RESUMO
Inflammasome activation and subsequent pyroptosis are critical defense mechanisms against microbes. However, overactivation of inflammasome leads to death of the host. Although recent studies have uncovered the mechanism of pyroptosis following inflammasome activation, how pyroptotic cell death drives pathogenesis, eventually leading to death of the host, is unknown. Here, we identified inflammasome activation as a trigger for blood clotting through pyroptosis. We have shown that canonical inflammasome activation by the conserved type III secretion system (T3SS) rod proteins from Gram-negative bacteria or noncanonical inflammasome activation by lipopolysaccharide (LPS) induced systemic blood clotting and massive thrombosis in tissues. Following inflammasome activation, pyroptotic macrophages released tissue factor (TF), an essential initiator of coagulation cascades. Genetic or pharmacological inhibition of TF abolishes inflammasome-mediated blood clotting and protects against death. Our data reveal that blood clotting is the major cause of host death following inflammasome activation and demonstrate that inflammasome bridges inflammation with thrombosis.
Assuntos
Coagulação Sanguínea , Inflamassomos/metabolismo , Piroptose , Trombose/etiologia , Trombose/metabolismo , Animais , Infecções Bacterianas/complicações , Infecções Bacterianas/microbiologia , Biomarcadores , Caspases/metabolismo , Micropartículas Derivadas de Células/imunologia , Micropartículas Derivadas de Células/metabolismo , Modelos Animais de Doenças , Humanos , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Monócitos/imunologia , Monócitos/metabolismo , Transdução de Sinais , Tromboplastina/metabolismo , Trombose/sangue , Trombose/mortalidadeRESUMO
ABSTRACT: Hereditary angioedema (HAE), caused by C1 inhibitor protein deficiency, was recently shown to be associated with an increased risk for venous thromboembolism (VTE). To our knowledge, this is the first national family study of HAE, which aimed to determine the familial risk of VTE. The Swedish Multi-Generation Register was linked to the Swedish National Patient Register for the period of 1964 to 2018. Only patients with HAE with a validated diagnosis were included in the study and were linked to their family members. Hazard ratios (HRs) and 95% confidence intervals (CIs) for VTE were calculated for patients with HAE in comparison with relatives without HAE. Among 2006 individuals (from 276 pedigrees of 365 patients with HAE), 103 individuals were affected by VTE. In total, 35 (9.6%) patients with HAE were affected by VTE, whereas 68 (4.1%) non-HAE relatives were affected (P < .001). The adjusted HR for VTE among patients with HAE was 2.51 (95% CI, 1.67-3.77). Patients with HAE were younger at the first VTE than their non-HAE relatives (mean age, 51 years vs 63 years; P < .001). Before the age of 70 years, the HR for VTE among patients with HAE was 3.62 (95% CI, 2.26-5.80). The HR for VTE for patients with HAE born after 1964 was 8.29 (95% CI, 2.90-23.71). The HR for VTE for patients with HAE who were born in 1964 or earlier was 1.82 (95% CI, 1.14-2.91). HAE is associated with VTE among young and middle-aged individuals in Swedish families with HAE. The effect size of the association is in the order of other thrombophilias. We suggest that HAE may be considered a new rare thrombophilia.
Assuntos
Angioedemas Hereditários , Tromboembolia Venosa , Humanos , Angioedemas Hereditários/genética , Angioedemas Hereditários/epidemiologia , Angioedemas Hereditários/complicações , Feminino , Masculino , Tromboembolia Venosa/epidemiologia , Tromboembolia Venosa/genética , Tromboembolia Venosa/etiologia , Pessoa de Meia-Idade , Adulto , Suécia/epidemiologia , Fatores de Risco , Idoso , Linhagem , Sistema de Registros , Adulto Jovem , Família , AdolescenteRESUMO
C1 inhibitor (C1INH) is a multifunctional serine protease inhibitor that functions as a major negative regulator of several biological pathways, including the contact pathway of blood coagulation. In humans, congenital C1INH deficiency results in a rare episodic bradykinin-mediated swelling disorder called hereditary angioedema (HAE). Patients with C1INH deficiency-associated HAE (C1INH-HAE) have increased circulating markers of activation of coagulation. Furthermore, we recently reported that patients with C1INH-HAE had a moderate but significant increased risk of venous thromboembolism. To further investigate the impact of C1INH deficiency on activation of coagulation and thrombosis, we conducted studies using patient samples and mouse models. Plasmas from patients with C1INH-HAE had significantly increased contact pathway-mediated thrombin generation. C1INH-deficient mice, which have been used as a model of C1INH-HAE, had significantly increased baseline circulating levels of prothrombin fragment 1+2 and thrombin-antithrombin complexes. In addition, whole blood from C1INH-deficient mice supported significantly increased contact pathway-mediated thrombin generation. Importantly, C1INH-deficient mice exhibited significantly enhanced venous, but not arterial, thrombus formation. Furthermore, purified human C1INH normalized contact pathway-mediated thrombin generation and venous thrombosis in C1INH-deficient mice. These findings highlight a key role for endogenous C1INH as a negative regulator of contact pathway-mediated coagulation in humans and mice. Further, this work identifies endogenous C1INH as an important negative regulator of venous thrombus formation in mice, complementing the phenotype associated with C1INH-HAE.
Assuntos
Angioedemas Hereditários , Trombose , Trombose Venosa , Humanos , Animais , Camundongos , Angioedemas Hereditários/genética , Trombina , Proteína Inibidora do Complemento C1/genética , Coagulação Sanguínea , Trombose/etiologia , Trombose Venosa/etiologiaRESUMO
The blood-clotting protein fibrinogen has been implicated in host defense following Staphylococcus aureus infection, but precise mechanisms of host protection and pathogen clearance remain undefined. Peritonitis caused by staphylococci species is a complication for patients with cirrhosis, indwelling catheters, or undergoing peritoneal dialysis. Here, we sought to characterize possible mechanisms of fibrin(ogen)-mediated antimicrobial responses. Wild-type (WT) (Fib+) mice rapidly cleared S. aureus following intraperitoneal infection with elimination of â¼99% of an initial inoculum within 15 min. In contrast, fibrinogen-deficient (Fib-) mice failed to clear the microbe. The genotype-dependent disparity in early clearance resulted in a significant difference in host mortality whereby Fib+ mice uniformly survived whereas Fib- mice exhibited high mortality rates within 24 h. Fibrin(ogen)-mediated bacterial clearance was dependent on (pro)thrombin procoagulant function, supporting a suspected role for fibrin polymerization in this mechanism. Unexpectedly, the primary host initiator of coagulation, tissue factor, was found to be dispensable for this antimicrobial activity. Rather, the bacteria-derived prothrombin activator vWbp was identified as the source of the thrombin-generating potential underlying fibrin(ogen)-dependent bacterial clearance. Mice failed to eliminate S. aureus deficient in vWbp, but clearance of these same microbes in WT mice was restored if active thrombin was administered to the peritoneal cavity. These studies establish that the thrombin/fibrinogen axis is fundamental to host antimicrobial defense, offer a possible explanation for the clinical observation that coagulase-negative staphylococci are a highly prominent infectious agent in peritonitis, and suggest caution against anticoagulants in individuals susceptible to peritoneal infections.
Assuntos
Fibrinogênio/metabolismo , Peritonite/metabolismo , Protrombina/metabolismo , Animais , Antibacterianos/metabolismo , Anti-Infecciosos/metabolismo , Coagulação Sanguínea , Coagulase/metabolismo , Feminino , Fibrina/metabolismo , Fibrinogênio/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , TromboplastinaRESUMO
[Figure: see text].
Assuntos
Antineoplásicos/efeitos adversos , Arteriopatias Oclusivas/induzido quimicamente , Coagulação Sanguínea/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Trombose/induzido quimicamente , Trombose Venosa/induzido quimicamente , Animais , Arteriopatias Oclusivas/sangue , Arteriopatias Oclusivas/prevenção & controle , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Humanos , Terapia de Alvo Molecular/efeitos adversos , Neoplasias/sangue , Ativação Plaquetária/efeitos dos fármacos , Medição de Risco , Fatores de Risco , Transdução de Sinais , Trombose/sangue , Trombose/prevenção & controle , Trombose Venosa/sangue , Trombose Venosa/prevenção & controleRESUMO
OBJECTIVE: Patients with coronavirus disease 2019 (COVID-19) have a high rate of thrombosis. We hypothesized that severe acute respiratory syndrome coronavirus 2 infection leads to induction of TF (tissue factor) expression and increased levels of circulating TF-positive extracellular vesicles (EV) that may drive thrombosis. Approach and Results: We measured levels of plasma EV TF activity in 100 patients with COVID-19 with moderate and severe disease and 28 healthy controls. Levels of EV TF activity were significantly higher in patients with COVID-19 compared with controls. In addition, levels of EV TF activity were associated with disease severity and mortality. Finally, levels of EV TF activity correlated with several plasma markers, including D-dimer, which has been shown to be associated with thrombosis in patients with COVID-19. CONCLUSIONS: Our results indicate that severe acute respiratory syndrome coronavirus 2 infection induces the release of TF-positive EVs into the circulation that are likely to contribute to thrombosis in patients with COVID-19. EV TF activity was also associated with severity and mortality.
Assuntos
COVID-19/sangue , COVID-19/complicações , Vesículas Extracelulares/metabolismo , Idoso , Anticoagulantes/uso terapêutico , COVID-19/mortalidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , SARS-CoV-2 , Índice de Gravidade de Doença , Trombose/prevenção & controle , Trombose/virologiaRESUMO
OBJECTIVE: Quantitative relationships between the extent of injury and thrombus formation in vivo are not well understood. Moreover, it has not been investigated how increased injury severity translates to blood-flow modulation. Here, we investigated interconnections between injury length, clot growth, and blood flow in a mouse model of laser-induced thrombosis. Approach and Results: Using intravital microscopy, we analyzed 59 clotting events collected from the cremaster arteriole of 14 adult mice. We regarded injury length as a measure of injury severity. The injury caused transient constriction upstream and downstream of the injury site resulting in a 50% reduction in arteriole diameter. The amount of platelet accumulation and fibrin formation did not depend on arteriole diameter or deformation but displayed an exponentially increasing dependence on injury length. The height of the platelet clot depended linearly on injury length and the arteriole diameter. Upstream arteriolar constriction correlated with delayed upstream velocity increase, which, in turn, determined downstream velocity. Before clot formation, flow velocity positively correlated with the arteriole diameter. After the onset of thrombus growth, flow velocity at the injury site negatively correlated with the arteriole diameter and with the size of the above-clot lumen. CONCLUSIONS: Injury severity increased platelet accumulation and fibrin formation in a persistently steep fashion and, together with arteriole diameter, defined clot height. Arterial constriction and clot formation were characterized by a dynamic change in the blood flow, associated with increased flow velocity.
Assuntos
Músculos Abdominais/irrigação sanguínea , Arteríolas/patologia , Coagulação Sanguínea , Trombose/patologia , Lesões do Sistema Vascular/patologia , Animais , Arteríolas/lesões , Arteríolas/fisiopatologia , Velocidade do Fluxo Sanguíneo , Plaquetas/metabolismo , Constrição Patológica , Modelos Animais de Doenças , Fibrina/metabolismo , Microscopia Intravital , Masculino , Camundongos , Microscopia de Fluorescência , Índice de Gravidade de Doença , Trombose/sangue , Trombose/fisiopatologia , Fatores de Tempo , Lesões do Sistema Vascular/sangue , Lesões do Sistema Vascular/fisiopatologiaRESUMO
BACKGROUND: Puumala orthohantavirus (PUUV) causes hemorrhagic fever with renal syndrome (HFRS). Patients with HFRS have an activated coagulation system with increased risk of disseminated intravascular coagulation (DIC) and venous thromboembolism (VTE). The aim of the study was to determine whether circulating extracellular vesicle tissue factor (EVTF) activity levels associates with DIC and VTE (grouped as intravascular coagulation) in HFRS patients. METHODS: Longitudinal samples were collected from 88 HFRS patients. Patients were stratified into groups of those with intravascular coagulation (n = 27) and those who did not (n = 61). We measured levels of circulating EVTF activity, fibrinogen, activated partial prothrombin time, D-dimer, tissue plasminogen activator (tPA), plasminogen activator inhibitor 1 (PAI-1), and platelets. RESULTS: Plasma EVTF activity was transiently increased during HFRS. Levels of EVTF activity were significantly associated with plasma tPA and PAI-1, suggesting that endothelial cells could be a potential source. Patients with intravascular coagulation had significantly higher peak EVTF activity levels compared with those who did not, even after adjustment for sex and age. The peak EVTF activity value predicting intravascular coagulation was 0.51 ng/L with 63% sensitivity and 61% specificity with area under the curve = 0.63 (95% confidence interval, 0.51-0.76) and P = .046. CONCLUSIONS: Plasma EVTF activity during HFRS is associated with intravascular coagulation.
Assuntos
Coagulação Intravascular Disseminada/sangue , Vesículas Extracelulares/metabolismo , Febre Hemorrágica com Síndrome Renal/sangue , Tromboplastina/metabolismo , Adulto , Biomarcadores/sangue , Coagulação Sanguínea , Feminino , Fibrinólise , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Inibidor 1 de Ativador de Plasminogênio/sangue , Virus Puumala/patogenicidade , Sensibilidade e Especificidade , Ativador de Plasminogênio Tecidual/sangue , Tromboembolia Venosa/sangueRESUMO
Pancreatic cancer is associated with a high incidence of venous thromboembolism. Neutrophils have been shown to contribute to thrombosis in part by releasing neutrophil extracellular traps (NET). A recent study showed that increased plasma levels of the NET biomarker, citrullinated histone H3 (H3Cit), are associated with venous thromboembolism in patients with pancreatic and lung cancer but not in those with other types of cancer, including breast cancer. In this study, we examined the contribution of neutrophils and NET to venous thrombosis in nude mice bearing human pancreatic tumors. We found that tumor-bearing mice had increased circulating neutrophil counts and levels of granulocyte-colony stimulating factor, neutrophil elastase, H3Cit and cell-free DNA compared with controls. In addition, thrombi from tumor-bearing mice contained increased levels of the neutrophil marker Ly6G, as well as higher levels of H3Cit and cell-free DNA. Thrombi from tumor-bearing mice also had denser fibrin with thinner fibers consistent with increased thrombin generation. Importantly, either neutrophil depletion or administration of DNase I reduced the thrombus size in tumor-bearing but not in control mice. Our results, together with clinical data, suggest that neutrophils and NET contribute to venous thrombosis in patients with pancreatic cancer.
Assuntos
Armadilhas Extracelulares , Neoplasias Pancreáticas , Trombose Venosa , Animais , Humanos , Camundongos , Camundongos Nus , Neutrófilos , Trombose Venosa/etiologiaRESUMO
Activation of the intrinsic pathway of coagulation contributes to the pathogenesis of arterial and venous thrombosis. Critical insights into the involvement of intrinsic pathway factors have been derived from the study of gene-specific knockout animals and targeted inhibitors. Importantly, preclinical studies have indicated that targeting components of this pathway, including FXI (factor XI), FXII, and PKK (prekallikrein), reduces thrombosis with no significant effect on protective hemostatic pathways. This review highlights the advances made from studying the intrinsic pathway using gene-specific knockout animals and inhibitors in models of arterial and venous thrombosis. Development of inhibitors of activated FXI and FXII may reduce thrombosis with minimal increases in bleeding compared with current anticoagulant drugs.
Assuntos
Coagulação Sanguínea/fisiologia , Trombose/fisiopatologia , Animais , Anticoagulantes/efeitos adversos , Anticoagulantes/farmacologia , Anticoagulantes/uso terapêutico , Tempo de Sangramento , Coagulação Sanguínea/efeitos dos fármacos , Fatores de Coagulação Sanguínea/antagonistas & inibidores , Fatores de Coagulação Sanguínea/genética , Fatores de Coagulação Sanguínea/fisiologia , Modelos Animais de Doenças , Desenho de Fármacos , Ativação Enzimática , Hemorragia/induzido quimicamente , Humanos , Camundongos Knockout , Primatas , Coelhos , Ratos , Trombose/tratamento farmacológico , Trombose/epidemiologia , Trombose/prevenção & controleRESUMO
Activation of the blood coagulation cascade leads to fibrin deposition and platelet activation that are required for hemostasis. However, aberrant activation of coagulation can lead to thrombosis. Thrombi can cause tissue ischemia, and fibrin degradation products and activated platelets can enhance inflammation. In addition, coagulation proteases activate cells by cleavage of PARs (protease-activated receptors), including PAR1 and PAR2. Direct oral anticoagulants have recently been developed to specifically inhibit the coagulation proteases FXa (factor Xa) and thrombin. Administration of these inhibitors to wild-type mice can be used to determine the roles of FXa and thrombin in different inflammatory diseases. These results can be compared with the phenotypes of mice with deficiencies of either Par1 (F2r) or Par2 (F2rl1). However, inhibition of coagulation proteases will have effects beyond reducing PAR signaling, and a deficiency of PARs will abolish signaling from all proteases that activate these receptors. We will summarize studies that examine the roles of coagulation proteases, particularly FXa and thrombin, and PARs in different mouse models of inflammatory disease. Targeting FXa and thrombin or PARs may reduce inflammatory diseases in humans.
Assuntos
Coagulação Sanguínea , Modelos Animais de Doenças , Fator Xa/fisiologia , Inflamação/etiologia , Receptores Ativados por Proteinase/fisiologia , Trombina/fisiologia , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/etiologia , Animais , Apolipoproteínas E/fisiologia , Aterosclerose/tratamento farmacológico , Aterosclerose/etiologia , Inibidores do Fator Xa/uso terapêutico , Inflamação/tratamento farmacológico , Camundongos , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/etiologia , Trombina/antagonistas & inibidoresRESUMO
Murine models are widely used valuable tools to study deep vein thrombosis. Leading experts in venous thrombosis research came together through the American Venous Forum to develop a consensus on maximizing the utility and application of available mouse models of venous thrombosis. In this work, we provide an algorithm for model selection, with discussion of the advantages, disadvantages, and applications of the main mouse models of venous thrombosis. Additionally, we provide a detailed surgical description of the models with guidelines to validate surgical technique.
Assuntos
Modelos Animais de Doenças , Camundongos , Trombose Venosa , Algoritmos , Animais , Cloretos/toxicidade , Eletrólise , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/patologia , Compostos Férricos/toxicidade , Radicais Livres , Hemorreologia , Ligadura , Recidiva , Projetos de Pesquisa , Veias/cirurgia , Trombose Venosa/induzido quimicamente , Trombose Venosa/etiologia , Trombose Venosa/fisiopatologia , VênulasRESUMO
The ferric chloride models of arterial thrombosis are useful tools with which to investigate the cellular and molecular mechanisms that contribute to arterial thrombosis. Recent insights have, however, revealed the complex and multifaceted mechanism by which ferric chloride induces thrombus formation. Here, we discuss the strengths and weaknesses of the ferric chloride models of arterial thrombosis. Particular focus is given to the phenotypes of different knockout mice in the ferric chloride models and how these compare to other models with independent modes of initiation. Further, we discuss the relevance of the ferric chloride models to the human pathology of atherothrombotic disease.
Assuntos
Trombose das Artérias Carótidas/metabolismo , Cloretos/toxicidade , Modelos Animais de Doenças , Eritrócitos/metabolismo , Compostos Férricos/toxicidade , Animais , Anticoagulantes/farmacologia , Anticoagulantes/uso terapêutico , Plaquetas/patologia , Trombose das Artérias Carótidas/induzido quimicamente , Cloretos/metabolismo , Compostos Férricos/metabolismo , Humanos , Camundongos , Camundongos KnockoutAssuntos
Aterosclerose , Trombose Venosa , Animais , Camundongos , Trombose Venosa/etiologia , InflamaçãoRESUMO
Tissue factor (TF) is the high-affinity receptor and cofactor for factor (F)VII/VIIa. The TF-FVIIa complex is the primary initiator of blood coagulation and plays an essential role in hemostasis. TF is expressed on perivascular cells and epithelial cells at organ and body surfaces where it forms a hemostatic barrier. TF also provides additional hemostatic protection to vital organs, such as the brain, lung, and heart. Under pathological conditions, TF can trigger both arterial and venous thrombosis. For instance, atherosclerotic plaques contain high levels of TF on macrophage foam cells and microvesicles that drives thrombus formation after plaque rupture. In sepsis, inducible TF expression on monocytes leads to disseminated intravascular coagulation. In cancer patients, tumors release TF-positive microvesicles into the circulation that may contribute to venous thrombosis. TF also has nonhemostatic roles. For instance, TF-dependent activation of the coagulation cascade generates coagulation proteases, such as FVIIa, FXa, and thrombin, which induce signaling in a variety of cells by cleavage of protease-activated receptors. This review will focus on the roles of TF in protective hemostasis and pathological thrombosis.
Assuntos
Hemostasia , Tromboplastina/metabolismo , Trombose/sangue , Animais , Aterosclerose/sangue , Aterosclerose/complicações , Coagulação Sanguínea , Fator IX/metabolismo , Fator VIIa/metabolismo , Fator X/metabolismo , Fibrinolíticos/uso terapêutico , Regulação da Expressão Gênica , Hemostasia/efeitos dos fármacos , Humanos , Neoplasias/sangue , Neoplasias/complicações , Fatores de Risco , Sepse/sangue , Sepse/complicações , Transdução de Sinais , Tromboplastina/antagonistas & inibidores , Tromboplastina/genética , Trombose/tratamento farmacológico , Trombose/etiologia , Trombose/genéticaRESUMO
OBJECTIVE: The anti-cancer anthracycline drug Doxorubicin (Dox) causes cardiotoxicity. We investigated the role of protease-activated receptor 1 (PAR-1) in Dox-induced cardiotoxicity. METHODS AND RESULTS: In vitro experiments revealed that PAR-1 enhanced Dox-induced mitochondrial dysfunction, reactive oxygen species and cell death of cardiac myocytes and cardiac fibroblasts. The contribution of PAR-1 to Dox-induced cardiotoxicity was investigated by subjecting PAR-1-/- mice and PAR-1+/+ mice to acute and chronic exposure to Dox. Heart function was measured by echocardiography. PAR-1-/- mice exhibited significant less cardiac injury and dysfunction compared to PAR-1+/+ mice after acute and chronic Dox administration. PAR-1-/- mice had reduced levels of nitrotyrosine, apoptosis and inflammation in their heart compared to PAR-1+/+ mice. Furthermore, inhibition of PAR-1 in wild-type mice with vorapaxar significantly reduced the acute Dox-induced cardiotoxicity. CONCLUSION: Our results indicate that activation of PAR-1 contributes to Dox-induced cardiotoxicity. Inhibition of PAR-1 may be a new approach to reduce Dox-induced cardiotoxicity in cancer patients.
Assuntos
Antibióticos Antineoplásicos/efeitos adversos , Cardiotoxicidade/etiologia , Cardiotoxicidade/metabolismo , Doxorrubicina/efeitos adversos , Receptor PAR-1/metabolismo , Análise de Variância , Animais , Apoptose/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ecocardiografia , Fibroblastos/metabolismo , Traumatismos Cardíacos/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/citologia , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Thiol isomerases such as protein-disulfide isomerase (PDI) direct disulfide rearrangements required for proper folding of nascent proteins synthesized in the endoplasmic reticulum. Identifying PDI substrates is challenging because PDI catalyzes conformational changes that cannot be easily monitored (e.g. compared with proteolytic cleavage or amino acid phosphorylation); PDI has multiple substrates; and it can catalyze either oxidation, reduction, or isomerization of substrates. Kinetic-based substrate trapping wherein the active site motif CGHC is modified to CGHA to stabilize a PDI-substrate intermediate is effective in identifying some substrates. A limitation of this approach, however, is that it captures only substrates that are reduced by PDI, whereas many substrates are oxidized by PDI. By manipulating the highly conserved -GH- residues in the CGHC active site of PDI, we created PDI variants with a slowed reaction rate toward substrates. The prolonged intermediate state allowed us to identify protein substrates that have biased affinities for either oxidation or reduction by PDI. Because extracellular PDI is critical for thrombus formation but its extracellular substrates are not known, we evaluated the ability of these bidirectional trapping PDI variants to trap proteins released from platelets and on the platelet surface. Trapped proteins were identified by mass spectroscopy. Of the trapped substrate proteins identified by mass spectroscopy, five proteins, cathepsin G, glutaredoxin-1, thioredoxin, GP1b, and fibrinogen, showed a bias for oxidation, whereas annexin V, heparanase, ERp57, kallekrein-14, serpin B6, tetranectin, and collagen VI showed a bias for reduction. These bidirectional trapping variants will enable more comprehensive identification of thiol isomerase substrates and better elucidation of their cellular functions.
Assuntos
Plaquetas/enzimologia , Isomerases de Dissulfetos de Proteínas/química , Domínio Catalítico , Humanos , Cinética , Isomerases de Dissulfetos de Proteínas/metabolismo , Especificidade por SubstratoRESUMO
Deep vein thrombosis and common complications, including pulmonary embolism and post-thrombotic syndrome, represent a major source of morbidity and mortality worldwide. Experimental models of venous thrombosis have provided considerable insight into the cellular and molecular mechanisms that regulate thrombus formation and subsequent resolution. Here, we critically appraise the ex vivo and in vivo techniques used to assess venous thrombosis in these models. Particular attention is paid to imaging modalities, including magnetic resonance imaging, micro-computed tomography, and high-frequency ultrasound that facilitate longitudinal assessment of thrombus size and composition.
Assuntos
Veias , Trombose Venosa/diagnóstico , Animais , Biomarcadores/metabolismo , Velocidade do Fluxo Sanguíneo , Diagnóstico por Imagem/métodos , Valor Preditivo dos Testes , Prognóstico , Fluxo Sanguíneo Regional , Veias/metabolismo , Veias/patologia , Veias/fisiopatologia , Trombose Venosa/metabolismo , Trombose Venosa/patologia , Trombose Venosa/fisiopatologiaRESUMO
Surgery is associated with an increased risk of venous thromboembolic events (VTE) including deep vein thrombosis and pulmonary embolism. Although the current treatment regiments such as mechanical manipulation and administration of pharmacological prophylaxis significantly reduced the incidence of postsurgical VTE, they remain a major cause of postoperative morbidity and mortality worldwide. The pathophysiology of venous thrombosis traditionally emphasizes the series of factors that constitute Virchow triad of factors. However, inflammation can also be a part of this by giving rise to a hypercoagulable state and endothelial damage. The inflammatory response after surgery, which is initiated by a cytokine "storm" and occurs within hours of surgery, creates a prothrombotic environment that is further accentuated by several cellular processes including neutrophil extracellular traps formation, platelet activation, and the generation of tissue factor-bearing microparticles. Although such inflammatory markers are elevated in undergoing surgery, the precise mechanism by which they give rise to venous thrombosis is poorly understood. Here, we discuss the potential mechanisms linking inflammation to thrombosis, and highlight strategies that may minimize surgical inflammation and reduce the incidence of postoperative VTE.