RESUMO
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.
Assuntos
Afibrinogenemia/metabolismo , Fibrina/biossíntese , Fibrinogênio/biossíntese , Técnicas de Silenciamento de Genes , Lipossomos/farmacologia , RNA Interferente Pequeno , Afibrinogenemia/genética , Animais , Plaquetas/metabolismo , Modelos Animais de Doenças , Feminino , Fibrina/genética , Fibrinogênio/genética , Humanos , Masculino , Camundongos , Nanopartículas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologiaRESUMO
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.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/patologia , Fator XIII/fisiologia , Fibrina/metabolismo , Fibrinogênio/metabolismo , Polimerização , Trombina/metabolismo , Acetaminofen/toxicidade , Analgésicos não Narcóticos/toxicidade , Animais , Coagulação Sanguínea , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Fibrina/química , Fibrinogênio/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Platelets play a pivotal role in stimulating liver regeneration after partial hepatectomy in rodents and humans. Liver regeneration in rodents is delayed when platelets are inhibited. However, the exact mechanisms whereby platelets accumulate and promote liver regeneration remain uncertain. Thrombin-dependent intrahepatic fibrin(ogen) deposition was recently reported after partial hepatectomy (PHx) in mice, but the role of fibrin(ogen) deposits in liver regeneration has not been investigated. We tested the hypothesis that fibrin(ogen) contributes to liver regeneration by promoting intrahepatic platelet accumulation and identified the trigger of rapid intrahepatic coagulation after PHx. PHx in wild-type mice triggered rapid intrahepatic coagulation, evidenced by intrahepatic fibrin(ogen) deposition. Intrahepatic fibrin(ogen) deposition was abolished in mice with liver-specific tissue factor deficiency, pinpointing the trigger of coagulation after PHx. Direct thrombin activation of platelets through protease-activated receptor-4 did not contribute to hepatocyte proliferation after PHx, indicating that thrombin contributes to liver regeneration primarily by driving intrahepatic fibrin(ogen) deposition. Fibrinogen depletion with ancrod reduced both intrahepatic platelet accumulation and hepatocyte proliferation after PHx, indicating that fibrin(ogen) contributes to liver regeneration after PHx by promoting intrahepatic platelet accumulation. Consistent with the protective function of fibrin(ogen) in mice, low postoperative plasma fibrinogen levels were associated with liver dysfunction and mortality in patients undergoing liver resection. Moreover, increased intrahepatic fibrin(ogen) deposition was evident in livers of patients after liver resection but was remarkably absent in patients displaying hepatic dysfunction postresection. The results suggest a novel mechanism whereby coagulation-dependent intrahepatic fibrin(ogen) deposition drives platelet accumulation and liver regeneration after PHx.
Assuntos
Coagulação Sanguínea , Fibrinogênio/metabolismo , Hepatectomia/métodos , Hepatopatias/cirurgia , Regeneração Hepática , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Seguimentos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Ativação Plaquetária , Prognóstico , Estudos Prospectivos , Receptores de Trombina/metabolismo , Trombina/metabolismo , Tromboplastina/metabolismoRESUMO
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 (300â¯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 24â¯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 48â¯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 48â¯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 αIIbß3 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.
Assuntos
Acetaminofen/efeitos adversos , Analgésicos não Narcóticos/efeitos adversos , Plaquetas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Fígado/metabolismo , Fator de von Willebrand/metabolismo , Acetaminofen/administração & dosagem , Analgésicos não Narcóticos/administração & dosagem , Animais , Coagulação Sanguínea/efeitos dos fármacos , Humanos , Fígado/patologia , Masculino , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necrose , Agregação Plaquetária/efeitos dos fármacos , Fator de von Willebrand/administração & dosagem , Fator de von Willebrand/genética , Fator de von Willebrand/farmacocinéticaRESUMO
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.
Assuntos
Hemostasia/fisiologia , Hepatopatias/complicações , Regeneração Hepática/fisiologia , Humanos , Hepatopatias/patologiaRESUMO
The ratios between von Willebrand factor propeptide (VWFpp) or factor VIII activity ( FVIII: C) and VWF antigen (VWF:Ag) reflect synthesis, secretion, and clearance of VWF. We aimed to define the pathophysiology of 658 patients with type 1, 2, or 3 von Willebrand disease (VWD) with VWF levels ≤30 U/dL from the Willebrand in The Netherlands (WiN) study using the VWFpp/VWF:Ag and FVIII: C/VWF:Ag ratios. We evaluated the use of VWFpp in the classification and diagnosis of VWD. On the basis of the ratios, reduced VWF synthesis was observed in 18% of type 1 and only 2% of type 2 patients. A significant proportion of type 3 patients had detectable VWFpp (41%). These patients had a lower bleeding score than type 3 patients who had a complete absence of VWF:Ag and VWFpp (14.0 vs 19.5; P = .025). The majority of these patients had missense mutations with rapid VWF clearance, whereas type 3 patients with no VWFpp were homozygous for null alleles. In conclusion, VWFpp identified severe type 1 VWD with very low VWF levels in patients who had previously been classified as type 3 VWD. This study underlines the clinical significance of the VWFpp assay in the diagnosis and classification of VWD.
Assuntos
Hemorragia/patologia , Mutação/genética , Precursores de Proteínas/genética , Doenças de von Willebrand/classificação , Doenças de von Willebrand/genética , Fator de von Willebrand/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Estudos Transversais , Feminino , Seguimentos , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Países Baixos , Fenótipo , Prognóstico , Adulto Jovem , Doenças de von Willebrand/diagnóstico , Fator de von Willebrand/metabolismoRESUMO
Liver diseases are associated with complex changes in the hemostatic system and elevated levels of the platelet-adhesive protein Von Willebrand factor (VWF) are reported in patients with acute and chronic liver damage. Although elevated levels of VWF are associated with fibrosis in the general population, the role of VWF in acute and chronic liver injury has not been examined in depth in experimental settings. We tested the hypothesis that VWF deficiency inhibits experimental liver injury and fibrosis. Wild-type (WT) and VWF-deficient mice were challenged with carbon tetrachloride (CCl4) and the impact of VWF deficiency on acute liver injury and chronic liver fibrosis was determined. VWF deficiency did not significantly affect acute CCl4-induced hepatocellular necrosis in mice. Chronic CCl4 challenge, twice weekly for 6weeks, significantly increased hepatic stellate cell activation and collagen deposition in livers of WT mice. Interestingly, hepatic induction of several profibrogenic and stellate cell activation genes was attenuated in VWF-deficient mice. Moreover, birefringent sirius red staining (indicating type I and III collagens) and type I collagen immunofluorescence indicated a reduction in hepatic collagen deposition in CCl4-exposed VWF-deficient mice compared to CCl4-exposed WT mice. The results indicate that VWF deficiency attenuates chronic CCl4-induced liver fibrosis without affecting acute hepatocellular necrosis. The results are the first to demonstrate that VWF deficiency reduces the progression of liver fibrosis, suggesting a mechanistic role of elevated plasma VWF levels in cirrhosis.
Assuntos
Cirrose Hepática/patologia , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo , Actinas/metabolismo , Animais , Intoxicação por Tetracloreto de Carbono/patologia , Doença Crônica , Colágeno Tipo I/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Von Willebrand disease (VWD) is a bleeding disorder characterized by reduced plasma von Willebrand factor (VWF) levels or functionally abnormal VWF. Low VWF plasma levels in VWD patients are the result of mutations in the VWF gene that lead to decreased synthesis, impaired secretion, increased clearance or a combination thereof. However, expression studies of variants located in the A domains of VWF are limited. We therefore characterized the biosynthesis of VWF mutations, located in the VWF A1-A3 domains, that were found in families diagnosed with VWD. Human Embryonic Kidney 293 (HEK293) cells were transiently transfected with plasmids encoding full-length wild-type VWF or mutant VWF. Six mutations in the A1-A3 domains were expressed. We found that all mutants, except one, showed impaired formation of elongated pseudo-Weibel-Palade bodies (WPB). In addition, two mutations also showed reduced numbers of pseudo-WPB, even in the heterozygous state, and increased endoplasmic reticulum retention, which is in accordance with the impaired regulated secretion seen in patients. Regulated secretion upon stimulation of transfected cells reproduced the in vivo situation, indicating that HEK293 cells expressing VWF variants found in patients with VWD can be used to properly assess defects in regulated secretion.
Assuntos
Mutação , Corpos de Weibel-Palade/metabolismo , Doenças de von Willebrand/metabolismo , Fator de von Willebrand/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Estrutura Terciária de Proteína , Corpos de Weibel-Palade/genética , Doenças de von Willebrand/genética , Doenças de von Willebrand/patologia , Fator de von Willebrand/genéticaRESUMO
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.
RESUMO
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.
Assuntos
Hepatectomia , Hepatopatias , Humanos , Camundongos , Animais , Hepatectomia/efeitos adversos , Hepatectomia/métodos , Regeneração Hepática/fisiologia , Fator XIII , Fígado/cirurgia , FibrinaRESUMO
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 NRD-21, 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.
RESUMO
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, â¼10 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.
Assuntos
Transtornos da Coagulação Sanguínea , Doença Hepática Induzida por Substâncias e Drogas , Falência Hepática , Camundongos , Animais , Acetaminofen/metabolismo , Trombina/metabolismo , Falência Hepática/metabolismo , Falência Hepática/patologia , Fígado/metabolismo , Fibrina/metabolismo , Transtornos da Coagulação Sanguínea/induzido quimicamente , Transtornos da Coagulação Sanguínea/metabolismo , Fibrinogênio/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Camundongos Endogâmicos C57BLRESUMO
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 αIIbß3 binding domain in APAP-induced liver injury. METHODS: Mice expressing mutant fibrinogen incapable of engaging integrin αIIbß3 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 αIIbß3. 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.
Assuntos
Acetaminofen , Doença Hepática Crônica Induzida por Substâncias e Drogas , Animais , Camundongos , Fibrina/metabolismo , Fibrinogênio/genética , Fibrinogênio/metabolismo , Integrinas , LisinaRESUMO
Lipids contribute to hematopoiesis and membrane properties and dynamics, however, little is known about the role of lipids in megakaryopoiesis. Here, a lipidomic analysis of megakaryocyte progenitors, megakaryocytes, and platelets revealed a unique lipidome progressively enriched in polyunsaturated fatty acid (PUFA)-containing phospholipids. In vitro, inhibition of both exogenous fatty acid functionalization and uptake and 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 thrombocytopenia. 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 novel and viable target to modulate platelet counts.
RESUMO
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.
Assuntos
Plaquetas , Antígenos CD36 , Ácidos Graxos Insaturados , Megacariócitos , Fosfolipídeos , Trombopoese , Animais , Plaquetas/metabolismo , Trombopoese/fisiologia , Antígenos CD36/metabolismo , Antígenos CD36/genética , Fosfolipídeos/metabolismo , Megacariócitos/metabolismo , Megacariócitos/citologia , Humanos , Ácidos Graxos Insaturados/metabolismo , Camundongos Knockout , Trombocitopenia/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Contagem de Plaquetas , Células Cultivadas , Feminino , Camundongos , Lipidômica , Células Progenitoras de Megacariócitos/metabolismo , Células Progenitoras de Megacariócitos/citologiaRESUMO
BACKGROUND: Mutations of cysteine residues in von Willebrand factor are known to reduce the storage and secretion of this factor, thus leading to reduced antigen levels. However, one cysteine mutation, p.Cys2773Ser, has been found in patients with type 2A(IID) von Willebrand's disease who have normal plasma levels of von Willebrand factor. We hypothesize that disruption of either intra- or interchain disulfide bonds by cysteine mutations in von Willebrand factor has different effects on the biogenesis of Weibel-Palade bodies. DESIGN AND METHODS: The effect of specific cysteine mutations that either disrupt intrachain (p.Cys1130Phe and p.Cys2671Tyr) or interchain (p.Cys2773Ser) disulfide bonds on storage and secretion of von Willebrand factor was studied by transient transfection of human embryonic kidney cell line 293. Upon expression of von Willebrand factor these cells formed endothelial Weibel-Palade body-like organelles called pseudo-Weibel-Palade bodies. Storage of von Willebrand factor was analyzed with both confocal immunofluorescence and electron microscopy. Regulated secretion of von Willebrand factor was induced by phorbol 12-myristate 13-acetate. RESULTS: p.Cys1130Phe and p.Cys2671Tyr reduced the storage of von Willebrand factor into pseudo-Weibel-Palade bodies with notable retention of von Willebrand factor in the endoplasmic reticulum, whereas p.Cys2773Ser-von Willebrand factor was stored normally. As expected, wild-type von Willebrand factor formed proteinaceous tubules that were seen under electron microscopy as longitudinal striations in pseudo-Weibel-Palade bodies. p.Cys2773Ser caused severe defects in von Willebrand factor multimerization but the factor formed normal tubules. Furthermore, the basal and regulated secretion of von Willebrand factor was drastically impaired by p.Cys1130Phe and p.Cys2671Tyr, but not by p.Cys2773Ser. CONCLUSIONS: We postulate that natural mutations of cysteines involved in the formation of interchain disulfide bonds do not affect either the storage in Weibel-Palade bodies or secretion of von Willebrand factor, whereas mutations of cysteines forming intrachain disulfide bonds lead to reduced von Willebrand factor storage and secretion because the von Willebrand factor is retained in the endoplasmic reticulum.
Assuntos
Retículo Endoplasmático/genética , Mutação , Corpos de Weibel-Palade/genética , Fator de von Willebrand/genética , Cisteína/química , Cisteína/genética , Dissulfetos/química , Dissulfetos/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Plasmídeos , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética , Serina/química , Serina/genética , Transfecção , Tirosina/química , Tirosina/genética , Corpos de Weibel-Palade/química , Corpos de Weibel-Palade/patologia , Doenças de von Willebrand/genética , Doenças de von Willebrand/metabolismo , Doenças de von Willebrand/patologia , Fator de von Willebrand/químicaRESUMO
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.
Assuntos
Hepatopatias , Trombose , Doenças de von Willebrand , Proteína ADAMTS13 , Plaquetas , Hemostasia , Humanos , Hepatopatias/tratamento farmacológico , Fator de von WillebrandRESUMO
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.
Assuntos
Colestase/fisiopatologia , Cirrose Hepática/fisiopatologia , Fator de von Willebrand/genética , 1-Naftilisotiocianato , Doença Aguda , Animais , Plaquetas/metabolismo , Colestase/genética , Doença Crônica , Modelos Animais de Doenças , Feminino , Cirrose Hepática/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Inhibition of von Willebrand factor (VWF) expression in endothelial cells results in enhanced, possible dysfunctional angiogenesis, consistent with observations of severe gastrointestinal bleedings caused by vascular malformations in patients with von Willebrand disease (VWD). VWF is stored in endothelial Weibel-Palade bodies (WPB) with several other mediators of angiogenesis, like angiopoietin-2, osteoprotegerin and galectin-3. Increased release of angiopoietin-2 has been observed in medium of endothelial cells lacking VWF, but data on circulating levels of angiogenic factors in patients with VWD are lacking. The aim of this study was therefore to investigate plasma levels of angiogenic factors in patients with various types of VWD to obtain more insight into the pathogenesis of vascular malformations in these patients. We hypothesized that VWF deficiency leads to increased circulating levels of other WPB components. We therefore measured plasma levels of the WPB components angiopoietin-2, osteoprotegerin and galectin-3 as well as two other angiogenic factors (angiopoietin-1 and vascular endothelial growth factor [VEGF]) that are not stored within WPB. We observed that various angiogenic mediators are significantly different between types of VWD patients. Type 2A VWD patients had higher angiopoietin-1 levels compared with type 2B patients. Patients who have increased VWF clearance had higher angiopoietin-2 levels, whereas patients who have impaired VWF synthesis had higher galectin-3 levels. VEGF levels were negatively associated with VWF levels as type 3 VWD patients had the highest VEGF levels. However, complete VWF deficiency did not lead to increased circulating levels of other WPB components.