RESUMO
Neuroinflammation is assumed as the critical pathophysiologic mechanism of white matter lesions (WMLs), and infiltrated peripheral monocyte-derived macrophages are implicated in the development of neuroinflammation. This study sought to explore the blood molecules that promote the migration of peripheral monocytes to the sites of WMLs. The serum protein expression profiles of patients and Sprague-Dawley rat models with WMLs were detected by data-independent acquisition (DIA) proteomics technique. Compared with corresponding control groups, we acquired 62 and 41 differentially expressed proteins (DEPs) in the serum of patients and model rats with WMLs respectively. Bioinformatics investigations demonstrated that these DEPs were linked to various Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms involved in neuroinflammation. Afterward, we identified thrombin-activatable fibrinolysis inhibitor (TAFI) as a shared and overexpressed protein in clinical and animal serum samples, which was further verified by enzyme-linked immunosorbent assay. Additionally, an upregulation of TAFI was also observed in the white matter of rat models, and the inhibition of TAFI impeded the migration of peripheral monocytes to the area of WMLs. In vitro experiments suggested that TAFI could enhance the migration ability of RAW264.7 cells and increase the expression of Ccr2. Our study demonstrates that neuroinflammatory signals can be detected in the peripheral blood of WMLs patients and model rats. TAFI may serve as a potential protein that promotes the migration of peripheral monocytes to WMLs regions, thereby providing a novel molecular target for further investigation into the interaction between the central and peripheral immune systems.
Assuntos
Carboxipeptidase B2 , Substância Branca , Humanos , Ratos , Animais , Fibrinólise/fisiologia , Carboxipeptidase B2/genética , Carboxipeptidase B2/metabolismo , Doenças Neuroinflamatórias , Monócitos/metabolismo , Proteômica , Substância Branca/metabolismo , Ratos Sprague-Dawley , Trombina/metabolismo , Trombina/farmacologiaRESUMO
BACKGROUND: Thrombin is a critical protease modulating thrombosis as well as inflammation, which are one of the main pathophysiological mechanisms in sickle vasculopathy, and its levels were reported to be high in sickle cell disease (SCD). The thrombin-thrombomodulin complex activates the TAFI inhibitor of fibrinolysis, which acts by reducing plasmin affinity for its substrate thus hindering fibrinolysis. OBJECTIVE: We aimed to determine the influence of the Thr325Ile single nucleotide polymorphism (SNP) on TAFI antigen levels and potential effects on the severity of SCD in a cohort of Egyptian patients. METHODS: Genotyping of Thr325lle polymorphism using Taq-Man SNP genotyping assay and TAFI level measurement using an enzyme-linked immunosorbent assay were performed for 80 SCD patients (45 homozygous HbSS, 16 S/ß0 and 19 Sß+) as well as 80 age- and gender-matched healthy control subjects. RESULTS: Plasma TAFI levels were higher in SCD patients with Thr325Ile polymorphism, yet the difference was not statistically significant (p = .204). SCD patients with polymorphic genotypes had a greater number of hospital admissions (p = .03). Ten patients with acute chest syndrome had the homozygous polymorphic genotype (GG), and all patients with pulmonary hypertension had the polymorphic genotype (six were homozygous [GG] and five were heterozygous [GA]). Patients with SCD complicated with pulmonary hypertension showed significantly higher plasma TAFI levels (p = .044). CONCLUSION: The analysis of Thr325Ile polymorphisms combined with plasma TAFI levels suggests that the analyzed SNP could influence plasma TAFL levels and SCD disease severity and hospitalization rates, which could be predictors for complex disease.
Assuntos
Anemia Falciforme , Carboxipeptidase B2 , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Adulto Jovem , Anemia Falciforme/genética , Anemia Falciforme/sangue , Carboxipeptidase B2/genética , Carboxipeptidase B2/sangue , Estudos de Casos e Controles , Estudos de Coortes , Egito , Genótipo , Prognóstico , Índice de Gravidade de DoençaRESUMO
Rivaroxaban is a direct factor Xa inhibitor, recently implemented as a favorable alternative to warfarin in anticoagulation therapy. Rivaroxaban effectively reduces thrombin generation, which plays a major role in the activation of thrombin activatable fibrinolysis inhibitor (TAFI) to TAFIa. Based on the antifibrinolytic role of TAFIa, we hypothesized that rivaroxaban would consequently induce more rapid clot lysis. In vitro clot lysis assays were used to explore this hypothesis and additionally determine the effects of varying TAFI levels and a stabilizing Thr325Ile polymorphism (rs1926447) in the TAFI protein on the effects of rivaroxaban. Rivaroxaban was shown to decrease thrombin generation, resulting in less TAFI activation, thus enhancing lysis. These effects were also shown to be less substantial in the presence of greater TAFI levels or the more stable Ile325 enzyme. These findings suggest a role for TAFI levels and the Thr325Ile polymorphism in the pharmacodynamics and pharmacogenomics of rivaroxaban.
Assuntos
Carboxipeptidase B2 , Humanos , Carboxipeptidase B2/genética , Carboxipeptidase B2/farmacologia , Rivaroxabana/farmacologia , Fibrinólise , Trombina/metabolismo , MutaçãoRESUMO
Carboxypeptidase U (CPU, TAFIa, CPB2) is a potent attenuator of fibrinolysis that is mainly synthesized by the liver as its inactive precursor proCPU. Aside from its antifibrinolytic properties, evidence exists that CPU can modulate inflammation, thereby regulating communication between coagulation and inflammation. Monocytes and macrophages play a central role in inflammation and interact with coagulation mechanisms resulting in thrombus formation. The involvement of CPU and monocytes/macrophages in inflammation and thrombus formation, and a recent hypothesis that proCPU is expressed in monocytes/macrophages, prompted us to investigate human monocytes and macrophages as a potential source of proCPU. CPB2 mRNA expression and the presence of proCPU/CPU protein were studied in THP-1, PMA-stimulated THP-1 cells and primary human monocytes, M-CSF-, IFN-γ/LPS-, and IL-4-stimulated-macrophages by RT-qPCR, Western blotting, enzyme activity measurements, and immunocytochemistry. CPB2 mRNA and proCPU protein were detected in THP-1 and PMA-stimulated THP-1 cells as well as in primary monocytes and macrophages. Moreover, CPU was detected in the cell medium of all investigated cell types and it was demonstrated that proCPU can be activated into functionally active CPU in the in vitro cell culture environment. Comparison of CPB2 mRNA expression and proCPU concentrations in the cell medium between the different cell types provided evidence that CPB2 mRNA expression and proCPU secretion in monocytes and macrophages is related to the degree to which these cells are differentiated. Our results indicate that primary monocytes and macrophages express proCPU. This sheds new light on monocytes and macrophages as local proCPU sources.
Assuntos
Carboxipeptidase B2 , Macrófagos , Monócitos , Humanos , Carboxipeptidase B2/genética , Carboxipeptidase B2/metabolismo , Diferenciação Celular/genética , Inflamação , Ativação de Macrófagos/genética , Macrófagos/metabolismo , Monócitos/metabolismo , RNA MensageiroRESUMO
Patients with hemophilia A display varied bleeding phenotypes not correlated with degree of deficiency of factor VIII level. We investigated Plasminogen Activator Inhibitor 1(PAI1) level and Thrombin Activatable Fibrinolysis Inhibitor (TAFI) also known as Carboxypeptidase B2 (CPB2) level in Patients with hemophilia A and their possible correlation with bleeding tendency. Twenty-six patients attending in hematology unit of pediatric department were included in this study. In addition, fourteen apparently healthy subjects matched ages and genders were included as control group. The International Society of Thrombosis Bleeding Assessment Tool (ISTH/BAT) was used to assess bleeding score in patients. Plasma levels of Plasminogen Activator Fibrinolysis Inhibitor (PAI1) and Thrombin Activatable Fibrinolysis Inhibitor (TAFI) zymogen were measured by enzyme-linked immunosorbent assay (ELIZA). As compared to controls, hemophilic patients had significantly high bleeding score, low PAI 1 level and high TAFI level. There was no significant correlation between bleeding score by ISTH/BAT and patient severity. PAI 1 and TAFI level have no significant correlation with patient severity. PAI 1 level was statistically significant different between intense and non-intense hemorrhagic groups, while TAFI level has no significant correlation with bleeding phenotype. PAI 1 and TAFI levels had significantly correlation between patients and controls. PAI-1 level had statistically significant correlation with bleeding phenotype, while TAFI level failed to show any correlation between intense and non-intense hemorrhagic groups. So, PAI-1 levels may have predictive value of bleeding tendency in hemophiliacs.
Assuntos
Carboxipeptidase B2 , Hemofilia A , Trombose , Carboxipeptidase B2/genética , Egito , Feminino , Fibrinólise , Hemorragia , Humanos , Masculino , Inibidor 1 de Ativador de Plasminogênio , TrombinaRESUMO
Thrombus formation remains a major cause of morbidity and mortality worldwide. Current antiplatelet and anticoagulant therapies have been effective at reducing vascular events, but at the expense of increased bleeding risk. Targeting proteins that interact with fibrinogen and which are involved in hypofibrinolysis represents a more specific approach for the development of effective and safe therapeutic agents. The antifibrinolytic proteins alpha-2 antiplasmin (α2AP), thrombin activatable fibrinolysis inhibitor (TAFI), complement C3 and plasminogen activator inhibitor-2 (PAI-2), can be incorporated into the fibrin clot by FXIIIa and affect fibrinolysis by different mechanisms. Therefore, these antifibrinolytic proteins are attractive targets for the development of novel therapeutics, both for the modulation of thrombosis risk, but also for potentially improving clot instability in bleeding disorders. This review summarises the main properties of fibrinogen-bound antifibrinolytic proteins, their effect on clot lysis and association with thrombotic or bleeding conditions. The role of these proteins in therapeutic strategies targeting the fibrinolytic system for thrombotic diseases or bleeding disorders is also discussed.
Assuntos
Carboxipeptidase B2/genética , Fibrinogênio/genética , Hemorragia/terapia , alfa 2-Antiplasmina/genética , Anticoagulantes , Complemento C3/genética , Fibrinólise/genética , Hemorragia/genética , Humanos , Inibidor 2 de Ativador de Plasminogênio/genética , Trombose/genéticaRESUMO
Procarboxypeptidase U (proCPU, TAFI, proCPB2) is a basic carboxypeptidase zymogen that is converted by thrombin(-thrombomodulin) or plasmin into the active carboxypeptidase U (CPU, TAFIa, CPB2), a potent attenuator of fibrinolysis. As CPU forms a molecular link between coagulation and fibrinolysis, the development of CPU inhibitors as profibrinolytic agents constitutes an attractive new concept to improve endogenous fibrinolysis or to increase the efficacy of thrombolytic therapy in thromboembolic diseases. Furthermore, extensive research has been conducted on the in vivo role of CPU in (the acute phase of) thromboembolic disease, as well as on the hypothesis that high proCPU levels and the Thr/Ile325 polymorphism may cause a thrombotic predisposition. In this paper, an overview is given of the methods available for measuring proCPU, CPU, and inactivated CPU (CPUi), together with a summary of the clinical data generated so far, ranging from the current knowledge on proCPU concentrations and polymorphisms as potential thromboembolic risk factors to the positioning of different CPU forms (proCPU, CPU, and CPUi) as diagnostic markers for thromboembolic disease, and the potential benefit of pharmacological inhibition of the CPU pathway.
Assuntos
Carboxipeptidase B2/metabolismo , Carboxipeptidase B2/fisiologia , Tromboembolia/metabolismo , Coagulação Sanguínea/fisiologia , Carboxipeptidase B2/genética , Fibrinolisina/metabolismo , Fibrinólise/fisiologia , Genótipo , Humanos , Trombina/metabolismo , Tromboembolia/fisiopatologia , Terapia Trombolítica/métodos , Trombose/metabolismoRESUMO
Joint bleeds are common in congenital hemophilia but rare in acquired hemophilia A (aHA) for reasons unknown. To identify key mechanisms responsible for joint-specific bleeding in congenital hemophilia, bleeding phenotypes after joint injury and tail transection were compared in aHA wild-type (WT) mice (receiving an anti-factor VIII [FVIII] antibody) and congenital HA (FVIII-/-) mice. Both aHA and FVIII-/- mice bled severely after tail transection, but consistent with clinical findings, joint bleeding was notably milder in aHA compared with FVIII-/- mice. Focus was directed to thrombin-activatable fibrinolysis inhibitor (TAFI) to determine its potentially protective effect on joint bleeding in aHA. Joint bleeding in TAFI-/- mice with anti-FVIII antibody was increased, compared with WT aHA mice, and became indistinguishable from joint bleeding in FVIII-/- mice. Measurements of circulating TAFI zymogen consumption after joint injury indicated severely defective TAFI activation in FVIII-/- mice in vivo, consistent with previous in vitro analyses in FVIII-deficient plasma. In contrast, notable TAFI activation was observed in aHA mice, suggesting that TAFI protected aHA joints against bleeding. Pharmacological inhibitors of fibrinolysis revealed that urokinase-type plasminogen activator (uPA)-induced fibrinolysis drove joint bleeding, whereas tissue-type plasminogen activator-mediated fibrinolysis contributed to tail bleeding. These data identify TAFI as an important modifier of hemophilic joint bleeding in aHA by inhibiting uPA-mediated fibrinolysis. Moreover, our data suggest that bleed protection by TAFI was absent in congenital FVIII-/- mice because of severely defective TAFI activation, underscoring the importance of clot protection in addition to clot formation when considering prohemostatic strategies for hemophilic joint bleeding.
Assuntos
Carboxipeptidase B2/metabolismo , Hemartrose/etiologia , Hemartrose/metabolismo , Hemofilia A/complicações , Animais , Carboxipeptidase B2/genética , Modelos Animais de Doenças , Deleção de Genes , Hemartrose/genética , Hemofilia A/genética , Hemofilia A/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
RATIONALE: Pulmonary hypertension is a fatal disease; however, its pathogenesis still remains to be elucidated. Thrombin-activatable fibrinolysis inhibitor (TAFI) is synthesized by the liver and inhibits fibrinolysis. Plasma TAFI levels are significantly increased in chronic thromboembolic pulmonary hypertension (CTEPH) patients. OBJECTIVE: To determine the role of activated TAFI (TAFIa) in the development of CTEPH. METHODS AND RESULTS: Immunostaining showed that TAFI and its binding partner thrombomodulin (TM) were highly expressed in the pulmonary arteries (PAs) and thrombus in patients with CTEPH. Moreover, plasma levels of TAFIa were increased 10-fold in CTEPH patients compared with controls. In mice, chronic hypoxia caused a 25-fold increase in plasma levels of TAFIa with increased plasma levels of thrombin and TM, which led to thrombus formation in PA, vascular remodeling, and pulmonary hypertension. Consistently, plasma clot lysis time was positively correlated with plasma TAFIa levels in mice. Additionally, overexpression of TAFIa caused organized thrombus with multiple obstruction of PA flow and reduced survival rate under hypoxia in mice. Bone marrow transplantation showed that circulating plasma TAFI from the liver, not in the bone marrow, was activated locally in PA endothelial cells through interactions with thrombin and TM. Mechanistic experiments demonstrated that TAFIa increased PA endothelial permeability, smooth muscle cell proliferation, and monocyte/macrophage activation. Importantly, TAFIa inhibitor and peroxisome proliferator-activated receptor-α agonists significantly reduced TAFIa and ameliorated animal models of pulmonary hypertension in mice and rats. CONCLUSIONS: These results indicate that TAFIa could be a novel biomarker and realistic therapeutic target of CTEPH.
Assuntos
Pressão Arterial , Carboxipeptidase B2/metabolismo , Hipertensão Pulmonar/etiologia , Fígado/metabolismo , Artéria Pulmonar/metabolismo , Tromboembolia/complicações , Adulto , Animais , Permeabilidade Capilar , Carboxipeptidase B2/deficiência , Carboxipeptidase B2/genética , Estudos de Casos e Controles , Proliferação de Células , Doença Crônica , Modelos Animais de Doenças , Feminino , Células Hep G2 , Humanos , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Hipertensão Pulmonar/prevenção & controle , Hipóxia/complicações , Fígado/efeitos dos fármacos , Ativação de Macrófagos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , PPAR alfa/agonistas , PPAR alfa/metabolismo , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , Pirimidinas/farmacologia , Ratos Sprague-Dawley , Transdução de Sinais , Trombina/metabolismo , Tromboembolia/metabolismo , Tromboembolia/fisiopatologia , Tromboembolia/prevenção & controle , Trombomodulina/metabolismo , Transfecção , Regulação para CimaRESUMO
AIM: As angiogenesis is an essential step for chorionic villi formation. Vascular endothelial growth factor (VEGF) is essential for endothelial cell proliferation. Endothelial nitric oxide synthase (eNOS) is a powerful playmaker in hypoxia-induced angiogenesis. Thrombin-activatable fibrinolysis inhibitor (TAFI) regulates both fibrinolysis and inflammation. Genetic alterations of these factors may lead to recurrent spontaneous abortion (RSA). We aimed to investigate the combined genetic variants of VEGF G-1154A and two eNOS genetic variants: T-786C promoter region and intron 4 variable number of tandom repeats in addition to TAFI C-1040T among RSA patients. METHODS: The study included 50 patients with RSA and 50 healthy controls. Polymerase chain reaction and restriction fragment length polymorphism were used for genotyping. RESULTS: Both genetic alterations of eNOS confirmed at least a sixfold increase of RSA risk. Interestingly, they were associated with TAFI C-1040Tgenetic variant in 21 patients, eight of them had both studied eNOS genetic alterations and TAFI C-1040Tgenetic variant, while each eNOS genetic variant associated with TAFI C-1040Tconfirmed an almost one and half fold increase risk of RSA. CONCLUSION: These findings highlighted the role of eNOS and nitric oxide metabolism in RSA and opened the gate to investigate the interaction of vasoconstrictive and fibrinolytic inhibitor systems.
Assuntos
Aborto Habitual/genética , Carboxipeptidase B2/genética , Óxido Nítrico Sintase Tipo III/genética , Fator A de Crescimento do Endotélio Vascular/genética , Adolescente , Adulto , Estudos de Casos e Controles , Egito , Feminino , Humanos , Gravidez , Adulto JovemRESUMO
Factor XIII(a) [FXIII(a)] stabilizes clots and increases resistance to fibrinolysis and mechanical disruption. FXIIIa also mediates red blood cell (RBC) retention in contracting clots and determines venous thrombus size, suggesting FXIII(a) is a potential target for reducing thrombosis. However, the mechanism by which FXIIIa retains RBCs in clots is unknown. We determined the effect of FXIII(a) on human and murine clot weight and composition. Real-time microscopy revealed extensive RBC loss from clots formed in the absence of FXIIIa activity, and RBCs exhibited transient deformation as they exited the clots. Fibrin band-shift assays and flow cytometry did not reveal crosslinking of fibrin or FXIIIa substrates to RBCs, suggesting FXIIIa does not crosslink RBCs directly to the clot. RBCs were retained in clots from mice deficient in α2-antiplasmin, thrombin-activatable fibrinolysis inhibitor, or fibronectin, indicating RBC retention does not depend on these FXIIIa substrates. RBC retention in clots was positively correlated with fibrin network density; however, FXIIIa inhibition reduced RBC retention at all network densities. FXIIIa inhibition reduced RBC retention in clots formed with fibrinogen that lacks γ-chain crosslinking sites, but not in clots that lack α-chain crosslinking sites. Moreover, FXIIIa inhibitor concentrations that primarily block α-, but not γ-, chain crosslinking decreased RBC retention in clots. These data indicate FXIIIa-dependent retention of RBCs in clots is mediated by fibrin α-chain crosslinking. These findings expose a newly recognized, essential role for fibrin crosslinking during whole blood clot formation and consolidation and establish FXIIIa activity as a key determinant of thrombus composition and size.
Assuntos
Fatores de Coagulação Sanguínea/metabolismo , Coagulação Sanguínea/fisiologia , Eritrócitos/metabolismo , gama-Glutamiltransferase/metabolismo , Animais , Fatores de Coagulação Sanguínea/genética , Carboxipeptidase B2/genética , Carboxipeptidase B2/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Transtornos Hemorrágicos/genética , Transtornos Hemorrágicos/metabolismo , Humanos , Camundongos , Camundongos Knockout , alfa 2-Antiplasmina/deficiência , alfa 2-Antiplasmina/genética , alfa 2-Antiplasmina/metabolismo , gama-Glutamiltransferase/genéticaRESUMO
OBJECTIVE: The pathogenesis of chronic thromboembolic pulmonary hypertension (CTEPH) remains to be elucidated. Thrombin-activatable fibrinolysis inhibitor (TAFI) inhibits fibrinolysis. It remains to be elucidated whether TAFI is directly involved in the pathogenesis of CTEPH. We examined potential involvement of TAFI in the pathogenesis of CTEPH in humans. APPROACH AND RESULTS: We enrolled 68 consecutive patients undergoing right heart catheterization in our hospital, including those with CTEPH (n=27), those with pulmonary arterial hypertension (n=22), and controls (non-pulmonary hypertension, n=19). Whole blood clot lysis assay showed that the extent of clot remaining after 4 hours was significantly higher in CTEPH compared with pulmonary arterial hypertension or controls (41.9 versus 26.5 and 24.6%, both P<0.01). Moreover, plasma levels of TAFI were significantly higher in CTEPH than in pulmonary arterial hypertension or controls (19.4±4.2 versus 16.1±4.5 or 16.3±3.3 µg/mL, both P<0.05), which remained unchanged even after hemodynamic improvement by percutaneous transluminal pulmonary angioplasty. Furthermore, the extent of clot remaining after 4 hours was significantly improved with CPI-2KR (an inhibitor of activated TAFI) or prostaglandin E1 (an inhibitor of activation of platelets). Importantly, plasma levels of TAFI were significantly correlated with the extent of clot remaining after 4 hours. In addition, the extent of clot remaining after 4 hours was improved with an activated TAFI inhibitor. CONCLUSIONS: These results indicate that plasma levels of TAFI are elevated in patients with CTEPH and are correlated with resistance to clot lysis in those patients.
Assuntos
Plaquetas/enzimologia , Carboxipeptidase B2/sangue , Fibrinólise , Hipertensão Pulmonar/sangue , Embolia Pulmonar/sangue , Adulto , Idoso , Biomarcadores/sangue , Testes de Coagulação Sanguínea , Plaquetas/efeitos dos fármacos , Carboxipeptidase B2/antagonistas & inibidores , Carboxipeptidase B2/genética , Cateterismo Cardíaco , Estudos de Casos e Controles , Doença Crônica , Feminino , Fibrinólise/efeitos dos fármacos , Frequência do Gene , Humanos , Hipertensão Pulmonar/diagnóstico , Hipertensão Pulmonar/enzimologia , Hipertensão Pulmonar/etiologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Inibidores de Proteases/farmacologia , Embolia Pulmonar/complicações , Embolia Pulmonar/diagnóstico , Embolia Pulmonar/enzimologia , Fatores de Tempo , Regulação para CimaRESUMO
The mechanisms of hepatitis C virus (HCV)-associated hepatocarcinogenesis and disease progression are unclear. We previously observed that the expression level of carboxypeptidase B2 (CPB2) gene was remarkably suppressed by persistent HCV RNA replication in human hepatoma cell line Li23- derived cells. The results of the present study demonstrated that the CPB2 expression in patients with chronic hepatitis C was inversely correlated with several risk factors of hepatic fibrosis or steatosis, although ectopic CPB2 expression did not suppress the expression of fibrogenic or lipogenic genes. The suppressed CPB2 expression was restored by treatment with 5-azacytidine. To clarify the mechanism underlying this phenomenon, we analyzed the CPB2 promoter, and the results revealed that (1) hepatocyte nuclear factor 1 (HNF1), especially HNF1α, was essential for the CPB2 promoter, and (2) CPB2 promoter was not methylated by persistent HCV RNA replication. The expression levels of HNF1α and HNF1ß were also not changed by persistent HCV RNA replication. These results suggest the existence of 5-azacytidine-inducible or -reducible unknown factor(s) that can control the CPB2 expression. To evaluate this idea we performed a microarray analysis, and several gene candidates corresponding to the suggested factor(s) were identified.
Assuntos
Carboxipeptidase B2/metabolismo , Hepacivirus/fisiologia , Hepatite C/metabolismo , RNA Viral/fisiologia , Replicação Viral/fisiologia , Azacitidina/farmacologia , Carboxipeptidase B2/genética , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Hepatite C/patologia , Hepatite C/virologia , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 1-beta Nuclear de Hepatócito/genética , Fator 1-beta Nuclear de Hepatócito/metabolismo , Humanos , MicroRNAs , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras GenéticasRESUMO
Cardiovascular and cerebrovascular diseases (CCVDs) are common and have high rates of morbidity, mortality, and recurrence. Thrombin-activatable fibrinolysis inhibitor (TAFI) is also known as carboxypeptidase B2 and is encoded by the CPB2 gene; CPB2 polymorphisms have been explored in a variety of studies, but their correlation to the risk of CCVDs remains ambiguous. We examined the hypothesized associations between CPB2 mutations and CCVDs in a general population. We searched, Embase, the Cumulative Index to Nursing and Allied Health Literature, the Science Citation Index, and several Chinese databases without applying any language restrictions. Nine case-control studies were analyzed in the current meta-analysis, and odds ratios (ORs) with their 95% confidence intervals were calculated. The pooled ORs indicated that the CPB2 rs3742264 G>A polymorphism was associated with an increased risk of CCVDs in the allele model (all P values < 0.05). A similar result for the CPB2 rs1926447 C>T polymorphism and CCVDs risk was detected in the allele model (P < 0.05). Ethnicity subgroup analysis implied that the rs3742264 G>A polymorphism was more likely to lead to the development of cerebrovascular disease in Asians (all P values < 0.05), whereas rs1926447 C>T was associated with cardiovascular disease among Africans (all P values < 0.05). These data suggest that the polymorphisms investigated, especially rs3742264 G>A and rs1926447 C>T, have a modest effect on susceptibility to CCVDs.
Assuntos
Carboxipeptidase B2/genética , Doenças Cardiovasculares/genética , Transtornos Cerebrovasculares/genética , Polimorfismo de Nucleotídeo Único , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Thrombin-activatable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like proenzyme biosynthesized in the liver and released into the blood circulation. Activated TAFI (TAFIa) has been implicated as an important player in maintaining the balance between blood coagulation and fibrinolysis. In the present study, regulation of TAFI (CPB2) gene expression was investigated using cultured human hepatoma HepG2 cells. HepG2 cells were treated with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, and the levels of TAFI antigen and CPB2 mRNA were measured. HepG2 cells treated with LY29400 decreased their release of TAFI antigen into the conditioned medium (CM). In parallel, there were decreased levels of CPB2 mRNA and TAFI antigen in the cells. However, CPB2 gene promoter activity was not influenced by treatment of the cells with LY294002. The half-life of the CPB2 transcript was shortened by treatment with LY294002 compared with control. The present results suggest that the PI3K inhibitor LY294002 suppresses expression of TAFI, a prothrombotic factor, by decreasing the stability of CPB2 transcripts.
Assuntos
Carboxipeptidase B2/genética , Cromonas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Células Hep G2 , Humanos , RNA Mensageiro/metabolismoRESUMO
A series of 4,5,6,7-tetrahydro-1H-benzimidazole-5-carboxylic acid and 5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-7-carboxylic acid derivatives designed as inhibitors of TAFIa has been prepared via a common hydrogenation-alkylation sequence starting from the appropriate benzimidazole and imidazopyridine system. We present a successful design strategy using a conformational restriction approach resulting in potent and selective inhibitors of TAFIa. The X-ray structure of compound 5 in complex with a H333Y/H335Q double mutant TAFI indicate that the conformational restriction is responsible for the observed potency increase.
Assuntos
Benzimidazóis/farmacologia , Carboxipeptidase B2/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Piridinas/farmacologia , Benzimidazóis/síntese química , Benzimidazóis/química , Células CACO-2 , Carboxipeptidase B2/genética , Carboxipeptidase B2/metabolismo , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Conformação Molecular , Piridinas/síntese química , Piridinas/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Atherosclerosis is the leading etiologic factor of Atherosclerotic Cerebral Infarction (ACI). Previous studies have shown that thrombin activatable fibrinolysis inhibitor (TAFI) may play an important role in the occurrence of acute cerebral infarction, and the levels of TAFI are affected by several single nucleotide polymorphisms (SNPs) located in the regulatory and coding regions of the gene encoding TAFI. The present study aimed to determine whether polymorphisms (TAFI -2345 2G/1G, -1690 A/G, -438 A/G, +1583 A/T) of the TAFI gene were associated with ACI in a Han Chinese population. METHODS: The variant genotypes were identified by restriction fragment length polymorphism (RFLP) and allele-specific polymerase chain reactions (AS-PCR) in 225 patients with ACI and 184 age-matched healthy individuals. RESULTS: There was a significant difference in the genotype and allele frequencies of TAFI -2345 2G/1G and -1690 A/G polymorphisms between the ACI and control subjects. Further stratification analysis by gender revealed that the presence of the -438 AA genotype and the A allele conferred a higher risk of developing ACI in male patients (p < 0.05). Haplotype analysis demonstrated that four haplotypes of TAFI are significantly associated with ACI. CONCLUSIONS: Our study provides preliminary evidence that the TAFI -2345 2G/1G and -1690 A/G polymorphisms are associated with ACI susceptibility in a Han Chinese population.
Assuntos
Carboxipeptidase B2/genética , Infarto Cerebral/genética , Polimorfismo Genético/genética , Idoso , Alelos , Povo Asiático/genética , Feminino , Frequência do Gene/genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
BACKGROUND: Procarboxypeptidase B2 (proCPB2 or TAFI) is a zymogen that after activation cleaves C-terminal basic residues from peptides or proteins with many identified targets. A splice variant of CPB2 has been found in the brain lacking essential residues for its carboxypeptidase function. The aim was to determine CPB2 expression in the brain and effects of CPB2 deficiency (Cpb2 -/-) on behavior. MATERIALS AND METHODS: Behavioral effects were tested by comparing Cpb2 -/- mice in short-term (open field and elevated zero maze tests) and long-term (Phenotyper) observations with wild-type (WT) controls. RESULTS: Long-term observation compared day 1 (acclimatizing to novel environment) to day 4 (fully acclimatized) with the inactive (day) and active (night) periods analyzed separately. Brain expression of CPB2 mRNA and protein was interrogated in publicly available databases. Long-term observation demonstrated differences between WT and Cpb2 -/- mice in several parameters. For example, Cpb2 -/- mice moved more frequently on both days 1 and 4, especially in the normally inactive periods. Cpb2 -/- mice spent more time on the shelter and less time in it. Differences were more pronounced on day 4 after the mice had fully acclimatized. In short-term observations, no differences were observed between Cpb2 -/- mice and WT mice. Brain expression of CBP2 was not detectable in the human protein atlas. Databases of single-cell RNAseq did not show expression of CPB2 mRNA in either human or mouse brain. CONCLUSION: Continuous observation of home-cage behavior suggests that Cpb2 -/- mice are more active than WT mice, show different day-night activity levels, and might have a different way of processing information.
Assuntos
Carboxipeptidase B2 , Humanos , Animais , Camundongos , Carboxipeptidase B2/genética , Encéfalo/metabolismo , RNA Mensageiro/genéticaRESUMO
BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) levels are positively correlated with the risk of thrombosis. The mechanism of how TAFI affects venous thromboembolism (VTE) remains uncertain. In addition, the role of sex on the risk of VTE has also been studied. However, their association also remains unclear. OBJECTIVES: To investigate how TAFI and/or sex affect venous thrombus stability and consequent pulmonary embolism (PE). METHODS: Ferric chloride-induced thrombi were formed within the femoral veins of male and female wild-type (WT) or TAFI-knockout (Cpb2-/-) mice. Thrombi were imaged over 2 hours using intravital videomicroscopy to quantify embolization and thrombus size over time. Lungs were examined by immunohistochemistry to quantify (a) emboli and (b) fibrin composition of these emboli. RESULTS: Embolization events in female mice were higher than in males (7.9-fold in WT and 3.1-fold in Cpb2-/- mice). Although the maximal thrombus sizes were not different across groups, Cpb2-/- mice had thrombi that were, on average, 24% smaller at the end of the 2-hour experiment than WT mice. Loss of TAFI led to a 4.0- and 2.8-fold increase in PE burden in males and females, respectively, while sex had no influence. Pulmonary emboli in Cpb2-/- mice had higher fibrin composition compared with WT mice. CONCLUSION: Female mice had less stable venous thrombi than male mice, suggesting a higher risk of PE in females with deep vein thrombosis. Mice lacking TAFI had more thrombus degradation and higher PE burden than WT mice. These results confirm the role of TAFI in venous thrombosis.
Assuntos
Carboxipeptidase B2 , Embolia Pulmonar , Trombose , Tromboembolia Venosa , Masculino , Feminino , Camundongos , Animais , Carboxipeptidase B2/genética , Modelos Animais de Doenças , Embolia Pulmonar/genética , Embolia Pulmonar/metabolismo , Fibrina , FibrinóliseRESUMO
Septic infections dysregulate hemostatic pathways, prompting coagulopathy. Nevertheless, anticoagulant therapies typically fail to protect humans from septic pathology. The data reported in this work may help to explain this discrepancy by demonstrating critical protective roles for coagulation leading to fibrin deposition during host defense against the Gram-negative bacterium Yersinia enterocolitica. After i.p. inoculation with Y. enterocolitica, fibrinogen-deficient mice display impaired cytokine and chemokine production in the peritoneal cavity and suppressed neutrophil recruitment. Moreover, both gene-targeted fibrinogen-deficient mice and wild-type mice treated with the anticoagulant coumadin display increased hepatic bacterial burden and mortality following either i.p. or i.v. inoculation with Y. enterocolitica. Mice with low tissue factor activity succumb to yersiniosis with a phenotype similar to fibrin(ogen)-deficient mice, whereas factor XI-deficient mice show wild-type levels of resistance. Mice deficient in plasminogen activator inhibitor-1 or thrombin-activatable fibrinolysis inhibitor display modest phenotypes, but mice deficient in both plasminogen activator inhibitor-1 and thrombin-activatable fibrinolysis inhibitor succumb to yersiniosis with a phenotype resembling fibrin(ogen)-deficient mice. These findings demonstrate critical protective roles for the tissue factor-dependent extrinsic coagulation pathway during host defense against bacteria and caution that therapeutics targeting major thrombin-generating or antifibrinolytic pathways may disrupt fibrin-mediated host defense during Gram-negative sepsis.