RESUMEN
Hereditary coagulation factor VII (FVII) deficiency is a rare autosomal recessive bleeding disorder. The aims of this study were to identify and verify the pathogenic mutation sites in a family with hereditary coagulation FVII deficiency, and preliminarily explore the underlying mechanisms. We identified a novel combination of compound heterozygous mutations, c.572-1G>A and c.1037A>C in F7 gene, associated with FVII deficiency. The splice site mutation c.572-1G>A led to a truncation, resulting in the loss of the essential catalytic domain of the FVII protein. The c.1037A>C missense mutation has not been previously reported. Our study revealed that this mutation leads to steric hindrance between residues, causing significant changes in the energy and structure of the FVII protein, ultimately affecting its function. These changes disrupt the normal function of the FVII protein, accelerating the development of inherited FVII deficiency. Moreover, the mRNA expression of the F7 gene and the protein expression of the FVII antigen (FVII: Ag) were significantly lower in the proband, as well as in the proband's parents, compared to the healthy control (P<0.05). Our findings not only elucidate the genetic underpinning of FVII deficiency in the family studied but also contribute a new mutation to the known disease spectrum, potentially assisting in future diagnostic and therapeutic approaches.
Asunto(s)
Pueblo Asiatico , Deficiencia del Factor VII , Factor VII , Heterocigoto , Linaje , Humanos , Deficiencia del Factor VII/genética , Factor VII/genética , Factor VII/metabolismo , Masculino , Femenino , Pueblo Asiatico/genética , Adulto , Mutación Missense , Mutación , China , Pueblos del Este de AsiaRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is considered a risk factor for cardiovascular and cerebrovascular disease owing to its close association with coagulant disturbances. However, the precise biological functions and mechanisms that connect coagulation factors to NAFLD pathology remain inadequately understood. Herein, with unbiased bioinformatics analyses followed by functional testing, we demonstrate that hepatic expression of coagulation factor VII (FVII) decreases in patients and mice with NAFLD/nonalcoholic steatohepatitis (NASH). By using adenovirus-mediated F7-knockdown and hepatocyte-specific F7-knockout mouse models, our mechanistic investigations unveil a noncoagulant function of hepatic FVII in mitigating lipid accumulation and lipotoxicity. This protective effect is achieved through the suppression of fatty acid uptake, orchestrated via the AKT-CD36 pathway. Interestingly, intracellular FVII directly interacts with AKT and PP2A, thereby promoting their association and triggering the dephosphorylation of AKT. Therapeutic intervention through adenovirus-mediated liver-specific overexpression of F7 results in noteworthy improvements in liver steatosis, inflammation, injury, and fibrosis in severely afflicted NAFLD mice. In conclusion, our findings highlight coagulation factor FVII as a critical regulator of hepatic steatosis and a potential target for the treatment of NAFLD and NASH.
Asunto(s)
Factor VII , Enfermedad del Hígado Graso no Alcohólico , Animales , Humanos , Ratones , Factor VII/genética , Factor VII/metabolismo , Ácidos Grasos/metabolismo , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Shade-intolerant plants sense changes in the light environment and trigger shade-avoidance syndrome in the presence of neighboring vegetation. Phytochrome-interacting factor 7 (PIF7) is an essential regulator that integrates shade signals into plant transcriptional networks. While the regulation of PIF7 under shade conditions has been well studied, the mechanism that represses PIF7 activity under white light remains ambiguous. Here, we report that PIF7 forms nuclear puncta containing phase-separated liquid-like condensates. Phytochrome B (phyB) then binds to dephosphorylated PIF7 and promotes its condensed phase of PIF7 under white light. The phyB-PIF7 condensate subsequently inhibits the DNA-binding activity of PIF7. However, shade inactivation of phyB causes the dissociation of phyB-PIF7 condensates and allows unbound PIF7 to promote the transcription of shade-induced genes. This reversible transcriptional condensation via phase separation provides sessile organisms with the flexibility of gene control to adapt to their surrounding environment.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Fitocromo , Fitocromo/metabolismo , Fitocromo B/genética , Fitocromo B/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Factor VII/genética , Factor VII/metabolismo , Separación de Fases , Luz , Regulación de la Expresión Génica de las Plantas , Proteínas de Unión al ADN/metabolismoRESUMEN
MAIN CONCLUSION: LBD18 and IAA14 antagonistically interact with ARF7 through the electrostatic faces in the ARF7PB1 domain, modulating ARF7 transcriptional activity. Auxin Response Factor 7 (ARF7)/ARF19 control lateral root development by directly activating Lateral Organ Boundaries Domain 16 (LBD16)/LBD18 genes in Arabidopsis. LBD18 upregulates ARF19 expression by binding to the ARF19 promoter. It also interacts with ARF7 through the Phox and Bem1 (PB1) domain to enhance the ARF7 transcriptional activity, forming a dual mode of positive feedback loop. LBD18 competes with the repressor indole-3-acetic acid 14 (IAA14) for ARF7 binding through the PB1 domain. In this study, we examined the molecular determinant of the ARF7 PB1 domain for interacting with LBD18 and showed that the electronic faces in the ARF7 PB1 domain are critical for interacting with LBD18 and IAA14/17. We used a luminescence complementation imaging assay to determine protein-protein interactions. The results showed that mutation of the invariant lysine residue and the OPCA motif in the PB1 domain in ARF7 significantly reduces the protein interaction between ARF7 and LBD18. Transient gene expression assays with Arabidopsis protoplasts showed that IAA14 suppressed transcription-enhancing activity of LBD18 on the LUC reporter gene fused to the ARF19 promoter harboring an auxin response element, but mutation of the invariant lysine residue and OPCA motif in the PB1 domain of IAA14 reduced the repression capability of IAA14 for transcription-enhancing activity of LBD18. We further showed that the same mutation in the PB1 domain of IAA14 reduces its repression capability, thereby increasing the LUC activity induced by both ARF7 and LBD18 compared with IAA14. These results suggest that LBD18 competes with IAA14 for ARF7 binding via the electrostatic faces of the ARF7 PB1 domain to modulate ARF7 transcriptional activity.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Factor VII/genética , Factor VII/metabolismo , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos/metabolismo , Lisina/metabolismo , Raíces de Plantas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Factor VII activating protease (FSAP) was first isolated from human plasma less than 30 years ago. Since then, many research groups have described the biological properties of this protease and its role in hemostasis and other processes in humans and other animals. With the progress of knowledge about the structure of FSAP, several of its relationships with other proteins or chemical compounds that may modulate its activity have been explained. These mutual axes are described in the present narrative review. The first part of our series of manuscripts on FSAP describes the structure of this protein and the processes leading to the enhancement and inhibition of its activities. The following parts, II and III, concern the role of FSAP in hemostasis and in the pathophysiology of human diseases, with particular emphasis on cardiovascular diseases.
Asunto(s)
Enfermedades Cardiovasculares , Factor VII , Animales , Humanos , Factor VII/metabolismo , Serina Endopeptidasas/metabolismo , Péptido Hidrolasas , Hemostasis/fisiologíaRESUMEN
INTRODUCTION: Acquired factor VII (FVII) deficiency is a rare condition with various causes, including acquired inhibitors to FVII, liver disease, and malignancies. Myxoid pleomorphic liposarcoma is a rare and aggressive form of soft tissue sarcoma that can cause a range of clinical manifestations, including bleeding and clotting disorders. PATIENT CONCERNS AND DIAGNOSIS: We present a case report of a 21-year-old man with severe acquired FVII deficiency due to mediastinal myxoid pleomorphic liposarcoma. The patient presented with elevated International normalized ratio (INR) and a severe reduction in FVII coagulant activity, unresponsive to conventional therapy. While an acquired inhibitor to FVII was initially suspected, negative results from laboratory testing, including protein G sepharose adsorption and a Bethesda assay using Immunoglobulin G purified from patient plasma, made the diagnosis of an acquired inhibitor to FVII uncertain. INTERVENTIONS AND OUTCOME: The patient underwent surgical resection of the tumor, supported by recombinant FVII infusion, leading to the normalization of coagulation parameters. However, a relapse of the disease was detected 6 months later when he was noted to have a decline in FVII levels. CONCLUSION: This case highlights the importance of considering rare causes of bleeding and clotting disorders, particularly in unresponsive or atypical presentations. It also underscores the need for close monitoring and follow-up in patients with acquired FVII deficiency, even after successful treatment.
Asunto(s)
Deficiencia del Factor VII , Liposarcoma , Masculino , Humanos , Adulto Joven , Adulto , Deficiencia del Factor VII/complicaciones , Deficiencia del Factor VII/diagnóstico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Factor VII/metabolismo , Hemorragia/etiología , Coagulación Sanguínea , Liposarcoma/complicacionesRESUMEN
We investigated the molecular basis of factor VII (FVII) deficiency in a Japanese patient and identified compound heterozygous mutations. Factor VII activity and antigen levels in the patient were less than 5.0% and 6.5% of controls, respectively. All exons, exon-intron boundaries, and the 5' promoter region of F7 from genomic DNA were amplified using polymerase chain reaction (PCR). Sequencing analysis of PCR fragments revealed that the patient was heterozygous for a known T to C substitution at nucleotide position 38, which resulted in the p.Leu13Pro missense mutation (Factor VII Morioka) in the signal peptide region, and a novel mutation in the 5' promoter region (-58G>C). An electrophoretic mobility shift assay showed that the mutation in the promoter region reduced the binding of hepatocyte nuclear factor (HNF). It is presumed that the reduced binding of HNF-4 to the F7 promoter region reduces F7 transcription and thus reduces the synthesis and expression of FVII.
Asunto(s)
Deficiencia del Factor VII , Humanos , Deficiencia del Factor VII/genética , Factor VII/genética , Factor VII/metabolismo , Mutación , Heterocigoto , Regiones Promotoras GenéticasRESUMEN
Factor VII Activating protease (FSAP) has a protective effect in diverse disease conditions as inferred from studies in FSAP-/- mice and humans deficient in FSAP activity due to single-nucleotide polymorphism. The zymogen form of FSAP in plasma is activated by extracellular histones that are released during tissue injury or inflammation or by positively charged surfaces. However, it is not clear whether this activation mechanism is specific and amenable to manipulation. Using a phage display approach, we have identified a Cys-constrained 11 amino acid peptide, NNKC9/41, that activates pro-FSAP in plasma. The synthetic linear peptide has a propensity to cyclize through the terminal Cys groups, of which the antiparallel cyclic dimer, but not the monocyclic peptide, is the active component. Other commonly found zymogens in the plasma, related to the hemostasis system, were not activated. Binding studies with FSAP domain deletion mutants indicate that the N-terminus of FSAP is the key interaction site of this peptide. In a monoclonal antibody screen, we identified MA-FSAP-38C7 that prevented the activation of pro-FSAP by the peptide. This antibody bound to the LESLDP sequence (amino acids 30-35) in an intrinsically disordered stretch in the N-terminus of FSAP. The plasma clotting time was shortened by NNKC9/41, and this was reversed by MA-FSAP-38C7, demonstrating the utility of this peptide. Peptide NNKC9/41 will be useful as a tool to delineate the molecular mechanism of activation of pro-FSAP, elucidate its biological role, and provide a starting point for the pharmacological manipulation of FSAP activity.
Asunto(s)
Bacteriófagos , Factor VII , Animales , Humanos , Ratones , Aminoácidos , Anticuerpos Monoclonales/metabolismo , Bacteriófagos/metabolismo , Precursores Enzimáticos/metabolismo , Factor VII/metabolismo , Histonas , Péptido Hidrolasas/metabolismo , Péptidos/metabolismo , Serina Endopeptidasas/metabolismoRESUMEN
Venoms are evolutionary novelties that have real-world implications due to their impact upon human health. However, relative to the abundant studies of elapid and viperid snake venoms, fewer investigations have been undertaken on those of rear-fanged snakes as they are more problematic for obtaining venom. While most rear-fanged venomous snakes are not considered to be of great medical importance, several species are capable of producing fatalities. Most notable among these are snakes from the genus Rhabdophis, the Asian "keelback" snakes. Prior work have described potent procoagulant toxicity suggesting Factor X and prothrombin activation, but did not investigate the ability to activate other clotting factors. Here we show that in addition to activating both Factor X and prothrombin (with prothrombin twice that of FX), the venom of Rhabdophis subminiatus is able to more potently activate Factor VII (ten times that of prothrombin), while also activating FXII and FIX equipotently to prothrombin, and with FXI also activated but at a much lower level. The ability to activate FVII represents a third convergent evolution of this trait. The Australian elapid clade of [Oxyuranus (taipans) + Pseudonaja (brown snakes)] was the first identified to have evolved this trait. and only recently was it shown to be independently present in another lineage (the Central American viperid species Porthidium volcanicum). In addition, the abilities to activate FXI and FXII are also convergent between R. subminiatus and P. volcanicum, but with R. subminiatus being much more potent. By testing across amphibian, avian, and mammalian plasmas we demonstrate that the venom is potently procoagulant across diverse plasma types. However, consistent with dietary preference, R. subminiatus venom was most potent upon amphibian plasma. While a Rhabdophis antivenom is produced in Japan to treat R. tigrinus envenomings, it is scarce even within Japan and is not exported. As this genus is very wide-ranging in Asia, alternate treatment options are in need of development. Hence we tested the ability of candidate, broad-spectrum enzyme inhibitors to neutralize R. subminiatus venom: marimastat was more effective than prinomastat but both marimastat and prinomastat were significantly more effective than DMPS (2,3-Dimercapto-1-propanesulfonic acid). The findings of this study shed light on the evolution of these fascinating rear-fanged snakes as well as explored their systemic effects upon blood coagulation and point to potential treatment options for the rare, but potentially lethal encounters.
Asunto(s)
Antivenenos , Colubridae , Animales , Antivenenos/farmacología , Australia , Coagulación Sanguínea , Factores de Coagulación Sanguínea/metabolismo , Factores de Coagulación Sanguínea/farmacología , Elapidae/metabolismo , Factor VII/metabolismo , Factor VII/farmacología , Factor X/metabolismo , Factor X/farmacología , Humanos , Ácidos Hidroxámicos , Mamíferos , Compuestos Orgánicos , Protrombina , Venenos de Serpiente/farmacología , Unitiol/metabolismo , Unitiol/farmacologíaRESUMEN
The size of plant organs is highly responsive to environmental conditions. The plant's embryonic stem, or hypocotyl, displays phenotypic plasticity, in response to light and temperature. The hypocotyl of shade avoiding species elongates to outcompete neighboring plants and secure access to sunlight. Similar elongation occurs in high temperature. However, it is poorly understood how environmental light and temperature cues interact to effect plant growth. We found that shade combined with warm temperature produces a synergistic hypocotyl growth response that dependent on PHYTOCHROME-INTERACTING FACTOR 7 (PIF7) and auxin. This unique but agriculturally relevant scenario was almost totally independent on PIF4 activity. We show that warm temperature is sufficient to promote PIF7 DNA binding but not transcriptional activation and we demonstrate that additional, unknown factor/s must be working downstream of the phyB-PIF-auxin module. Our findings will improve the predictions of how plants will respond to increased ambient temperatures when grown at high density.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Fitocromo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al ADN/metabolismo , Factor VII/genética , Factor VII/metabolismo , Regulación de la Expresión Génica de las Plantas , Hipocótilo/metabolismo , Ácidos Indolacéticos/metabolismo , Luz , Fitocromo/metabolismoRESUMEN
Interferon (IFN)-induced protein 35 (IFI35, also known as IFP35), a member of IFN induced genes (ISGs), participates in virus infection, cancer progression and the chronic inflammatory diseases. However, its roles during fish nodavirus infection still remained largely unknown. In the present study, a homolog of IFI35 from orange spotted grouper (Epinephelus coioides) (EcIFI35) was cloned and characterized. The open reading frame of EcIFI35 was composed of 1,128 bp, and encoded a 375 amino acid polypeptide, which contained two conserved N-myc-interactor (Nmi)/IFP35 domains (NIDs). Homology analysis indicated that EcIFI35 shared 95.73% and 31.96% identity with homologs of giant grouper (E. lanceolatus) and human (Homo sapiens), respectively. The transcription of EcIFI35 was significantly up-regulated in grouper spleen (GS) cells after challenged with red-spotted grouper nervous necrosis virus (RGNNV), polyinosinic:polycytidylic acid [poly(I:C)] or lipopolysaccharide (LPS). The subcellular localization analysis showed that EcIFI35 encoded a cytoplasmic protein. The ectopic expression of EcIFI35 inhibited RGNNV replication by reducing viral genes transcription and protein synthesis. Co-immunoprecipitation (Co-IP) assay demonstrated that EcIFI35 interacted with RGNNV coat protein (CP), and partly co-localized with CP. EcIFI35 overexpression promoted the expression of IFN-related molecules and pro-inflammatory factors, including IFN regulatory factor 7 (IRF7), mitochondrial antiviral signaling protein (MAVS) and myxovirus resistance gene I (MxI), nuclear factor κB (NF-κB), interleukin 6 (IL-6) and IL-8. Together, our results revealed that EcIFI35 interacted with CP and inhibited fish nodavirus replication through positively regulated host innate immune response.
Asunto(s)
Lubina , Infecciones por Virus ADN , Enfermedades de los Peces , Nodaviridae , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Animales , Antivirales , Factor VII/genética , Factor VII/metabolismo , Proteínas de Peces/química , Regulación de la Expresión Génica , Humanos , Inmunidad Innata/genética , Interferones/metabolismo , Interleucina-6/genética , Interleucina-8/genética , Lipopolisacáridos , FN-kappa B/metabolismo , Nodaviridae/fisiología , Poli I-C/farmacología , Alineación de SecuenciaRESUMEN
Factor VII (FVII) is a serine protease with a key role in initiating the coagulation cascade. It is part of a family of vitamin K-dependent clotting proteins, which require vitamin K for formation of their specialized membrane-binding domains (Gla domains). Membrane binding of the FVII Gla domain is critical to the activity of FVII, mediating the formation of its complex with other clotting factors. While Gla domains among coagulation factors are highly conserved in terms of amino acid sequence and structure, they demonstrate differential binding specificity toward anionic lipids. Although most Gla domain-containing clotting proteins display a strong preference for phosphatidylserine (PS), it has been demonstrated that FVII and protein C instead bind preferentially to phosphatidic acid (PA). We have developed the first model of the FVII Gla domain bound to PA lipids in membranes containing PA, the highly mobile membrane mimetic model, which accelerates slow diffusion of lipids in molecular dynamics simulations and therefore facilitates the membrane binding process and enhances sampling of lipid interactions. Simulations were performed using atomic level molecular dynamics, requiring a fixed charge to all atoms. The overall charge assigned to each PA lipid for this study was -1. We also developed an additional model of the FVII Gla domain bound to a 1:1 PS/PC membrane and compared the modes of binding of PS and PA lipids to FVII, allowing us to identify potential PA-specific binding sites.
Asunto(s)
Factor VII , Ácidos Fosfatidicos , Secuencia de Aminoácidos , Sitios de Unión , Factores de Coagulación Sanguínea , Factor VII/química , Factor VII/metabolismo , Fosfatidilserinas/metabolismo , Vitamina K/metabolismoRESUMEN
BACKGROUND: Recombinant human ADAMTS13 (rADAMTS13) is a key protein in fundamental research for investigating its mode of action and the pathophysiology of thrombotic thrombocytopenic purpura (TTP). However, the expression of rADAMTS13 is quite low in mammalian cells, which makes the production of the protein time-consuming and labor-intensive. OBJECTIVES: We aimed at increasing the yield of rADAMTS13 by (1) using a more optimal signal peptide (SP) and (2) constructing an N-terminal fusion protein of ADAMTS13 with human serum albumin domain 1 (AD1-ADAMTS13). METHODS: Six SPs were investigated to select the most optimal SP. Expression plasmids containing the most optimal SP and ADAMTS13 cDNA or the fusion construct AD1-ADAMTS13 were generated and transiently transfected into CHOEBNALT85 cell-line. Expression levels of rADAMTS13 in expression medium were analyzed and compared with the expression level of rADAMTS13 with native SP (nat-SP). RESULTS: Expression of rADAMTS13 with coagulation factor VII (FVII) SP was 3-fold higher (16.00 µg/ml) compared with the expression with nat-SP (5.03 µg/ml). The highest yields were obtained with AD1-ADAMTS13 protein with a 15-fold higher concentration (78.22 µg/ml) compared with the expression with nat-SP. The rADAMTS13 expressed with FVII-SP retained its activity (104.0%) to cleave von Willebrand factor, whereas AD1-ADAMTS13 demonstrated even higher activity (144.3%). CONCLUSION: We succeeded in generating expression vectors that yield (1) rADAMTS13 at higher levels because of more optimal FVII-SP and (2) high levels of AD1-ADAMTS13 N-terminal fusion protein. The highest expression levels were obtained with AD1-ADAMTS13 N-terminal fusion protein, which is paving the way for highly efficient protein production.
Asunto(s)
Púrpura Trombocitopénica Trombótica , Factor de von Willebrand , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS13/genética , Proteína ADAMTS13/metabolismo , Animales , ADN Complementario , Factor VII/metabolismo , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes/metabolismo , Albúmina Sérica Humana , Factor de von Willebrand/genética , Factor de von Willebrand/metabolismoRESUMEN
BACKGROUND: Inflammation and coagulation are linked and pathogenic in neuroinflammatory diseases. Protease-activated receptor 1 (PAR1) can be activated both by thrombin, inducing increased inflammation, and activated protein C (aPC), inducing decreased inflammation. Modulation of the aPC-PAR1 pathway may prevent the neuroinflammation associated with PAR1 over-activation. METHODS: We synthesized a group of novel molecules based on the binding site of FVII/aPC to the endothelial protein C receptor (EPCR). These molecules modulate the FVII/aPC-EPCR pathway and are therefore named FEAMs-Factor VII, EPCR, aPC Modulators. We studied the molecular and behavioral effects of a selected FEAM in neuroinflammation models in-vitro and in-vivo. RESULTS: In a lipopolysaccharide (LPS) induced in-vitro model, neuroinflammation leads to increased thrombin activity compared to control (2.7 ± 0.11 and 2.23 ± 0.13 mU/ml, respectively, p = 0.01) and decreased aPC activity (0.57 ± 0.01 and 1.00 ± 0.02, respectively, p < 0.0001). In addition, increased phosphorylated extracellular regulated kinase (pERK) (0.99 ± 0.13, 1.39 ± 0.14, control and LPS, p < 0.04) and protein kinase B (pAKT) (1.00 ± 0.09, 2.83 ± 0.81, control and LPS, p < 0.0002) levels indicate PAR1 overactivation, which leads to increased tumor necrosis factor-alpha (TNF-α) level (1.00 ± 0.04, 1.35 ± 0.12, control and LPS, p = 0.02). In a minimal traumatic brain injury (mTBI) induced neuroinflammation in-vivo model in mice, increased thrombin activity, PAR1 activation, and TNF-α levels were measured. Additionally, significant memory impairment, as indicated by a lower recognition index in the Novel Object Recognition (NOR) test and Y-maze test (NOR: 0.19 ± 0.06, -0.07 ± 0.09, p = 0.03. Y-Maze: 0.50 ± 0.03, 0.23 ± 0.09, p = 0.02 control and mTBI, respectively), as well as hypersensitivity by hot-plate latency (16.6 ± 0.89, 12.8 ± 0.56 s, control and mTBI, p = 0.01), were seen. FEAM prevented most of the molecular and behavioral negative effects of neuroinflammation in-vitro and in-vivo, most likely through EPCR-PAR1 interactions. CONCLUSION: FEAM is a promising tool to study neuroinflammation and a potential treatment for a variety of neuroinflammatory diseases.
Asunto(s)
Proteína C , Receptor PAR-1 , Animales , Receptor de Proteína C Endotelial/metabolismo , Factor VII/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Lipopolisacáridos/toxicidad , Ratones , Enfermedades Neuroinflamatorias , Proteína C/metabolismo , Proteína C/uso terapéutico , Receptor PAR-1/metabolismo , Transducción de Señal , Trombina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Tissue factor (TF) expression in cancers correlates with poor prognosis. Recently, the first TF-targeted therapy was approved by the U.S. Food and Drug Administration for cervical cancer. To unfold the potential of TF-targeted therapies, correct stratification and selection of patients eligible for treatments may become important for optimization of patient outcomes. TF-targeted PET imaging based on 18F-radiolabeled active-site inhibited versions of the TF natural ligand coagulation factor VII (18F-ASIS) has in preclinical models convincingly demonstrated its use for noninvasive quantitative measurements of TF expression in tumor tissue. 18F-ASIS PET imaging thus has the potential to act as a diagnostic companion for TF-targeted therapies in the clinical setting. Methods: In this first-in-humans trial, we included 10 cancer patients (4 pancreatic, 3 breast, 2 lung, and 1 cervical cancer) for 18F-ASIS PET imaging. The mean and SD of administered 18F-ASIS activity was 157 ± 35 MBq (range, 93-198 MBq). PET/CT was performed after 1, 2, and 4 h. The primary objectives were to establish the safety, biodistribution, pharmacokinetics, and dosimetry of 18F-ASIS. Secondary objectives included quantitative measurements of SUVs in tumor tissue with PET and evaluation of the correlation (Pearson correlation) between tumor SUVmax and ex vivo TF expression in tumor tissue. Results: Administration of 18F-ASIS was safe, and no adverse events were observed. No clinically significant changes in vital signs, electrocardiograms, or blood parameters were observed after injection of 18F-ASIS. Mean 18F-ASIS plasma half-life was 3.2 ± 0.6 h, and the radiotracer was predominantly excreted in the urine. For injection activity of 200 MBq of 18F-ASIS, effective whole-body dose was 4 mSv and no prohibitive organ-specific absorbed doses were found. Heterogeneous radiotracer uptake was observed across patients and within tumors. We found a trend of a positive correlation between tumor SUVmax and ex vivo TF expression (r = 0.84, P = 0.08, n = 5). Conclusion: 18F-ASIS can be safely administered to cancer patients for PET imaging of TF expression in tumors. The trial marks the first test of a TF-targeted PET radiotracer in humans (first-in-class). The findings represent important first steps toward clinical implementation of 18F-ASIS PET imaging of TF expression.
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Neoplasias Primarias Secundarias , Neoplasias del Cuello Uterino , Femenino , Humanos , Factor VII/metabolismo , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Tomografía de Emisión de Positrones , Radiometría , Tromboplastina/metabolismo , Distribución TisularRESUMEN
Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that induces malignant T-cell lymphomas in chickens, leading to great economic loss in poultry industry. The unique-short kinase 3 (Us3), a serine/threonine kinase encoded by three MDV types (MDV-1, MDV-2 and HVT), is important for MDV replication. However, the mechanism of Us3 facilitating MDV replication has not been completely elucidated. In the present study, we report that Us3 significantly facilities MDV replication via inhibition of ß interferon (IFN-ß) production at the late phase of MDV infection. Overexpression or interference of Us3 significantly promoted or inhibited the replication of MDV, and accordingly inhibited or promoted the expression of IFN-ß. Further, Us3 was shown to suppresses interferon stimulatory DNA (ISD)-triggered IFN-ß production by targeting IFN regulatory factor 7 (IRF7) rather than NF-κB signaling. Moreover, Us3 but not kinase-dead (KD) Us3 mutant K220A blocked the nuclear translocation of IRF7 by inhibiting dimerization. Importantly, Us3 phosphorylated and interacted with IRF7. Furthermore, Us3-deficient MDV weakened viral replication and increased IFN-ß production in infected cells or chickens. These results indicated that Us3 interrupts the cytosolic DNA sensing pathway, thereby leading to inhibition of IFN-ß production by targeting IRF7, promoting MDV replication. Our finding expands the knowledge about the role of Us3 in MDV replication.
Asunto(s)
Herpesvirus Gallináceo 2 , Enfermedad de Marek , Animales , Pollos , Factor VII/metabolismo , Herpesvirus Gallináceo 2/genética , Proteínas Serina-Treonina Quinasas/genética , Serina/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/genéticaRESUMEN
Fibroblast growth factor-21 (FGF-21) performs a wide range of biological functions in organisms. Here, we report for the first time that FGF-21 suppresses thrombus formation with no notable risk of bleeding. Prophylactic and therapeutic administration of FGF-21 significantly improved the degree of vascular stenosis and reduced the thrombus area, volume and burden. We determined the antithrombotic mechanism of FGF-21, demonstrating that FGF-21 exhibits an anticoagulant effect by inhibiting the expression and activity of factor VII (FVII). FGF-21 exerts an antiplatelet effect by inhibiting platelet activation. FGF-21 enhances fibrinolysis by promoting tissue plasminogen activator (tPA) expression and activation, while inhibiting plasminogen activator inhibitor 1 (PAI-1) expression and activation. We further found that FGF-21 mediated the expression and activation of tPA and PAI-1 by regulating the ERK1/2 and TGF-ß/Smad2 pathways, respectively. In addition, we found that FGF-21 inhibits the expression of inflammatory factors in thrombosis by regulating the NF-κB pathway.
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Coagulación Sanguínea/efectos de los fármacos , Fibrinolíticos/farmacología , Factores de Crecimiento de Fibroblastos/farmacología , Trombosis/prevención & control , Animales , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Línea Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor VII/genética , Factor VII/metabolismo , Fibrinólisis/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Masculino , Ratones Endogámicos ICR , FN-kappa B/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Activación Plaquetaria/efectos de los fármacos , Conejos , Transducción de Señal , Proteína Smad2/metabolismo , Trombosis/sangre , Trombosis/genética , Activador de Tejido Plasminógeno/genética , Activador de Tejido Plasminógeno/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Excessive bradykinin (BK) generation from high molecular weight kininogen (HK) by plasma kallikrein (PK) due to lack of protease inhibition is central to the pathophysiology of hereditary angioedema (HAE). Inadequate protease inhibition may contribute to HAE through a number of plasma proteases including factor VII activating protease (FSAP) that can also cleave HK. OBJECTIVE: To investigate the interaction between FSAP and C1 inhibitor (C1Inh) and evaluate the potential role of FSAP in HAE with C1Inh deficiency. MATERIALS AND METHODS: Plasma samples from 20 persons with HAE types 1 or 2 in remission were studied and compared to healthy controls. We measured and compared antigenic FSAP levels, spontaneous FSAP activity, FSAP generation potential, activation of plasma pre-kallikrein (PPK) by FSAP, and the formation of FSAP-C1Inh and FSAP-alpha2-antiplasmin (FSAP-α2AP) complexes. Furthermore, we measured HK cleavage and PK activation after activation of endogenous pro-FSAP and after addition of exogenous FSAP. RESULTS: In plasma from HAE patients, there is increased basal FSAP activity compared to healthy volunteers. HAE plasma exhibits decreased formation of FSAP-C1Inh complexes and increased formation of FSAP-α2AP complexes in histone-activated plasma. Although exogenous FSAP can cleave HK in plasma, this was not seen when endogenous plasma pro-FSAP was activated with histones in either group. PK was also not activated by FSAP in plasma. CONCLUSION: In this study, we established that FSAP activity is increased and the pattern of FSAP-inhibitor complexes is altered in HAE patients. However, we did not find evidence suggesting that FSAP contributes directly to HAE attacks.
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Angioedemas Hereditarios/fisiopatología , Proteína Inhibidora del Complemento C1/genética , Quininógeno de Alto Peso Molecular/metabolismo , Serina Endopeptidasas/metabolismo , Angioedemas Hereditarios/sangre , Angioedemas Hereditarios/genética , Antifibrinolíticos/metabolismo , Bradiquinina/biosíntesis , Factor VII/metabolismo , Humanos , Calicreínas/sangre , Calicreínas/metabolismo , Serina Endopeptidasas/genéticaRESUMEN
Proteolytic activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases is thought to contribute to renal sodium retention in nephrotic syndrome. However, the identity of the responsible proteases remains elusive. This study evaluated factor VII activating protease (FSAP) as a candidate in this context. We analyzed FSAP in the urine of patients with nephrotic syndrome and nephrotic mice and investigated its ability to activate human ENaC expressed in Xenopus laevis oocytes. Moreover, we studied sodium retention in FSAP-deficient mice (Habp2-/-) with experimental nephrotic syndrome induced by doxorubicin. In urine samples from nephrotic humans, high concentrations of FSAP were detected both as zymogen and in its active state. Recombinant serine protease domain of FSAP stimulated ENaC-mediated whole-cell currents in a time- and concentration-dependent manner. Mutating the putative prostasin cleavage site in γ-ENaC (γRKRK178AAAA) prevented channel stimulation by the serine protease domain of FSAP. In a mouse model for nephrotic syndrome, active FSAP was present in nephrotic urine of Habp2+/+ but not of Habp2-/- mice. However, Habp2-/- mice were not protected from sodium retention compared to nephrotic Habp2+/+ mice. Western blot analysis revealed that in nephrotic Habp2-/- mice, proteolytic cleavage of α- and γ-ENaC was similar to that in nephrotic Habp2+/+ animals. In conclusion, active FSAP is excreted in the urine of nephrotic patients and mice and activates ENaC in vitro involving the putative prostasin cleavage site of γ-ENaC. However, endogenous FSAP is not essential for sodium retention in nephrotic mice.
Asunto(s)
Canales Epiteliales de Sodio/metabolismo , Factor VII/metabolismo , Riñón/metabolismo , Síndrome Nefrótico/metabolismo , Péptido Hidrolasas/metabolismo , Sodio/metabolismo , Animales , Doxorrubicina/metabolismo , Doxorrubicina/farmacología , Humanos , Transporte Iónico/efectos de los fármacos , Transporte Iónico/fisiología , Riñón/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Proteolisis/efectos de los fármacos , Serina Endopeptidasas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Xenopus laevis/metabolismoRESUMEN
BACKGROUND: Hereditary factor VII deficiency (FVIID) is a rare congenital autosomal recessive bleeding disorder. In clinical manifestations, its onset is caused by variant of the F7 gene (NM_019616) with strong heterogeneity. We identified a family with hematuria caused by a novel F7 compound heterozygous variant and investigated the FVIID-dependent mechanism impacted by these variants. METHODS: Coagulation factors in the proband were functionally verified. We located pathogenic variants in relevant genes using next-generation sequencing after target enrichment and verified them by Sanger sequencing. We examined the coagulation activity and secretion pattern of recombinant FVII variants expressed in cells and observed their location and stability by immunofluorescence. RESULTS: We found a missense variant c.1207G>A (p.Gly403Ser) and a frameshift variant c.154_155del (p.Arg53fs) in the F7 gene of the proband. FVII activity tests showed that the variants significantly decreased its presence in the cell culture supernatant. Moreover, the R53fs mutant lacked the FVII functional domain and had no detectable activity. Immunofluorescence indicated that the p.Gly403Ser variant was distributed to the cell membrane and cytoplasm, whereas the FVII R53fs variant was not detected. Deficient FVII protein function and severe coagulation disorder are the likely causes of hematuria and other bleeding symptoms in the proband. CONCLUSIONS: The newly discovered F7 gene variants enrich the spectrum of hereditary FVII deficiency and provide a new foundation for the diagnosis and treatment of this type of coagulation disorder.