ABSTRACT:Pharmacological plasminogen reduction enhanced liver regeneration experimentally and clinically. Small interfering RNA-induced plasminogen deficiency promoted hepatocyte proliferation after partial hepatectomy in mice, contrasting genetic deficiency models. In the HeLiX trial, tranexamic acid reduced posthepatectomy liver failure odds, suggesting a novel therapeutic strategy.

Background: Protease-activated receptor-1 (PAR1) has emerged as an important link between coagulation and the complications of obesity including metabolic dysfunction- associated steatotic liver disease (MASLD). PAR1 is expressed by various cells and cleaved by different proteases to generate unique tethered agonists that activate distinct signaling pathways. Mice expressing PAR1 with an R41Q mutation have disabled canonical thrombin-mediated signaling, whereas R46Q mice express PAR1 resistant to noncanonical signaling by activated protein C. Methods: Mice with whole body and hepatocyte-selective PAR1 deficiency as well as PAR1 R41Q and R46Q mice were fed a high-fat diet (HFD) to induce MASLD. Results: HFD-fed R41Q mice displayed reduced hepatic steatosis and liver/body weight ratio. In contrast, HFD-fed R46Q mice displayed increased relative liver weight and hepatic steatosis alongside increased serum alanine aminotransferase activity. Surprisingly, despite the distinct impact of PAR1 mutations on steatosis, selective deletion of PAR1 in hepatocytes had no impact. To evaluate a viable PAR1-targeted approach, mice with HFD-induced obesity were treated with the allosteric PAR1 modulator NRD21, which inhibits canonical PAR1 inflammatory signaling but promotes PAR1 protective, noncanonical anti-inflammatory signaling. NRD-21 treatment reduced plasma tumor necrosis factor-alpha, serum alanine aminotransferase activity, hepatic steatosis, and insulin resistance (Homeostatic Model Assessment for Insulin Resistance) but increased plasma active glucagon-like peptide-1. Conclusion: The results suggest that nonhepatocellular canonical PAR1 cleavage drives MASLD in obese mice and provide translational proof-of-concept that selective pharmacologic modulation of PAR1 yields multiple metabolic benefits in experimental obesity.

Introduction Roctavian (AAV5-HLP-hFVIII-SQ) is an approved gene therapy in the EU for the treatment of severe hemophilia A. A single infusion provides therapeutic levels of FVIII expression in adult men. The precise mechanisms contributing to variability and durability in transgene expression are unknown. Multiple lines of evidence suggest low RNA production contributes to the decline of FVIII expression and low-response to AAV gene therapy. Previously, we have shown that decreased interaction of active histones with episomal genomes may mediate the decline in transgene expression. Additional studies suggested hepatocyte capacity to fold and secrete FVIII may contribute to variability. We hypothesize (1) modifying the chromatin interaction with AAV-episomes using epigenic regulators may increase accessibility of vector genomes and (2) the use of molecular chaperones may improve FVIII folding and secretion.

Background:Acetaminophen (APAP) overdose is a leading cause of drug-induced acute liver failure (ALF). Neutrophil activation has been associated with poor outcomes in patients with ALF and is proposed to amplify coagulation in this context. However, the precise role of neutrophils in APAP-induced liver injury is not known.Methods:We used a dual antibody-mediated neutrophil depletion strategy to determine the role of neutrophils in mice challenged with different doses of APAP (300 or 600 mg/kg) that produce hepatotoxicity and ALF-like pathology.Results:Flow cytometry confirmed depletion of neutrophils in whole blood prior to APAP challenge. Mice given isotype control and challenged with 300 mg/kg APAP developed marked hepatocellular necrosis and showed an increase in biomarkers of coagulation cascade activation. Neutrophil depletion (anti-Ly6G) did not affect either liver injury or coagulation activation in mice challenged with 300 mg/kg APAP. Mice given isotype control and challenged with 600 mg/kg APAP developed hepatic necrosis alongside marked hemorrhage and congestion indicative of vascular injury. Interestingly, hepatic neutrophil and platelet accumulation were increased in mice given 600 mg/kg APAP compared with those given the lower APAP dose. Neutrophil depletion significantly reduced the severity of liver necrosis in mice challenged with 600 mg/kg APAP, without significantly impacting biomarkers of coagulation activity. Notably, neutrophil depletion significantly reduced hepatic platelet accumulation in mice challenged with 600 mg/kg APAP.Conclusion:The results indicate a role of neutrophils in APAP-induced liver injury that is dependent on the APAP dose and suggest involvement of neutrophil-platelet interactions in promoting hepatic injury in experimental APAP-induced ALF.

Background: Hemophilia A is a recessive bleeding disorders with factor VIII (FVIII) deficiency.Half of the patients have severe hemophilia with <1% clotting factor activity, but variation of bleeding severity exists

Lipids contribute to hematopoiesis and membrane properties and dynamics; however, little is known about the role of lipids in megakaryopoiesis. Here we show that megakaryocyte progenitors, megakaryocytes and platelets present a unique lipidome progressively enriched in polyunsaturated fatty acid (PUFA)-containing phospholipids. In vitro, inhibition of both exogenous fatty acid functionalization and uptake as well as de novo lipogenesis impaired megakaryocyte differentiation and proplatelet production. In vivo, mice on a high saturated fatty acid diet had significantly lower platelet counts, which was prevented by eating a PUFA-enriched diet. Fatty acid uptake was largely dependent on CD36, and its deletion in mice resulted in low platelets. Moreover, patients with a CD36 loss-of-function mutation exhibited thrombocytopenia and increased bleeding. Our results suggest that fatty acid uptake and regulation is essential for megakaryocyte maturation and platelet production and that changes in dietary fatty acids may be a viable target to modulate platelet counts.

BACKGROUND:Hepatic deposition of cross-linked fibrin(ogen) occurs alongside platelet accumulation as a hallmark of acetaminophen (APAP)-induced liver injury.OBJECTIVES:We sought to define the precise role of the fibrinogen γ-chain C-terminal integrin αIIbβ3 binding domain in APAP-induced liver injury.METHODS:Mice expressing mutant fibrinogen incapable of engaging integrin αIIbβ3 due to a C-terminal fibrinogen γ-chain truncation (mutant fibrinogen-γΔ5 [FibγΔ5] mice) and wild-type mice were challenged with APAP (300 mg/kg, intraperitoneally).RESULTS:We observed an altered pattern of fibrin(ogen) deposition in the livers of APAP-challenged FibγΔ5 mice. This led to the unexpected discovery that fibrinogen γ-chain cross-linking was altered in the livers of APAP-challenged FibγΔ5 mice compared with that in wild-type mice, including absence of γ-γ dimer and accumulation of larger molecular weight cross-linked γ-chain complexes. This finding was not unique to the injured liver because activation of coagulation did not produce γ-γ dimer in plasma from FibγΔ5 mice or purified FibγΔ5 fibrinogen. Sanger sequencing predicted that the fibrinogen-γΔ5 γ-polypeptide would terminate at lysine residue 406, but liquid chromatography tandem mass spectrometry analysis revealed that this critical lysine residue was absent in purified fibrinogen-γΔ5 protein. Interestingly, hepatic deposition of this uniquely aberrantly cross-linked fibrin(ogen) in FibγΔ5 mice was associated with exacerbated hepatic injury, an effect not recapitulated by pharmacologic inhibition of integrin αIIbβ3.CONCLUSION:The results indicate that fibrinogen-γΔ5 lacks critical residues essential to form γ-γ dimer in response to thrombin and suggest that hepatic accumulation of abnormally cross-linked fibrin(ogen) can exacerbate hepatic injury.

BACKGROUND:Patients with acetaminophen (APAP)-induced acute liver failure (ALF) display both hyper- and hypocoagulable changes not necessarily recapitulated by standard hepatotoxic doses of APAP used in mice (eg, 300 mg/kg). OBJECTIVES:We sought to examine coagulation activation in vivo and plasma coagulation potential ex vivo in experimental settings of APAP-induced hepatotoxicity and repair (300-450 mg/kg) and APAP-induced ALF (600 mg/kg) in mice. RESULTS:APAP-induced ALF was associated with increased plasma thrombin-antithrombin complexes, decreased plasma prothrombin, and a dramatic reduction in plasma fibrinogen compared with lower APAP doses. Hepatic fibrin(ogen) deposits increased independent of APAP dose, whereas plasma fibrin(ogen) degradation products markedly increased in mice with experimental ALF. Early pharmacologic anticoagulation (+2 hours after 600 mg/kg APAP) limited coagulation activation and reduced hepatic necrosis. The marked coagulation activation evident in mice with APAP-induced ALF was associated with a coagulopathy detectable ex vivo in plasma. Specifically, prolongation of the prothrombin time and inhibition of tissue factor-initiated clot formation were evident even after restoration of physiological fibrinogen concentrations. Plasma endogenous thrombin potential was similarly reduced at all APAP doses. Interestingly, in the presence of ample fibrinogen, ∼10 times more thrombin was required to clot plasma from mice with APAP-induced ALF compared with plasma from mice with simple hepatotoxicity. CONCLUSION:The results indicate that robust pathologic coagulation cascade activation in vivo and suppressed coagulation ex vivo are evident in mice with APAP-induced ALF. This unique experimental setting may fill an unmet need as a model to uncover mechanistic aspects of the complex coagulopathy of ALF.

Background: Activation of coagulation and fibrin deposition in the regenerating liver appears to promote adequate liver regeneration in mice. In humans, perioperative hepatic fibrin deposition is reduced in patients who develop liver dysfunction after partial hepatectomy (PHx), but the mechanism underlying reduced fibrin deposition in these patients is unclear. Methods and Results: Hepatic deposition of cross-linked (ie, stabilized) fibrin was evident in livers of mice after two-thirds PHx. Interestingly, hepatic fibrin cross-linking was dramatically reduced in mice after 90% PHx, an experimental setting of failed liver regeneration, despite similar activation of coagulation after two-thirds or 90% PHx. Likewise, intraoperative activation of coagulation was not reduced in patients who developed liver dysfunction after PHx. Preoperative fibrinogen plasma concentration was not connected to liver dysfunction after PHx in patients. Rather, preoperative and postoperative plasma activity of the transglutaminase coagulation factor (F)XIII, which cross-links fibrin, was lower in patients who developed liver dysfunction than in those who did not. PHx-induced hepatic fibrin cross-linking and hepatic platelet accumulation were significantly reduced in mice lacking the catalytic subunit of FXIII (FXIII-/- mice) after two-thirds PHx. This was coupled with a reduction in both hepatocyte proliferation and liver-to-body weight ratio as well as an apparent reduction in survival after two-thirds PHx in FXIII-/-mice. Conclusion: The results indicate that FXIII is a critical driver of liver regeneration after PHx and suggest that perioperative plasma FXIII activity may predict posthepatectomy liver dysfunction. The results may inform strategies to stabilize proregenerative fibrin during liver resection.

Fibrinogen plays a pathologic role in multiple diseases. It contributes to thrombosis and modifies inflammatory and immune responses, supported by studies in mice expressing fibrinogen variants with altered function or with a germline fibrinogen deficiency. However, therapeutic strategies to safely and effectively tailor plasma fibrinogen concentration are lacking. Here, we developed a strategy to tune fibrinogen expression by administering lipid nanoparticle (LNP)-encapsulated small interfering RNA (siRNA) targeting the fibrinogen α chain (siFga). Three distinct LNP-siFga reagents reduced both hepatic Fga messenger RNA and fibrinogen levels in platelets and plasma, with plasma levels decreased to 42%, 16%, and 4% of normal within 1 week of administration. Using the most potent siFga, circulating fibrinogen was controllably decreased to 32%, 14%, and 5% of baseline with 0.5, 1.0, and 2.0 mg/kg doses, respectively. Whole blood from mice treated with siFga formed clots with significantly decreased clot strength ex vivo, but siFga treatment did not compromise hemostasis following saphenous vein puncture or tail transection. In an endotoxemia model, siFga suppressed the acute phase response and decreased plasma fibrinogen, D-dimer, and proinflammatory cytokine levels. In a sterile peritonitis model, siFga restored normal macrophage migration in plasminogen-deficient mice. Finally, treatment of mice with siFga decreased the metastatic potential of tumor cells in a manner comparable to that observed in fibrinogen-deficient mice. The results indicate that siFga causes robust and controllable depletion of fibrinogen and provides the proof-of-concept that this strategy can modulate the pleiotropic effects of fibrinogen in relevant disease models.

Intravascular fibrin clot formation follows a well-ordered series of reactions catalyzed by thrombin cleavage of fibrinogen leading to fibrin polymerization and cross-linking by factor XIIIa (FXIIIa). Extravascular fibrin(ogen) deposits are observed in injured tissues; however, the mechanisms regulating fibrin(ogen) polymerization and cross-linking in this setting are unclear. The objective of this study was to determine the mechanisms of fibrin polymerization and cross-linking in acute liver injury induced by acetaminophen (APAP) overdose. Hepatic fibrin(ogen) deposition and cross-linking were measured following APAP overdose in wild-type mice, mice lacking the catalytic subunit of FXIII (FXIII-/-), and in FibAEK mice, which express mutant fibrinogen insensitive to thrombin-mediated fibrin polymer formation. Hepatic fibrin(ogen) deposition was similar in APAP-challenged wild-type and FXIII-/- mice, yet cross-linking of hepatic fibrin(ogen) was dramatically reduced (>90%) by FXIII deficiency. Surprisingly, hepatic fibrin(ogen) deposition and cross-linking were only modestly reduced in APAP-challenged FibAEK mice, suggesting that in the APAP-injured liver fibrin polymerization is not strictly required for the extravascular deposition of cross-linked fibrin(ogen). We hypothesized that the oxidative environment in the injured liver, containing high levels of reactive mediators (eg, peroxynitrite), modifies fibrin(ogen) such that fibrin polymerization is impaired without impacting FXIII-mediated cross-linking. Notably, fibrin(ogen) modified with 3-nitrotyrosine adducts was identified in the APAP-injured liver. In biochemical assays, peroxynitrite inhibited thrombin-mediated fibrin polymerization in a concentration-dependent manner without affecting fibrin(ogen) cross-linking over time. These studies depict a unique pathology wherein thrombin-catalyzed fibrin polymerization is circumvented to allow tissue deposition and FXIII-dependent fibrin(ogen) cross-linking.

Acute and chronic liver disease are associated with substantial alterations in the hemostatic system, including elevated levels of the platelet-adhesive protein von Willebrand factor (VWF). Carbon tetrachloride-induced liver fibrosis is reduced in VWF-deficient mice, but it is unclear if VWF plays a pathologic role in all settings of liver fibrosis. Indeed, several studies suggest an anti-fibrotic role for components of the hemostatic system, including platelets, in experimental settings of bile duct fibrosis. However, the role of VWF in this specific pathology has not been examined. We tested the hypothesis that VWF exerts hepatoprotective effects in experimental bile duct injury. Wild-type and VWF-deficient (VWF-/-) mice were challenged with the bile duct toxicant alpha-naphthylisothiocyanate (ANIT) and the impact of VWF deficiency on acute cholestatic liver injury and chronic liver fibrosis was determined. Acute ANIT (60 mg/kg, po)-induced cholestatic liver injury was associated with increased VWF plasma antigen and activity levels. VWF deficiency enhanced ANIT-induced hepatocellular injury, evidenced by increased plasma ALT activity and area of hepatocellular necrosis. Surprisingly, platelet accumulation within necrotic areas was increased in ANIT-challenged VWF-/- mice compared to wild-type mice. Compared to acute ANIT challenge, hepatic platelet accumulation was modest and appeared to be VWF-dependent in mice exposed to ANIT diet (0.05 %) for 6 weeks. However, contrasting the role of VWF after acute ANIT challenge, VWF deficiency did not impact biliary fibrosis induced by chronic ANIT exposure. The results suggest that VWF plays dichotomous roles in experimental acute and chronic ANIT-induced cholestatic liver injury.

Background: Obesity is associated with an altered plasma lipid composition that impacts platelet activation, indicating that lipids directly affect platelet function and reactivity. However, very little is known about which essential lipids are important to enable platelet production from their precursors, megakaryocytes (MKs), and whether obesity influences lipid composition and consequently affects thrombopoiesis.

Acute and chronic liver disease are associated with substantial alterations in the hemostatic system. Evidence from both experimental and clinical studies suggests that anticoagulants slow the progression of liver disease. Efficacy of those anticoagulant drugs is, in part, attributed to a reduction of microthrombi formation within the liver. Although anticoagulant drugs show promising results, bleeding risk associated with these drugs is an obvious drawback, particularly in patients with a complex coagulopathy driven by decreased liver function. Identifying therapies that reduce intrahepatic thrombosis with minimal bleeding risk would significantly advance the field. Among the hemostatic alterations observed in patients are substantially increased levels of the platelet-adhesive protein von Willebrand factor (VWF). In contrast, levels of A Disintegrin and Metalloproteinase with Thrombospondin motifs, the enzyme that regulates VWF activity, are significantly reduced in patients with liver disease. Highly elevated VWF levels are proposed to accelerate intrahepatic thrombus formation and thus be a driver of disease progression. Strong clinical evidence suggesting a link between liver disease and changes in VWF is now being matched by emerging mechanistic data showing a detrimental role for VWF in the progression of liver disease. This review focuses on clinical and experimental evidence supporting a connection between VWF function and the progression of acute and chronic liver diseases. Furthermore, with the recent anticipated approval of several novel therapies targeting VWF, we discuss potential strategies and benefits of targeting VWF as an innovative therapy for patients with liver disease.

We are pleased to present a Special Column on "Drug-and Chemical-induced Liver Injury". The scope of articles in this issue is diverse, covering a wide range of unanswered questions in this research area faced by both clinicians and basic researchers. Liver injury remains a major obstacle in the development of new drugs and is a concern for otherwise incredibly efficacious drugs. Even for well-established hepatotoxic drugs,

The liver is unique in its remarkable regenerative capacity, which enables the use of liver resection as a treatment for specific liver diseases, including removal of neoplastic liver disease. After resection, the remaining liver tissue (i.e, liver remnant) regenerates to maintain normal hepatic function. In experimental settings as well as patients, removal of up to two-thirds of the liver mass stimulates a rapid and highly coordinated process resulting in the regeneration of the remaining liver. Mechanisms controlling the initiation and termination of regeneration continue to be discovered, and many of the fundamental signaling pathways controlling the proliferation of liver parenchymal cells (i.e., hepatocytes) have been uncovered. Interestingly, while hemostatic complications (i.e., bleeding and thrombosis) are primarily thought of as a complication of surgery itself, strong evidence suggests that components of the hemostatic system are, in fact, powerful drivers of liver regeneration. This review focuses on the clinical and translational evidence supporting a link between the hemostatic system and liver regeneration, and the mechanisms whereby the hemostatic system directs liver regeneration discovered using experimental settings.

Background & Aim: Acetaminophen (APAP)-induced acute liver failure is associated with substantial alterations in the hemostatic system. In mice, platelets accumulate in the liver after APAP overdose and appear to promote liver injury. Interestingly, patients with acute liver injury have highly elevated levels of the platelet-adhesive protein von Willebrand factor (VWF), but a mechanistic connection between VWF and progression of liver injury has not been established. We tested the hypothesis that VWF contributes directly to experimental APAP-induced acute liver injury. Methods: Wild-type mice and VWF-deficient (Vwf(-/-)) mice were given a hepatotoxic dose of APAP (300 mg/kg, i.p.) or vehicle (saline). VWF plasma levels were measured by ELISA, and liver necrosis or hepatocyte proliferation was measured by immunohistochemistry. Platelet and VWF deposition were measured by immunofluorescence. Results: In wild-type mice, VWF plasma levels, high molecular weight (HMW) VWF multimers, and VWF activity decreased 24 h after APAP challenge. These changes coupled to robust hepatic VWF and platelet deposition, although VWF deficiency had minimal effect on peak hepatic platelet accumulation or liver injury. VWF plasma levels were elevated 48 h after APAP challenge, but with relative reductions in HMW multimers and VWF activity. Whereas hepatic platelet aggregates persisted in livers of APAP-challenged wild-type mice, platelets were nearly absent in Vwf(-/-) mice 48 h after APAP challenge. The absence of platelet aggregates was linked to dramatically accelerated repair of the injured liver. Complementing observations in Vwf(-/-) mice, blocking VWF or the platelet integrin alpha(IIb)beta(3) during development of injury significantly reduced hepatic platelet aggregation and accelerated liver repair in APAP-challenged wild-type mice. Conclusion: These studies are the first to suggest a mechanistic link between VWF, hepatic platelet accumulation, and liver repair. Targeting VWF might provide a novel therapeutic approach to improve repair of the APAP-injured liver. Lay summary: Patients with acute liver injury due to acetaminophen overdose have highly elevated levels of the platelet-adhesive protein von Willebrand factor. It is not known whether von Willebrand factor plays a direct role in the progression of acute liver injury. We discovered that von Willebrand factor delays repair of the acetaminophen-injured liver in mice and that targeting von Willebrand factor, even in mice with established liver injury, accelerates liver repair. (C) 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Most of the blood supplied to the liver (~80%) enters through the portal vein, which serves to carry oxygen‐depleted, but nutrient‐rich, blood to the liver so that critical metabolic functions can be performed. Portal hypertension (PH) is a common consequence of chronic liver diseases, regardless of etiology. PH is characterized by an increase in portal venous pressure, leading to an increase in the pressure gradient between the portal vein and venous outflow from the liver to the inferior vena cava.1.Bosch J. Groszmann R.J. Shah V.H. Evolution in the understanding of the pathophysiological basis of portal hypertension: how changes in paradigm are leading to successful new treatments.J Hepatol. 2015; 62: S121-30Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar Increased portal vein pressure can cause serious complications, including the development of abnormal, enlarged veins (i.e., varices) within the gastrointestinal tract. If the wall tension becomes too high, these can rupture, resulting in potentially life‐threatening complications.2.Sanyal A.J. Bosch J. Blei A. Arroyo V. Portal hypertension and its complications.Gastroenterology. 2008; 134: 1715-28Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar The most common cause of PH is cirrhosis, the end‐stage of chronic liver diseases such as viral or alcoholic hepatitis and non‐alcoholic fatty liver disease.1.Bosch J. Groszmann R.J. Shah V.H. Evolution in the understanding of the pathophysiological basis of portal hypertension: how changes in paradigm are leading to successful new treatments.J Hepatol. 2015; 62: S121-30Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar The pathophysiology of PH is complex and has various etiologies. For example, mechanical obstruction of blood flow can occur because of hepatic fibrosis, wherein chronic liver damage results in excess deposition of collagens, which replace healthy liver tissue. Other etiologies causing disruption of intrahepatic blood flow and congestion, including extrahepatic causes, such as hepatic vein thrombosis (i.e., Budd‐Chiari disease) and congestive heart disease, can also cause PH. Notably, PH and hepatic congestion are cyclically related pathologies, insofar as chronic hepatic congestion also promotes hepatic fibrosis. Because PH has such negative consequences, there is a pressing demand to understand the mechanisms driving this condition and to establish novel approaches to limit PH and its consequences. Several years ago, the laboratory of Shah established a creative experimental approach to define the impact of chronic hepatic venous congestion on various liver pathologies, including hepatic fibrosis and PH.3.Simonetto D.A. Yang H.Y. Yin M. de Assuncao T.M. Kwon J.H. Hilscher M. et al.Chronic passive venous congestion drives hepatic fibrogenesis via sinusoidal thrombosis and mechanical forces.Hepatology. 2015; 61: 648-59Crossref PubMed Scopus (112) Google Scholar Specifically, after surgical induction of partial stenosis (~70%) of the suprahepatic caudal vena cava (partial inferior vena cava ligation, pIVCL), venous outflow from the liver is reduced, leading to chronic hepatic congestion.3.Simonetto D.A. Yang H.Y. Yin M. de Assuncao T.M. Kwon J.H. Hilscher M. et al.Chronic passive venous congestion drives hepatic fibrogenesis via sinusoidal thrombosis and mechanical forces.Hepatology. 2015; 61: 648-59Crossref PubMed Scopus (112) Google Scholar In this robust model, sinusoidal dilation and vascular congestion are evident 6 weeks after surgery. Moreover, profibrogenic changes occur, including activation of hepatic stellate cells, which constitute a primary source of collagen synthesis in the liver.4.Tsuchida T. Friedman S.L. Mechanisms of hepatic stellate cell activation.Nat Rev Gastroenterol Hepatol. 2017; 14: 397-411Crossref PubMed Scopus (1276) Google Scholar Indeed, hepatic fibrosis was significantly increased in mice after pIVCL, as was PH. This elegant model offers a fascinating opportunity to define mechanisms of liver pathology driven by congestive hepatopathy, and the connection of these processes to PH. Among other changes evident in the liver after pIVCL is the apparent activation of the blood coagulation cascade. Specifically, hepatic fibrin(ogen) deposition, largely restricted to the liver sinusoids and intima of larger hepatic vessels, was evident after pIVCL.3.Simonetto D.A. Yang H.Y. Yin M. de Assuncao T.M. Kwon J.H. Hilscher M. et al.Chronic passive venous congestion drives hepatic fibrogenesis via sinusoidal thrombosis and mechanical forces.Hepatology. 2015; 61: 648-59Crossref PubMed Scopus (112) Google Scholar This observation recapitulates findings in livers from patients with congestive hepatopathy (i.e., chronic passive congestion of the liver) caused by chronic heart failure.3.Simonetto D.A. Yang H.Y. Yin M. de Assuncao T.M. Kwon J.H. Hilscher M. et al.Chronic passive venous congestion drives hepatic fibrogenesis via sinusoidal thrombosis and mechanical forces.Hepatology. 2015; 61: 648-59Crossref PubMed Scopus (112) Google Scholar Intrahepatic thrombosis can be a major complicating factor in chronic liver disease, and it has been suggested that microthrombotic events within the liver vasculature may contribute to disease progression.5.Lisman T. Porte R.J. Pathogenesis, prevention, and management of bleeding and thrombosis in patients with liver diseases.Res Pract Thromb Haemost. 2017; 1: 150-61Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar Studies in experimental animal models and one randomized study in humans have indeed demonstrated that anticoagulants slow down the progression of fibrotic diseases,6.Pant A. Kopec A.K. Luyendyk J.P. Role of the blood coagulation cascade in hepatic fibrosis.Am J Physiol Gastrointest Liver Physiol. 2018; 315: G171-6Crossref Scopus (29) Google Scholar, 7.Villa E. Cammà C. Marietta M. Luongo M. Critelli R. Colopi S. et al.Enoxaparin prevents portal vein thrombosis and liver decompensation in patients with advanced cirrhosis.Gastroenterology. 2012; 143: 1253-60Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar, 8.Anstee Q.M. Goldin R.D. Wright M. Martinelli A. Cox R. Thursz M.R. Coagulation status modulates murine hepatic fibrogenesis: implications for the development of novel therapies.J Thromb Haemost. 2008; 6: 1336-43Crossref PubMed Scopus (91) Google Scholar, 9.Abe W. Ikejima K. Lang T. Okumura K. Enomoto N. Kitamura T. et al.Low molecular weight heparin prevents hepatic fibrogenesis caused by carbon tetrachloride in the rat.J Hepatol. 2007; 46: 286-94Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 10.Kopec A.K. Joshi N. Towery K.L. Kassel K.M. Sullivan B.P. Flick M.J. et al.Thrombin inhibition with dabigatran protects against high‐fat diet‐induced fatty liver disease in mice.J Pharmacol Exp Ther. 2014; 351: 288-97Crossref PubMed Scopus (42) Google Scholar most likely because of a reduction in intrahepatic thrombosis. The pIVCL model is among the models of chronic liver disease in which coagulation has been implicated in the pathogenesis. Specifically, Shah et al demonstrated that administration of warfarin dramatically reduced hepatic fibrosis in mice that underwent pIVCL, as did transgenic overexpression of tissue factor pathway inhibitor (TFPI).3.Simonetto D.A. Yang H.Y. Yin M. de Assuncao T.M. Kwon J.H. Hilscher M. et al.Chronic passive venous congestion drives hepatic fibrogenesis via sinusoidal thrombosis and mechanical forces.Hepatology. 2015; 61: 648-59Crossref PubMed Scopus (112) Google Scholar With each intervention, pharmacologic or genetic, fibrin(ogen) deposition in the liver was dramatically reduced, implying that the deposits observed were fully cross‐linked fibrin clots and not soluble fibrinogen accumulated in the sinusoids. Collectively, these studies suggest that intrahepatic coagulation contributes to liver fibrosis driven by chronic passive venous congestion. Important basic and clinically relevant mechanisms to be identified are the link between changes in sinusoidal pressure and hepatic congestion and the activation of coagulation, and the mechanisms linking these various processes to hepatic fibrosis. In an elegant follow‐up to their initial study, Shah et al began to fill this knowledge gap in a recent publication in Gastroenterology.11.Hilscher M.B. Sehrawat T. Arab Verdugo J.P. Zeng Z. Gao J. Liu M. et al.Mechanical stretch increases expression of CXCL1 in liver sinusoidal endothelial cells to recruit neutrophils, generate sinusoidal microthombi, and promote portal hypertension.Gastroenterology. 2019; 157: 193-209Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar In this article, Hilscher et al identify a mechanism whereby mechanical forces are detected by liver sinusoidal endothelial cells, leading to sinusoidal fibrin(ogen) deposition, PH, and hepatic fibrosis. Neutrophil extracellular traps (NETs) were identified as critical players in this stepwise process. NETs are networks of extracellular fibers consisting primarily of DNA and histones released from neutrophils into the extracellular space in combination with other neutrophil‐resident proteins, including the proteases neutrophil elastase (NE), myeloperoxidase (MPO), and cathepsin G.12.Kaplan M.J. Radic M. Neutrophil extracellular traps: double‐edged swords of innate immunity.J Immunol. 2012; 189: 2689-95Crossref PubMed Scopus (736) Google Scholar The initial observation of NET formation was in the context of innate immune responses to infections.13.Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. et al.Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-5Crossref PubMed Scopus (6162) Google Scholar A rapid evolution of studies has followed, with emerging evidence supporting a role for NETs in numerous pathophysiologic conditions. For example, NETs contribute to neutrophil‐driven coagulation in experimental models of thrombosis triggered by venous stasis,14.von Brühl M.L. Stark K. Steinhart A. Chandraratne S. Konrad I. Lorenz M. et al.Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo.J Exp Med. 2012; 209: 819-35Crossref PubMed Scopus (1189) Google Scholar and prevention of NET formation reduces thrombus formation in mice.15.Fuchs T.A. Brill A. Duerschmied D. Schatzberg D. Monestier M. Myers Jr, D.D. et al.Extracellular DNA traps promote thrombosis.Proc Natl Acad Sci USA. 2010; 107: 15880-5Crossref PubMed Scopus (1586) Google Scholar Furthermore, NETs were identified in thrombi isolated from patients with venous thromboembolism,16.Savchenko A.S. Martinod K. Seidman M.A. Wong S.L. Borissoff J.I. Piazza G. et al.Neutrophil extracellular traps form predominantly during the organizing stage of human venous thromboembolism development.J Thromb Haemost. 2014; 12: 860-70Crossref PubMed Scopus (175) Google Scholar and elevated levels of NET components, such as nucleosomes, histones, MPO, and cell‐free DNA, are evident in patients with thrombosis‐associated diseases.17.Jiménez‐Alcázar M. Kim N. Fuchs T.A. Circulating extracellular DNA: cause or consequence of thrombosis?.Semin Thromb Hemost. 2017; 43: 553-61Crossref PubMed Scopus (51) Google Scholar Recently, elevated levels of NET components were observed in patients undergoing liver transplantation, and were associated with coagulation activation.18.von Meijenfeldt F.A. Burlage L.C. Bos S. Adelmeijer J. Porte R.J. Lisman T. Elevated plasma levels of cell‐free DNA during liver transplantation are associated with activation of coagulation.Liver Transpl. 2018; 24: 1716-25Crossref PubMed Scopus (25) Google Scholar Interestingly, levels of MPO‐DNA complexes were substantially elevated before the start of surgery, suggesting that NETosis occurs in patients with liver disease. Indeed, elevated levels of MPO‐DNA complexes were observed in patients with non‐alcoholic steatohepatitis, and both MPO and NE have been reported to mediate damaging effects in various experimental liver disease models,19.Honda M. Kubes P. Neutrophils and neutrophil extracellular traps in the liver and gastrointestinal system.Nat Rev Gastroenterol Hepatol. 2018; 15: 206-21Crossref PubMed Scopus (114) Google Scholar in line with the findings of Hilscher et al.11.Hilscher M.B. Sehrawat T. Arab Verdugo J.P. Zeng Z. Gao J. Liu M. et al.Mechanical stretch increases expression of CXCL1 in liver sinusoidal endothelial cells to recruit neutrophils, generate sinusoidal microthombi, and promote portal hypertension.Gastroenterology. 2019; 157: 193-209Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar Hilscher et al11.Hilscher M.B. Sehrawat T. Arab Verdugo J.P. Zeng Z. Gao J. Liu M. et al.Mechanical stretch increases expression of CXCL1 in liver sinusoidal endothelial cells to recruit neutrophils, generate sinusoidal microthombi, and promote portal hypertension.Gastroenterology. 2019; 157: 193-209Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar showed that mechanical forces resembling those encountered in conditions of hepatic congestion elicit gene expression changes in liver sinusoidal endothelial cell activation secondary to activation of the mechanosensitive notch receptor pathway. Among the gene products induced was Cxcl1, a well‐appreciated neutrophil chemokine, prompting exploration of neutrophil and, more specifically, NET involvement in PH and hepatic fibrosis driven by pIVCL. Indeed, intravital microscopy revealed neutrophil‐platelet influx and NETosis in the liver sinusoids following pIVCL. Furthermore, the authors observed colocalization of fibrin and MPO deposits in livers from patients with congestive hepatopathy, suggesting that NET formation also occurs in patients. Using genetic and pharmacologic strategies to reduce NE activity, and using mice deficient in peptidylarginine deiminase, which is an enzyme central to NET formation, the authors demonstrated that NETs contribute to intrahepatic coagulation, PH and hepatic fibrosis after pIVCL.11.Hilscher M.B. Sehrawat T. Arab Verdugo J.P. Zeng Z. Gao J. Liu M. et al.Mechanical stretch increases expression of CXCL1 in liver sinusoidal endothelial cells to recruit neutrophils, generate sinusoidal microthombi, and promote portal hypertension.Gastroenterology. 2019; 157: 193-209Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar The study decisively links these changes to a pathway initiated by stretch‐activated signaling, by using mice with endothelial cell–selective notch deficiency and by examining notch signaling in cultured endothelial cells. Collectively, these studies represent multiple steps forward in identifying the mechanisms whereby hepatic congestion drives intrahepatic coagulation, and promotes PH and hepatic fibrosis. Strong experimental evidence and some clinical studies have linked blood coagulation protease activity to exacerbation of liver pathology. However, the mechanisms triggering and amplifying intrahepatic coagulation in the context of hepatic injury are not fully understood. In the case of pIVCL‐induced hepatic congestion, the studies by the Shah laboratory clearly show that NETs amplify intrahepatic coagulation activity. Prior studies have suggested that tissue factor (TF), expressed by parenchymal cells in the liver (i.e., hepatocytes), contributes to intrahepatic coagulation in diverse experimental settings.20.Rautou P.E. Tatsumi K. Antoniak S. Owens 3rd, A.P. Sparkenbaugh E. Holle L.A. et al.Hepatocyte tissue factor contributes to the hypercoagulable state in a mouse model of chronic liver injury.J Hepatol. 2016; 64: 53-9Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 21.Sullivan B.P. Kopec A.K. Joshi N. Cline H. Brown J.A. Bishop S.C. et al.Hepatocyte tissue factor activates the coagulation cascade in mice.Blood. 2013; 121: 1868-74Crossref Scopus (52) Google Scholar There is evidence to suggest that NETs amplify TF‐initiated coagulation reactions, but also that components of NETs activate the intrinsic pathway.22.Noubouossie D.F. Whelihan M.F. Yu Y.B. Sparkenbaugh E. Pawlinski R. Monroe D.M. et al.In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps.Blood. 2017; 129: 1021-9Crossref PubMed Scopus (144) Google Scholar The precise role of TF in the setting of pIVCL has not been determined, as the anticoagulant effect of TFPI overexpression cannot be ascribed only to inhibition of TF procoagulant activity.23.Mast A.E. Tissue factor pathway inhibitor: multiple anticoagulant activities for a single protein.Arterioscler Thromb Vasc Biol. 2016; 36: 9-14Crossref PubMed Scopus (100) Google Scholar The studies by the Shah laboratory were particularly intriguing to us, as our recent studies also identified sinusoidal fibrin deposition after partial hepatectomy,24.Groeneveld D. Pereyra D. Veldhuis Z. Adelmeijer J. Ottens P. Kopec A.K. et al.Intrahepatic fibrin(ogen) deposition drives liver regeneration after partial hepatectomy in mice and humans.Blood. 2019; 133: 1245-56Crossref PubMed Scopus (27) Google Scholar another setting in which hemodynamic changes are evident in the liver. It seems possible that shear or mechanical forces could be critical regulators of TF procoagulant activity in the liver vasculature. Another possibility is that mechanical forces applied to any number of TF‐expressing cells in the liver could prompt the release of TF‐expressing microvesicles, contributing to intrahepatic coagulation. Of specific interest would be studies determining whether NET formation and coagulation are sequential or concurrent synergistic processes in the pIVCL model. Whereas NET inhibition reduced coagulation, the comparative effect of anticoagulation on NET release was not investigated. Future studies determining the precise integration of NETs into pathways controlling intrahepatic coagulation may be informative, as novel strategies are developed to reduce pathologic coagulation in liver disease. Although intrahepatic coagulation clearly contributes to liver fibrosis after pIVCL, the precise downstream mechanisms are not yet known. It seems very reasonable to suggest that sinusoidal occlusion by fibrin drives liver pathology in the pIVCL model. Additional studies specifically targeting fibrin(ogen) and/or platelets could provide evidence for this mechanism. The identification of platelet‐neutrophil aggregates in the pIVCL model makes such studies of particular interest, especially as cooperative effects of NETs and platelets have been shown to promote coagulation in other settings.25.McDonald B. Davis R.P. Kim S.J. Tse M. Esmon C.T. Kolaczkowska E. et al.Platelets and neutrophil extracellular traps collaborate to promote intravascular coagulation during sepsis in mice.Blood. 2017; 129: 1357-67Crossref PubMed Scopus (345) Google Scholar Coagulation proteases, particularly thrombin and activated factor X, have been shown to contribute to the development of liver fibrosis. Indeed, several studies have documented an important role for protease‐activated receptors expressed by inflammatory cells and stellate cells in the pathogenesis of liver fibrosis.26.Kallis Y.N. Scotton C.J. Mackinnon A.C. Goldin R.D. Wright N.A. Iredale J.P. et al.Proteinase activated receptor 1 mediated fibrosis in a mouse model of liver injury: a role for bone marrow derived macrophages.PLoS One. 2014; 9: e86241Crossref Scopus (24) Google Scholar, 27.Rullier A. Gillibert‐Duplantier J. Costet P. Cubel G. Haurie V. Petibois C. et al.Protease‐activated receptor 1 knockout reduces experimentally induced liver fibrosis.Am J Physiol Gastrointest Liver Physiol. 2008; 294: G226-35Crossref PubMed Scopus (50) Google Scholar, 28.Sullivan B.P. Weinreb P.H. Violette S.M. Luyendyk J.P. The coagulation system contributes to alphaVbeta6 integrin expression and liver fibrosis induced by cholestasis.Am J Pathol. 2010; 177: 2837-49Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar Thus, it is possible that multiple mechanisms downstream of coagulation activation contribute to liver fibrosis after pIVCL. Multiple independent interventions interfering with NETs reduced PH in mice after pIVCL.11.Hilscher M.B. Sehrawat T. Arab Verdugo J.P. Zeng Z. Gao J. Liu M. et al.Mechanical stretch increases expression of CXCL1 in liver sinusoidal endothelial cells to recruit neutrophils, generate sinusoidal microthombi, and promote portal hypertension.Gastroenterology. 2019; 157: 193-209Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar These experiments provide a novel and clear link between NETs and a common end‐stage pathology of liver diseases, and suggest NETs as putative targets for novel therapies. However, precisely how NETs drive PH requires further study. Blocking NET formation reduced hepatic fibrin deposition after pIVCL, and this was associated with a reduction in PH. However, the development of PH after pIVCL was not affected by either warfarin or TFPI overexpression,3.Simonetto D.A. Yang H.Y. Yin M. de Assuncao T.M. Kwon J.H. Hilscher M. et al.Chronic passive venous congestion drives hepatic fibrogenesis via sinusoidal thrombosis and mechanical forces.Hepatology. 2015; 61: 648-59Crossref PubMed Scopus (112) Google Scholar although each intervention strongly reduced intrahepatic coagulation as shown by reduced amounts of sinusoidal fibrin deposits. Although NET‐amplified coagulation may drive hepatic fibrosis, this disconnect suggests the possibility that NETs may also drive PH through a mechanism independent of coagulation. Indeed, one could envision NET‐platelet interactions or NET‐dependent effects on endothelial cell function as offering an alternative path to disruption of hepatic blood flow, leading to hypertension. Interestingly, in the case of a separate fibrosis model induced by common bile duct ligation (BDL), Shah et al found that, unlike pIVCL, NET inhibition had no effect on liver fibrosis.11.Hilscher M.B. Sehrawat T. Arab Verdugo J.P. Zeng Z. Gao J. Liu M. et al.Mechanical stretch increases expression of CXCL1 in liver sinusoidal endothelial cells to recruit neutrophils, generate sinusoidal microthombi, and promote portal hypertension.Gastroenterology. 2019; 157: 193-209Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar This result is consistent with the prior finding that neutrophils do not contribute to hepatic fibrosis after BDL.29.Saito J.M. Bostick M.K. Campe C.B. Xu J. Maher J.J. Infiltrating neutrophils in bile duct‐ligated livers do not promote hepatic fibrosis.Hepatol Res. 2003; 25: 180-91Crossref Scopus (41) Google Scholar However, while having no effect on liver fibrosis, inhibition of NETs significantly reduced PH in the BDL model, further implying a unique mechanism linking NET function to regulation of hepatic blood flow. This point was particularly interesting to us, as it appears that modifying blood coagulation activity is but one way in which NETs may contribute to PH. As emerging data suggest that NETs are detrimental in a variety of liver diseases,19.Honda M. Kubes P. Neutrophils and neutrophil extracellular traps in the liver and gastrointestinal system.Nat Rev Gastroenterol Hepatol. 2018; 15: 206-21Crossref PubMed Scopus (114) Google Scholar identifying the finer mechanistic details will be important. The observation that endothelial notch deficiency reduced the pathologic changes in the liver after pIVCL affirms the presence of receptor‐mediated signal transduction pathways that detect and elicit a response to mechanical stretch within the liver microenvironment. Work by Shah et al provides compelling evidence that this response is tightly connected to NET formation and intrahepatic coagulation, and through these mechanisms exacerbates PH. Such a response seems to establish a pathologic amplification loop wherein mechanical forces applied to sinusoidal endothelial cells would worsen PH. Hilscher et al11.Hilscher M.B. Sehrawat T. Arab Verdugo J.P. Zeng Z. Gao J. Liu M. et al.Mechanical stretch increases expression of CXCL1 in liver sinusoidal endothelial cells to recruit neutrophils, generate sinusoidal microthombi, and promote portal hypertension.Gastroenterology. 2019; 157: 193-209Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar identified numerous genes induced by mechanical stretch in cultured sinusoidal endothelial cells. Although not within the scope of the specific hypothesis tested, mechanical stretch induced the expression of genes encoding products with anticoagulant activity (i.e., thrombomodulin and endothelial protein C receptor), profibrinolytic function (i.e., plasminogen), and synthesis of antiplatelet mediators (PTGIS, the gene encoding prostaglandin I2 [prostacyclin] synthase). It seems likely that these changes serve as a counterforce to the anticipated prothrombotic environment created by PH and its affiliated pathologies. Therapeutic modulation of the stretch‐induced induction of these antithrombotic factors also seems a plausible tactic to reduce fibrosis and PH. It is increasingly accepted that patients with chronic liver disease develop a precariously rebalanced hemostatic system. Moreover, experiments such as those from the Shah laboratory have contributed to the increasingly accepted concept that intrahepatic coagulation contributes to liver disease pathogenesis. In their recent article, Hilscher et al11.Hilscher M.B. Sehrawat T. Arab Verdugo J.P. Zeng Z. Gao J. Liu M. et al.Mechanical stretch increases expression of CXCL1 in liver sinusoidal endothelial cells to recruit neutrophils, generate sinusoidal microthombi, and promote portal hypertension.Gastroenterology. 2019; 157: 193-209Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar address numerous key issues within this area, including mechanisms linking mechanical stretch in the liver to coagulation, and cross‐talk between NETs and coagulation in pathologies associated with hepatic congestion. The studies raise numerous points for potential future study, and should inspire additional research to determine whether the mechanisms identified in the pIVCL model may serve as putative targets to reduce fibrosis or PH in patients with chronic liver disease. Neither author has any perceived conflicts of interest to disclose with respect to the manuscript. D. J. Groeneveld and J. P. Luyendyk both prepared and approved the final version of the manuscript. The preparation of this work was supported by grants from the National Institutes of Health to J. P. Luyendyk (R01 DK105099; R01 ES017537) and a European Hematology Association (EHA) Research Grant to D. J. Groeneveld. The content is solely the responsibility of the authors, and does not necessarily represent the official views of the National Institutes of Health.National Institutes of HealthR01 DK105099R01 ES017537