caused by highly pathogenic H5N1 and oseltamivir-resistant H1N1 viruses.

Abstract 18552: The Thrombin Inhibitor Dabigatran Inhibits High Fat Diet-induced Obesity and Fatty Liver Disease in Mice

Inflammation associated with non-alcoholic fatty liver disease (NALFD) increases the risk of adverse cardiovascular events. NAFLD is the hepatic manifestation of obesity and the metabolic syndrome and significantly contributes to liver-related morbidity. We have shown previously that the thrombin receptor protease activated receptor-1 (PAR-1) is essential for hepatic inflammation and steatosis in mice fed a high fat diet (HFD), but PAR-1 deficiency does not affect weight gain in mice fed a HFD. To determine whether pharmacologic inhibitors of thrombin could reduce the progression of NAFLD in mice on a HFD, we utilized the direct thrombin inhibitor, dabigatran etexilate. Wild type C57Bl/6 mice were fed a HFD formulated to contain dabigatran (10 mg/g chow) or an identical HFD with starch substituted for drug for 3 months. Plasma thrombin time was significantly prolonged in mice fed HFD+dabigatran, but no adverse bleeding events were observed. Despite consuming a similar amount of chow, mice fed HFD+dabigatran gained significantly less body weight compared to mice fed HFD alone. Induction of inflammatory genes including TNFα, MCP-1, and the lipogenic genes SCD-1 and CD36, was significantly reduced in livers of mice fed HFD+dabigatran compared to mice fed HFD alone. In agreement, hepatic steatosis was dramatically reduced by dabigatran in mice fed a HFD. In summary, pharmacologic inhibition of thrombin with dabigatran reduced various features of NAFLD in mice fed a HFD. In contrast to PAR-1-deficient mice, dabigatran administration also dramatically reduced diet-induced obesity. Taken together, the results indicate that thrombin contributes to NAFLD in mice fed a HFD, and that thrombin promotes diet-induced obesity in a PAR-1-independent manner. Clinical evaluation of the potential for thrombin inhibition to reduce systemic and hepatic inflammation in obese patients taking dabigatran for another indication, such as atrial fibrillation, could reveal novel beneficial effects of oral thrombin inhibitors in metabolic disease.

Protease-Activated Receptor 1 and Hematopoietic Cell Tissue Factor Are Required for Hepatic Steatosis in Mice Fed a Western Diet

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of obesity and metabolic syndrome and contributes to increased risk of cardiovascular disease and liver-related morbidity and mortality. Indeed, obese patients with metabolic syndrome generate greater amounts of thrombin, an indication of coagulation cascade activation. However, the role of the coagulation cascade in Western diet-induced NAFLD has not been investigated. Using an established mouse model of Western diet-induced NAFLD, we tested whether the thrombin receptor protease-activated receptor 1 (PAR-1) and hematopoietic cell-derived tissue factor (TF) contribute to hepatic steatosis. In association with hepatic steatosis, plasma thrombin-antithrombin levels and hepatic fibrin deposition increased significantly in C57Bl/6J mice fed a Western diet for 3 months. PAR-1 deficiency reduced hepatic inflammation, particularly monocyte chemoattractant protein-1 expression and macrophage accumulation. In addition, PAR-1 deficiency was associated with reduced steatosis in mice fed a Western diet, including reduced liver triglyceride accumulation and CD36 expression. Similar to PAR-1 deficiency, hematopoietic cell TF deficiency was associated with reduced inflammation and reduced steatosis in livers of low-density lipoprotein receptor-deficient mice fed a Western diet. Moreover, hematopoietic cell TF deficiency reduced hepatic fibrin deposition. These studies indicate that PAR-1 and hematopoietic cell TF are required for liver inflammation and steatosis in mice fed a Western diet.

The Coagulation System Contributes to αVβ6 Integrin Expression and Liver Fibrosis Induced by Cholestasis

Chronic injury to intrahepatic bile duct epithelial cells (BDECs) elicits expression of various mediators, including the αVβ6 integrin, promoting liver fibrosis. We tested the hypothesis that tissue factor (TF)-dependent thrombin generation and protease activated receptor-1 (PAR-1) activation contribute to liver fibrosis induced by cholestasis via induction of αVβ6 expression. To test this hypothesis, mice deficient in either TF or PAR-1 were fed a diet containing 0.025% α-naphthylisothiocyanate (ANIT), a BDEC-selective toxicant. In genetically modified mice with a 50% reduction in liver TF activity fed an ANIT diet, coagulation cascade activation and liver fibrosis were reduced. Similarly, liver fibrosis was significantly reduced in PAR-1(-/-) mice fed an ANIT diet. Hepatic integrin β6 mRNA induction, expression of αVβ6 protein by intrahepatic BDECs, and SMAD2 phosphorylation were reduced by TF deficiency and PAR-1 deficiency in mice fed the ANIT diet. Treatment with either an anti-αVβ6 blocking antibody or soluble transforming growth factor-β receptor type II reduced liver fibrosis in mice fed the ANIT diet. PAR-1 activation enhanced transforming growth factor-β1-induced integrin β6 mRNA expression in both transformed human BDECs and primary rat BDECs. Interestingly, TF and PAR-1 mRNA levels were increased in livers from patients with cholestatic liver disease. These results indicate that a TF-PAR-1 pathway contributes to liver fibrosis induced by chronic cholestasis by increasing expression of the αVβ6 integrin, an important regulator of transforming growth factor-β1 activation.

Protease-Activated Receptor-1 Contributes to Cardiac Remodeling and Hypertrophy

Protease-activated receptor-1 (PAR-1) is the high-affinity receptor for the coagulation protease thrombin. It is expressed by a variety of cell types in the heart, including cardiomyocytes and cardiac fibroblasts. We have shown that tissue factor (TF) and thrombin contribute to infarct size after cardiac ischemia-reperfusion (I/R) injury. Moreover, in vitro studies have shown that PAR-1 signaling induces hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. The purpose of the present study was to investigate the role of PAR-1 in infarction, cardiac remodeling, and hypertrophy after I/R injury. In addition, we analyzed the effect of overexpression of PAR-1 on cardiomyocytes. We found that PAR-1 deficiency reduced dilation of the left ventricle and reduced impairment of left ventricular function 2 weeks after I/R injury. Activation of ERK1/2 was increased in injured PAR-1(-/-) mice compared with wild-type mice; however, PAR-1 deficiency did not affect infarct size. Cardiomyocyte-specific overexpression of PAR-1 in mice induced eccentric hypertrophy (increased left ventricular dimension and normal left ventricular wall thickness) and dilated cardiomyopathy. Deletion of the TF gene in cardiomyocytes reduced the eccentric hypertrophy in mice overexpressing PAR-1. Our results demonstrate that PAR-1 contributes to cardiac remodeling and hypertrophy. Moreover, overexpression of PAR-1 on cardiomyocytes induced eccentric hypertrophy. Inhibition of PAR-1 after myocardial infarction may represent a novel therapy to reduce hypertrophy and heart failure in humans.

PAR-1 Deficiency Protects against Neuronal Damage and Neurologic Deficits after Unilateral Cerebral Hypoxia/Ischemia

Cardiovascular and neurologic surgeries often involve a temporary reduction in cerebral blood flow. In these conditions, as well as during cerebral ischemia and traumatic brain injury, the temporary loss of oxygen and glucose initiates a cascade of cellular events that culminate in neuronal death and damage. Understanding the mechanisms that contribute to neuronal death after hypoxia/ischemia is critically important for treatment of such brain injury. Here, we use a model of combined cerebral hypoxia/ischemia (H/I) to examine the role of protease-activated receptor-1 (PAR-1) in hypoxic/ischemic neuronal damage. Our data show that PAR-1-deficient mice have smaller lesion volumes than wild-type controls after 45 minutes of H/I. The results of the genetic block of PAR-1 were corroborated using a PAR-1 antagonist, which decreased infarct volume in wild-type C57Bl6 mice. Examination of cellular responses to H/I reveals that PAR-1 -/- animals have less cellular death and diminished glial fibrillary acidic protein expression. Additionally, PAR-1 -/- mice exhibit less motor behavior impairment in rotorod and inverted wire-hang tests. These data suggest that PAR-1 contributes to hypoxic/ischemic brain injury and are consistent with other studies that implicate serine proteases and their receptors in neuropathology after cerebral insults.

Sol Sherry lecture in thrombosis: how thrombin 'talks' to cells: molecular mechanisms and roles in vivo.

This article is a summary of the Sol Sherry Lecture of the Council on Arteriosclerosis, Thrombosis, and Vascular Biology, which was presented at the 70th Scientific Sessions of the American Heart Association in November 1997. It highlights work from our laboratory addressing the molecular mechanisms by which the coagulation protease thrombin elicits cellular responses, notes some of the novel issues that protease signaling raises, and cites recent work on the role of thrombin signaling in vivo. Thrombin is a multifunctional serine protease. In adult animals, active thrombin is generated in the context of vascular injury when activation of the coagulation cascade triggers conversion of the circulating zymogen prothrombin to active protease. Thrombin generation may also be important in other contexts, as will be shown below. Several of thrombin’s functions involve cleavage of circulating protein substrates, eg, conversion of fibrinogen to fibrin monomer or activation of protein C. However, thrombin also has important actions on cells. It is the most potent activator of platelets.1 It causes endothelial cells to deliver the leukocyte adhesion molecule P-selectin to their surfaces,2 to secrete von Willebrand factor,2 and to elaborate growth factors and cytokines.3 4 It is also a mitogen for fibroblasts and vascular smooth muscle cells.5 Such cellular actions of thrombin raised several important questions. How does thrombin, a protease, act like a hormone to control cellular behaviors? And what are the roles of thrombin-regulated cellular events in vivo? Thrombin’s actions on platelets are of particular interest. Arterial thrombosis underlies most cases of unstable angina and myocardial infarction. Studies in animal models and clinical trials suggest that these events are both platelet dependent and thrombin dependent, but the relative contributions of and interactions between thrombin-induced platelet activation and fibrin formation in acute coronary syndromes are not known. By elucidating …