Search

The chemotherapeutic drug doxorubicin is cardiotoxic and can cause irreversible heart failure. In addition to being cardiotoxic, doxorubicin also induces the activation of coagulation. We determined the effect of thrombin-mediated activation of protease-activated receptor 1 (PAR1) on doxorubicin-induced cardiac injury. Administration of doxorubicin to mice resulted in a significant increase in plasma prothrombin fragment 1+2, thrombin-antithrombin complexes, and extracellular vesicle tissue factor activity. Doxorubicin-treated mice expressing low levels of tissue factor, but not factor XII-deficient mice, had reduced plasma thrombin-antithrombin complexes compared to controls. To evaluate the role of thrombin-mediated activation of PAR1, transgenic mice insensitive to thrombin (Par1R41Q) or activated protein C (Par1R46Q) were subjected to acute and chronic models of doxorubicin-induced cardiac injury and compared with Par1 wild-type (Par1+/+) and PAR1 deficient (Par1-/-) mice. Par1R41Q and Par1-/- mice, but not Par1R46Q mice, demonstrated similar reductions in the cardiac injury marker cardiac troponin I, preserved cardiac function, and reduced cardiac fibrosis compared to Par1+/+ controls after administration of doxorubicin. Furthermore, inhibition of Gαq signaling downstream of PAR1 with the small molecule inhibitor Q94 significantly preserved cardiac function in Par1+/+ mice, but not in Par1R41Q mice subjected to the acute model of cardiac injury when compared to vehicle controls. In addition, mice with PAR1 deleted in either cardiomyocytes or cardiac fibroblasts demonstrated reduced cardiac injury compared to controls. Taken together, these data suggest that thrombin-mediated activation of PAR1 contributes to doxorubicin-induced cardiac injury.

Protease-activated receptor 1 (PAR1) is widely expressed in humans and mice, and is activated by a variety of proteases, including thrombin. Recently, we showed that PAR1 contributes to the innate immune response to viral infection. Mice with a global deficiency of PAR1 expressed lower levels of CXCL10 and had increased Coxsackievirus B3 (CVB3)-induced myocarditis compared with control mice. In this study, we determined the effect of cell type-specific deletion of PAR1 in cardiac myocytes (CMs) and cardiac fibroblasts (CFs) on CVB3-induced myocarditis. Mice lacking PAR1 in either CMs or CFs exhibited increased CVB3 genomes, inflammatory infiltrates, macrophages and inflammatory mediators in the heart and increased CVB3-induced myocarditis compared with wild-type controls. Interestingly, PAR1 enhanced poly I:C induction of CXCL10 in rat CFs but not in rat neonatal CMs. Importantly, activation of PAR1 reduced CVB3 replication in murine embryonic fibroblasts and murine embryonic cardiac myocytes. In addition, we showed that PAR1 reduced autophagy in murine embryonic fibroblasts and rat H9c2 cells, which may explain how PAR1 reduces CVB3 replication. These data suggest that PAR1 on CFs protects against CVB3-induced myocarditis by enhancing the anti-viral response whereas PAR1 on both CMs and fibroblasts inhibits viral replication.

Background Protease‐activated receptor 1 (PAR1) is expressed in various immune cells and in the lung. We showed that PAR1 plays a role in Coxsackievirus B3 infection by enhancing toll‐like receptor 3‐dependent interferon‐β expression in cardiac fibroblasts. Objectives We investigated the role of PAR1 in a mouse model of influenza A virus (IAV) infection. Methods We used mice with either a global deficiency of PAR1, cell type‐specific deficiencies of PAR1, or mutation of PAR1 at the R41 or R46 cleavage sites. Results PAR1‐deficient mice had increased CXCL1 expression in the lung, increased neutrophil recruitment, increased protein levels in the bronchoalveolar lavage fluid, and increased mortality after IAV infection compared with control mice infected with IAV. Results from mice with cell type‐specific deletion of PAR1 indicated that PAR1 expression by hematopoietic cells suppressed CXCL1 expression, whereas PAR1 expression by endothelial cells enhanced CXCL1 expression in response to IAV infection. PAR1 activation also enhanced polyinosinic:polycytodylic acid induction of interleukin‐8 in a human endothelial cell line. Mutation of the R46 cleavage site of PAR1 was associated with increased CXCL1 expression in the lung in response to IAV infection, which suggested that R46 signaling suppresses CXCL1 expression. Conclusions These results indicate that PAR1 expression by different cell types and activation by different proteases modulates the immune response during IAV infection.

Proteinase-activated receptor 2 (PAR2) was shown to enhance proinflammatory toll-like receptor 3 (TLR3) responses, while dampening TLR3-dependent antiviral response in epithelial cells. Moreover, Par2 -/- mice exhibited improved survival after influenza A virus (IAV) infection. The objective of the study is to identify PAR2 expressing cells contributing to IAV pathology, and test if PAR2 inhibition is a potential new target to treat IAV. Infection with H1N1 IAV (PR8) was analyzed in global ( Par2 -/- ), myeloid ( Par2 fl/fl ;LysM Cre+ ), neutrophil ( Par2 fl/fl ;MRP8 Cre+ ) and lung epithelial cell (EpC) Par2 deficient ( Par2 fl/fl ;SPC Cre+ ) mice and respective controls ( Par2 +/+ and Par2 fl/fl ). The effect of PAR2 activation on poly I:C activation of TLR3 was analyzed in bone marrow-derived macrophages (BMDM) and murine macrophage-like J774A.1 cells. Lastly, we analyzed PAR2 inhibition in wild-type (WT) mice using mouse-anti PAR2 antibody (SAM11) and small molecule PAR2 inhibitor ENMD-1068. Par2 -/- and Par2 fl/fl ;LysM Cre+ mice exhibited increased survival, reduced proinflammatory mediators and cellular infiltration in bronchoalveolar lavage fluid (BALF) 3 days post infection (dpi) compared to control mice. Reduced IAV pathology resulted in significantly better lung function (PenH, airway resistance) in Par2 -/- mice compared to Par2 +/+ mice 7 dpi. Neither Par2 fl/fl ;MRP8 Cre+ or Par2 fl/fl ;SPC Cre+ mice showed any survival benefit compared to Par2 fl/fl . In vitro studies showed that Par2 -/- BMDM produced less IL6 and IL12p40 than Par2 +/+ BMDM after poly I:C stimulation. J774A.1 cells stimulated with PAR2 agonist and poly I:C showed an increase in NFκB activation compared to poly I:C alone. Prophylactic PAR2 inhibition with SAM11 before and daily after IAV infection, decreased BALF inflammation and lung virus load compared to control IgG treated mice 3dpi. Therapeutic PAR2 inhibition with ENMD-1068 daily from 3 dpi resulted in improved lung function (PenH) compared to vehicle treated mice 7 dpi. Global or macrophage cell but not neutrophil or lung EpC Par2 deficiency was associated with reduced IAV-induced lung pathology and mortality. Additionally, PAR2 inhibition experiments showed PAR2 may be a therapeutic target to reduce IAV pathology.

The influenza A virus (IAV) causes a seasonal respiratory tract infection and is characterized by excessive inflammation and tissue pathology in the lungs. Protease-activated receptor 4 (PAR4) is expressed megakaryocytes/platelets, immune cells, and lung epithelial cells and it is the only functional thrombin receptor on murine platelets. Studies showed that platelets contribute to the pathogenesis of IAV. However, the mechanisms are unknown. While platelet inhibition in IAV infection was protective, a global PAR4 deficiency in mice was associated with increased IAV pathology including mortality. The objective of this study is to investigate the contribution of platelet PAR4 to IAV pathology. Mice (both sex) with PAR4 deficiency on megakaryocytes/platelets (PAR4 fl/fl ;PF4 Cre+ ) and controls (PAR4 fl/fl ) were infected with mouse adapted IAV (H1N1/PR8) at a dose, which induces 20% mortality in control mice. Inflammatory mediators and cellular inflammation in bronchoalveolar lavage fluid (BALF) was analyzed before, 3, 5 and 7 days post infection (dpi). Body weight changes were analyzed over 14 dpi and survival calculated with a human endpoint of a body weight loss of ≥25% initial weight. Lastly, lung function was analyzed by noninvasive whole-body plethysmography at 0 and 7 dpi. There were no significant differences in the analyzed inflammatory mediators, except for IFNγ, IL6, and protein, or any immune cell numbers in the BALF of both genotypes at any time point after infection. Both genotypes exhibited similar body weight changes over the course of infection which resulted that mice of both genotypes exhibited a mortality of approx. 20%. However, the lung function parameters calculated as PenH (airway resistance) and EF50 (flow rate at 50% expired volume) were significant higher in PAR4 fl/fl ;PF4 Cre+ compared to PAR4 fl/fl mice 7 dpi. Platelet PAR4 does not influence pathology in a mild IAV infection model. However, the more severe pulmonary dysfunction in PAR4 fl/fl ;PF4 Cre+ compared to PAR4 fl/fl mice suggest a protective role of platelet PAR4 in maintaining lung function during IAV infection. More studies are needed to investigate if the contribution of platelet PAR4 to IAV pathology is dependent on disease severity as recently suggested.

We report the unique case of a patient who recovered cardiac function despite a history of doxorubicin-induced cardiomyopathy, chest radiation therapy, high dose chemotherapy post-allogeneic stem cell transplant, and triplet pregnancy. Data are sparse on doxorubicin-induced cardiomyopathy in pregnant patients, calling for further studies to help formulate management or surveillance recommendations. (Level of Difficulty: Advanced.), We report the unique case of a patient who recovered cardiac function despite a history of doxorubicin-induced cardiomyopathy, chest radiation therapy…

BackgroundInnate immune responses to influenza A virus (IAV) infection are initiated in part by toll-like receptor 3 (TLR3). TLR3-dependent signaling induces an antiviral immune response and an NFκB-dependent inflammatory response. Protease-activated receptor 2 (PAR2) inhibits the antiviral response and enhances the inflammatory response. PAR2 deficiency protected mice during IAV infection. However, the PAR2 expressing cell-types contributing to IAV pathology in mice and the mechanism by which PAR2 contributes to IAV infection is unknown.MethodsIAV infection was analyzed in global (Par2-/-), myeloid (Par2fl/fl;LysMCre+) and lung epithelial cell (EpC) Par2 deficient (Par2fl/fl;SPCCre+) mice and their respective controls (Par2+/+ and Par2fl/fl). In addition, the effect of PAR2 activation on polyinosinic-polycytidylic acid (poly I:C) activation of TLR3 was analyzed in bone marrow-derived macrophages (BMDM). Lastly, we determined the effect of PAR2 inhibition in wild-type (WT) mice.ResultsAfter IAV infection, Par2-/- and mice with myeloid Par2 deficiency exhibited increased survival compared to infected controls. The improved survival was associated with reduced proinflammatory mediators and reduced cellular infiltration in bronchoalveolar lavage fluid (BALF) of Par2-/- and Par2fl/fl;LysMCre+ 3 days post infection (dpi) compared to infected control mice. Interestingly, Par2fl/fl;SPCCre+ mice showed no survival benefit compared to Par2fl/fl. In vitro studies showed that Par2-/- BMDM produced less IL6 and IL12p40 than Par2+/+ BMDM after poly I:C stimulation. In addition, activation of PAR2 on Par2+/+ BMDM increased poly I:C induction of IL6 and IL12p40 compared to poly I:C stimulation alone. Importantly, PAR2 inhibition prior to IAV infection protect WT mice.ConclusionGlobal Par2 or myeloid cell but not lung EpC Par2 deficiency was associated with reduced BALF inflammatory markers and reduced IAV-induced mortality. Our study suggests that PAR2 may be a therapeutic target to reduce IAV pathology.

BACKGROUND Protease-activated receptor 4 (PAR4) is expressed by a wide variety of cells, including megakaryocytes/platelets, immune cells, cardiomyocytes, and lung epithelial cells. It is the only functional thrombin receptor on murine platelets. A global deficiency of PAR4 is associated with impaired hemostasis and reduced thrombosis. OBJECTIVE We aimed to generate a mouse line with a megakaryocyte/platelet-specific deletion of PAR4 (PAR4fl/fl ;PF4Cre+ ) and use the mouse line to investigate the role of platelet PAR4 in hemostasis and thrombosis in mice. METHODS Platelets from PAR4fl/fl ;PF4Cre+ were characterized in vitro. Arterial and venous thrombosis was analyzed. Hemostatic plug formation was analyzed using a saphenous vein laser injury model in mice with global or megakaryocyte/platelet-specific deletion of PAR4 or wild-type mice treated with thrombin or glycoprotein VI (GPVI) inhibitors. RESULTS PAR4fl/fl ;PF4Cre+ platelets were unresponsive to thrombin or specific PAR4 stimulation but not to other agonists. PAR4-/- and PAR4fl/fl ;PF4Cre+ mice both exhibited a similar reduction in arterial thrombosis compared to their respective controls. More importantly, we show for the first time that platelet PAR4 is critical for venous thrombosis in mice. In addition, PAR4-/- mice and PAR4fl/fl ;PF4Cre+ mice exhibited a similar impairment in hemostatic plug stability in a saphenous vein laser injury model. Inhibition of thrombin in wild-type mice gave a similar phenotype. Combined PAR4 deficiency on platelets with GPVI inhibition did not impair hemostatic plug formation but further reduced plug stability. CONCLUSION We generated a novel PAR4fl/fl ;PF4Cre+ mouse line. We used this mouse line to show that PAR4 signaling in platelets is critical for arterial and venous thrombosis and hemostatic plug stability.

Introduction: Protease-activated receptor 4 (PAR4) is expressed by a wide variety of cells, including megakaryocytes/platelets, immune cells, cardiomyocytes and lung epithelial cells, and activated by multiple ligands including thrombin and cathepsin G. Importantly, PAR4 is the only functional thrombin receptor on murine platelets. A global deficiency of PAR4 is associated with impaired hemostasis and protection from thrombosis which are attributed to loss of platelet PAR4, but this has not been specifically demonstrated in mice. Additionally, global PAR4 deficiency increases mortality after influenza A virus infection, but the cell type/s responsible for the enhanced mortality have not been determined. Here, we describe the generation of PAR4 floxed (PAR4 fl/fl) mice that can be used to delete PAR4 in a cell type-specific manner, and examine the effect of megakaryocyte/platelet-specific deletion of PAR4 on hemostasis, thrombosis and viral infection using PAR4 fl/fl;PF4Cre + mice. Methods: PAR4global knockout (PAR4 -/-), MK/platelet-specific knockout (PAR4 fl/fl;PF4Cre +) and appropriate littermate control mice were used for experiments. Platelet function was determined by light transmission aggregometry and flow cytometry. Hemostasis was assessed in the saphenous vein laser injury model. Platelet plug formation was visualized by intravital microscopy following saphenous vein laser ablation (~50 μm diameter injury), followed by 2 subsequent ablations to reinjure the same site unless on-going bleeding was occurring. Mice were treated with ibrutinib (12.5 mg/kg) to inhibit GPVI signaling, or dabigatran etexilate (chow containing 10 mg/g) or recombinant hirudin (50 mg/kg) to inhibit thrombin activity. Thrombosis was assessed in the carotid artery FeCl 3 model. The carotid artery was exposed and 8% FeCl 3 applied for 3 mins. Blood flow was observed for 30 mins and occlusion was defined as no blood flow for 2 mins. To study susceptibility to viral infection, mice were challenged intranasally with a mouse-adapted H1N1 influenza A virus (H1N1 IAV PR8; 0.02 hemagglutination assay units), which induces mortality in 20% of WT mice. Mortality was defined as body weight loss greater than 25%, which required euthanasia. Results: As expected, PAR4 fl/fl;PF4Cre + platelets were unresponsive to thrombin or PAR4-specific stimulation, while the response to other agonists was retained. In the saphenous vein laser injury hemostasis model, PAR4 fl/fl;PF4Cre + mice were able to rapidly form a hemostatic platelet plug, but the majority of plugs (7/8) were unstable and re-opened after several minutes, leading to severely prolonged total bleeding times. We observed similar findings in global PAR4 -/- mice with 8/12 plugs re-opening. To investigate the mechanism mediating initial platelet plug formation, we inhibited GPVI signaling in PAR4 fl/fl;PF4Cre + mice using the Btk inhibitor ibrutinib. Ibrutinib administration shortened time to plug re-opening in PAR4 fl/fl;PF4Cre + mice but plugs were still able to form, which is likely mediated by GPIbα/VWF. We observed a similar phenotype to PAR4 fl/fl;PF4Cre + mice in mice treated with the direct thrombin inhibitor hirudin, suggesting thrombin is the primary activator of PAR4 during hemostatic plug formation. In the FeCl 3-induced carotid artery thrombosis model, both PAR4 fl/fl;PF4Cre + and PAR4 -/- mice were significantly protected compared to controls. Finally, when challenged with the mouse-adapted H1N1 IAV PR8, PAR4 fl/fl;PF4Cre + mice demonstrated similar body weight loss and survival as littermate controls. Conclusions: Our results in mice demonstrate that 1) platelet PAR4 is not required for initial hemostatic plug formation but is necessary for maintaining hemostatic plug stability, 2) loss of platelet PAR4 protects from arterial thrombosis, and 3) platelet PAR4 does not alter the course of H1N1 IAV infection, at least at the virus dose used in this study. In summary, we generated a novel mouse line carrying a floxed PAR4 allele which can be used to investigate cell-specific roles of PAR4 in disease. Disclosures No relevant conflicts of interest to declare.

Doxorubicin is an important but cardiotoxic chemotherapy used to treat and/or cure various malignancies. There are no guidelines for management of doxorubicin-induced cardiotoxicity in pregnant patients due to minimal data in this population. A 31 year old female with a history of nasal NK/T cell