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1.
FASEB J ; 36(10): e22564, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36165219

RESUMO

The outcome of ischemic stroke can be improved by further refinements of thrombolysis and reperfusion strategies. Factor VII activating protease (FSAP) is a circulating serine protease that could be important in this context. Its levels are raised in patients as well as mice after stroke and a single nucleotide polymorphism (SNP) in the coding sequence, which results in an inactive enzyme, is linked to an increased risk of stroke. In vitro, FSAP cleaves fibrinogen to promote fibrinolysis, activates protease-activated receptors, and decreases the cellular cytotoxicity of histones. Based on these facts, we hypothesized that FSAP can be used as a treatment for ischemic stroke. A combination of tissue plasminogen activator (tPA), a thrombolytic drug, and recombinant serine protease domain of FSAP (FSAP-SPD) improved regional cerebral perfusion and neurological outcome and reduced infarct size in a mouse model of thromboembolic stroke. FSAP-SPD also improved stroke outcomes and diminished the negative consequences of co-treatment with tPA in the transient middle cerebral artery occlusion model of stroke without altering cerebral perfusion. The inactive MI-isoform of FSAP had no impact in either model. FSAP enhanced the lysis of blood clots in vitro, but in the tail transection model of hemostasis, FSAP-SPD treatment provoked a faster clotting time indicating that it also has pro-coagulant actions. Thus, apart from enhancing thrombolysis, FSAP has multiple effects on stroke progression and represents a promising novel therapeutic strategy in the treatment of ischemic stroke.


Assuntos
Coagulantes , AVC Isquêmico , Acidente Vascular Cerebral , Animais , Modelos Animais de Doenças , Fator VII , Fibrinogênio , Fibrinolíticos/farmacologia , Fibrinolíticos/uso terapêutico , Histonas , Camundongos , Peptídeo Hidrolases , Receptores Ativados por Proteinase , Serina Endopeptidases/genética , Acidente Vascular Cerebral/tratamento farmacológico , Ativador de Plasminogênio Tecidual/genética
2.
Pflugers Arch ; 474(2): 217-229, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34870751

RESUMO

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.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Fator VII/metabolismo , Rim/metabolismo , Síndrome Nefrótica/metabolismo , Peptídeo Hidrolases/metabolismo , Sódio/metabolismo , Animais , Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Humanos , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Rim/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteólise/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Xenopus laevis/metabolismo
3.
Int J Mol Sci ; 23(22)2022 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-36430180

RESUMO

Factor-VII-activating protease (FSAP) is involved in the regulation of hemostasis and inflammation. Extracellular histones play a role in inflammation and the conversion of latent pro-FSAP into active FSAP. FSAP has been shown to regulate endothelial permeability, but the mechanisms are not clear. Here, we have investigated the effects of FSAP on endothelial permeability in vitro. A mixture of histones from calf thymus stimulated permeability, and the wild-type (WT) serine protease domain (SPD) of FSAP blocked this effect. WT-SPD-FSAP did not influence permeability on its own, nor that stimulated by thrombin or vascular endothelial growth factor (VEGF)-A165. Histones induced a large-scale rearrangement of the junction proteins VE-cadherin and zona occludens-1 from a clear junctional distribution to a diffuse pattern. The presence of WT-SPD-FSAP inhibited these changes. Permeability changes by histones were blocked by both TLR-2 and TLR4 blocking antibodies. Histones upregulated the expression of TLR-2, but not TLR-4, in HUVEC cells, and WT-SPD-FSAP abolished the upregulation of TLR-2 expression. An inactive variant, Marburg I (MI)-SPD-FSAP, did not have any of these effects. The inhibition of histone-mediated permeability may be an important function of FSAP with relevance to sepsis, trauma, and stroke and the need to be investigated further in in vivo experiments.


Assuntos
Histonas , Fator A de Crescimento do Endotélio Vascular , Humanos , Inflamação , Permeabilidade , Serina Endopeptidases/metabolismo , Receptor 2 Toll-Like/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
4.
EMBO J ; 36(3): 260-273, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28007894

RESUMO

The F-box protein FBXW7 is the substrate-recruiting subunit of an SCF ubiquitin ligase and a major tumor-suppressor protein that is altered in several human malignancies. Loss of function of FBXW7 results in the stabilization of numerous proteins that orchestrate cell proliferation and survival. Little is known about proteins that directly regulate the function of this protein. In the current work, we have mapped the interactome of the enigmatic pseudophosphatase STYX We reasoned that a catalytically inactive phosphatase might have adopted novel mechanisms of action. The STYX interactome contained several F-box proteins, including FBXW7. We show that STYX binds to the F-box domain of FBXW7 and disables its recruitment into the SCF complex. Therefore, STYX acts as a direct inhibitor of FBXW7, affecting the cellular levels of its substrates. Furthermore, we find that levels of STYX and FBXW7 are anti-correlated in breast cancer patients, which affects disease prognosis. We propose the STYX-FBXW7 interaction as a promising drug target for future investigations.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas F-Box/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Ligases SKP Culina F-Box/antagonistas & inibidores , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Proteína 7 com Repetições F-Box-WD , Humanos
5.
FASEB J ; 34(1): 1079-1090, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914657

RESUMO

Factor VII activating protease (FSAP) is a circulating serine protease implicated in thrombosis, atherosclerosis, stroke, and cancer. Using an overexpression strategy, we have systematically investigated the role of protease activated receptors (PAR)-1, -2, -3, and -4 on FSAP-mediated signaling in HEK293T and A549 cells. Cleavage of PAR-reporter constructs and MAPK phosphorylation was used to monitor receptor activation. FSAP cleaved PAR-2 and to a lesser degree PAR-1, but not PAR-3 or PAR-4 in both cell types. Robust MAPK activation in response to FSAP was observed after PAR-2, but not PAR-1 overexpression in HEK293T. Recombinant serine protease domain of wild type FSAP, but not the Marburg I isoform of FSAP, could reproduce the effects of plasma purified FSAP. Canonical cleavage of both PARs was suggested by mass spectrometric analysis of synthetic peptide substrates from the N-terminus of PARs and site directed mutagenesis studies. Surprisingly, knockdown of endogenous PAR-1, but not PAR-2, prevented the apoptosis-inhibitory effect of FSAP, suggesting that PAR1 is nevertheless a direct or indirect target in some cell types. This molecular characterization of PAR-1 and -2 as cellular receptors of FSAP will help to define the actions of FSAP in the context of cancer and vascular biology.


Assuntos
Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Serina Endopeptidases/metabolismo , Apoptose , Linhagem Celular Tumoral , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Mutagênese Sítio-Dirigida , Peptídeos/química , Fosforilação , Isoformas de Proteínas , Transdução de Sinais , Trombose
6.
Chembiochem ; 21(13): 1875-1884, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32180321

RESUMO

We describe a novel, easy and efficient combinatorial phage display peptide substrate-mining method to map the substrate specificity of proteases. The peptide library is displayed on the pVII capsid of the M13 bacteriophage, which renders pIII necessary for infectivity and efficient retrieval, in an unmodified state. As capture module, the 3XFLAG was chosen due to its very high binding efficiency to anti-FLAG mAbs and its independency of any post-translational modification. This library was tested with Factor-VII activating protease (WT-FSAP) and its single-nucleotide polymorphism variant Marburg-I (MI)-FSAP. The WT-FSAP results confirmed the previously reported Arg/Lys centered FSAP cleavage site consensus as dominant, as well as reinforcing MI-FSAP as a loss-of-function mutant. Surprisingly, rare substrate clones devoid of basic amino acids were also identified. Indeed one of these peptides was cleaved as free peptide, thus suggesting a broader range of WT-FSAP substrates than previously anticipated.


Assuntos
Peptídeos/metabolismo , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Humanos , Espectrometria de Massas , Biblioteca de Peptídeos , Peptídeos/análise , Peptídeos/química , Especificidade por Substrato
7.
J Biol Chem ; 291(40): 21020-21028, 2016 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-27462075

RESUMO

Deletion of the Habp2 gene encoding Factor VII-activating protease (FSAP) increases liver fibrosis in mice. A single nucleotide polymorphism (G534E) in HABP2 leads to lower enzymatic activity and is associated with enhanced liver fibrosis in humans. Liver fibrosis is associated with a decrease in FSAP expression but, to date, nothing is known about how this might be regulated. Primary mouse hepatocytes or the hepatocyte cell line, AML12, were treated with different factors, and expression of FSAP was determined. Of the various regulatory factors tested, only transforming growth factor-ß (TGF-ß) demonstrated a concentration- and time-dependent inhibition of FSAP expression at the mRNA and protein level. The TGF-ß-Type I receptor (ALK-5) antagonist SB431542 and Smad2 siRNA, but neither SIS3, which inhibits SMAD3, nor siRNA against Smad3 could block this effect. Various regions of the HABP2 promoter region were cloned into reporter constructs, and the promoter activity was determined. Accordingly, the promoter activity, which could phenocopy changes in Habp2 mRNA in response to TGF-ß, was found to be located in the 177-bp region upstream of the transcription start site, and this region did not contain any SMAD binding sites. Mutation analysis of the promoter and chromatin immunoprecipitation assays were performed to identify an important role for the ATF3 binding element. Thus, TGF-ß is the most likely mediator responsible for the decrease in FSAP expression in liver fibrosis.


Assuntos
Regulação Enzimológica da Expressão Gênica , Hepatócitos/metabolismo , Cirrose Hepática/metabolismo , Serina Endopeptidases/biossíntese , Fator de Crescimento Transformador beta/metabolismo , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Animais , Linhagem Celular , Hepatócitos/patologia , Humanos , Cirrose Hepática/genética , Cirrose Hepática/patologia , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Elementos de Resposta , Serina Endopeptidases/genética , Proteínas Smad/genética , Proteínas Smad/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/genética
9.
Stroke ; 47(2): 307-16, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26732560

RESUMO

BACKGROUND AND PURPOSE: Although a genetic contribution to ischemic stroke is well recognized, only a handful of stroke loci have been identified by large-scale genetic association studies to date. Hypothesizing that genetic effects might be stronger for early- versus late-onset stroke, we conducted a 2-stage meta-analysis of genome-wide association studies, focusing on stroke cases with an age of onset <60 years. METHODS: The discovery stage of our genome-wide association studies included 4505 cases and 21 968 controls of European, South-Asian, and African ancestry, drawn from 6 studies. In Stage 2, we selected the lead genetic variants at loci with association P<5×10(-6) and performed in silico association analyses in an independent sample of ≤1003 cases and 7745 controls. RESULTS: One stroke susceptibility locus at 10q25 reached genome-wide significance in the combined analysis of all samples from the discovery and follow-up stages (rs11196288; odds ratio =1.41; P=9.5×10(-9)). The associated locus is in an intergenic region between TCF7L2 and HABP2. In a further analysis in an independent sample, we found that 2 single nucleotide polymorphisms in high linkage disequilibrium with rs11196288 were significantly associated with total plasma factor VII-activating protease levels, a product of HABP2. CONCLUSIONS: HABP2, which encodes an extracellular serine protease involved in coagulation, fibrinolysis, and inflammatory pathways, may be a genetic susceptibility locus for early-onset stroke.


Assuntos
Isquemia Encefálica/genética , Serina Endopeptidases/genética , Acidente Vascular Cerebral/genética , Adulto , Idade de Início , Idoso , Povo Asiático/genética , População Negra/genética , Isquemia Encefálica/complicações , Cromossomos Humanos Par 10 , Simulação por Computador , DNA Intergênico/genética , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Polimorfismo de Nucleotídeo Único , Serina Endopeptidases/metabolismo , Acidente Vascular Cerebral/etiologia , População Branca/genética
10.
Blood ; 120(25): 5073-83, 2012 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-23065156

RESUMO

Protein S is a vitamin K-dependent glycoprotein, which, besides its anticoagulant function, acts as an agonist for the tyrosine kinase receptors Tyro3, Axl, and Mer. The endothelium expresses Tyro3, Axl, and Mer and produces protein S. The interaction of protein S with endothelial cells and particularly its effects on angiogenesis have not yet been analyzed. Here we show that human protein S, at circulating concentrations, inhibited vascular endothelial growth factor (VEGF) receptor 2-dependent vascularization of Matrigel plugs in vivo and the capacity of endothelial cells to form capillary-like networks in vitro as well as VEGF-A-induced endothelial migration and proliferation. Furthermore, protein S inhibited VEGF-A-induced endothelial VEGFR2 phosphorylation and activation of mitogen-activated kinase-Erk1/2 and Akt. Protein S activated the tyrosine phosphatase SHP2, and the SHP2 inhibitor NSC 87877 reversed the observed inhibition of VEGF-A-induced endothelial proliferation. Using siRNA directed against Tyro3, Axl, and Mer, we demonstrate that protein S-mediated SHP2 activation and inhibition of VEGF-A-stimulated proliferation were mediated by Mer. Our report provides the first evidence for the existence of a protein S/Mer/SHP2 axis, which inhibits VEGFR2 signaling, regulates endothelial function, and points to a role for protein S as an endogenous angiogenesis inhibitor.


Assuntos
Inibidores da Angiogênese/metabolismo , Neovascularização Fisiológica , Proteína S/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Inibidores da Angiogênese/administração & dosagem , Animais , Proliferação de Células , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Ativação Enzimática , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Proteína S/administração & dosagem , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/genética , Receptores Proteína Tirosina Quinases/genética , c-Mer Tirosina Quinase
11.
J Immunol ; 188(6): 2858-65, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22308306

RESUMO

Severe tissue injury results in early activation of serine protease systems including the coagulation and complement cascade. In this context, little is known about factor VII-activating protease (FSAP), which is activated by substances released from damaged cells such as histones and nucleosomes. Therefore, we have measured FSAP activation in trauma patients and have identified novel FSAP substrates in human plasma. Mass spectrometry-based methods were used to identify FSAP binding proteins in plasma. Anaphylatoxin generation was measured by ELISA, Western blotting, protein sequencing, and chemotaxis assays. Plasma samples from trauma patients were analyzed for FSAP Ag and activity, nucleosomes, C5a, and C3a. Among others, we found complement components C3 and C5 in FSAP coimmunoprecipitates. C3 and C5 were cleaved by FSAP in a dose- and time-dependent manner generating functional C3a and C5a anaphylatoxins. Activation of endogenous FSAP in plasma led to increased C5a generation, but this was not the case in plasma of a homozygous carrier of Marburg I single nucleotide polymorphism with lower FSAP activity. In multiple trauma patients there was a large increase in circulating FSAP activity and nucleosomes immediately after the injury. A high correlation between FSAP activity and C5a was found. These data suggest that activation of FSAP by tissue injury triggers anaphylatoxin generation and thereby modulates the posttraumatic inflammatory response in vivo. A strong link between C5a, nucleosomes, and FSAP activity indicates that this new principle might be important in the regulation of inflammation.


Assuntos
Complemento C5a/imunologia , Traumatismo Múltiplo/imunologia , Serina Endopeptidases/imunologia , Adulto , Idoso , Western Blotting , Complemento C5a/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoprecipitação , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Traumatismo Múltiplo/sangue , Serina Endopeptidases/sangue , Adulto Jovem
12.
J Hepatol ; 58(1): 104-11, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22989567

RESUMO

BACKGROUND & AIMS: Factor VII activating protease (FSAP) is a circulating serine protease produced in the liver. A single nucleotide polymorphism (G534E, Marburg I, MI-SNP) in the gene encoding FSAP (HABP2) leads to lower enzymatic activity and is associated with enhanced liver fibrosis in humans. FSAP is activated by damaged cells and its substrates include growth factors and hemostasis proteins. METHODS: We have investigated the progression of liver fibrosis in FSAP deficient mice and FSAP expression in human liver fibrosis. RESULTS: Serum FSAP concentrations declined in patients with end-stage liver disease, and hepatic FSAP expression was decreased in patients with advanced liver fibrosis and liver inflammation. Moreover, there was an inverse correlation between hepatic FSAP expression and inflammatory chemokines, chemokine receptors as well as pro-fibrotic mediators. Upon experimental bile duct ligation, FSAP(-/-) mice showed enhanced liver fibrosis in comparison to wild type mice, alongside increased expression of α-smooth muscle actin, collagen type I and fibronectin that are markers of stellate cell activation. Microarray analyses indicated that FSAP modulates inflammatory pathways. CONCLUSIONS: Lower FSAP expression is associated with enhanced liver fibrosis and inflammation in patients with chronic hepatic disorders and murine experimental liver injury. This strengthens the concept that FSAP is a "protective factor" in liver fibrosis and explains why carriers of the Marburg I SNP have more pronounced liver fibrosis.


Assuntos
Hepatite/imunologia , Cirrose Hepática/imunologia , Fígado/enzimologia , Fígado/imunologia , Serina Endopeptidases/imunologia , Adolescente , Adulto , Idoso , Animais , Modelos Animais de Doenças , Ativação Enzimática/genética , Feminino , Hepatite/genética , Hepatite/metabolismo , Humanos , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/metabolismo , Serina Endopeptidases/sangue , Serina Endopeptidases/genética , Transcriptoma , Adulto Jovem
13.
Arterioscler Thromb Vasc Biol ; 32(2): 427-33, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22116096

RESUMO

OBJECTIVE: Factor VII-activating protease (FSAP) activates both factor VII and pro-urokinase and inhibits platelet-derived growth factor-BB, thus regulating hemostasis- and remodeling-associated processes in the vasculature. A genetic variant of FSAP (Marburg I polymorphism) results in low enzymatic activity and is associated with an enhanced risk of carotid stenosis and stroke. We postulate that there are additional substrates for FSAP that will help to explain its role in vascular biology and have searched for such a substrate. METHODS AND RESULTS: Using screening procedures to determine the influence of FSAP on various hemostasis-related processes on endothelial cells, we discovered that FSAP inhibited tissue factor pathway inhibitor (TFPI), a major anticoagulant secreted by these cells. Proteolytic degradation of TFPI by FSAP could also be demonstrated by Western blotting, and the exact cleavage sites were determined by N-terminal sequencing. The Marburg I variant of FSAP had a diminished ability to inhibit TFPI. A monoclonal antibody to FSAP that specifically inhibited FSAP binding to TFPI reversed the inhibitory effect of FSAP on TFPI. CONCLUSIONS: The identification of TFPI as a sensitive substrate for FSAP increases our understanding of its role in regulating hemostasis and proliferative remodeling events in the vasculature.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Lipoproteínas/antagonistas & inibidores , Proteólise/efeitos dos fármacos , Serina Endopeptidases/farmacologia , Anticorpos Monoclonais/farmacologia , Proliferação de Células , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Hemostasia/fisiologia , Humanos , Lipoproteínas/efeitos dos fármacos , Serina Endopeptidases/efeitos dos fármacos , Serina Endopeptidases/imunologia
14.
J Exp Med ; 203(13): 2801-7, 2006 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-17145954

RESUMO

The G534E polymorphism (Marburg I [MI]) of factor VII-activating protease (FSAP) is associated with carotid stenosis and cardiovascular disease. We have previously demonstrated that FSAP is present in atherosclerotic plaques and it is a potent inhibitor of vascular smooth muscle proliferation and migration in vitro. The effect of wild-type (WT)- and MI-FSAP on neointima formation in the mouse femoral artery after wire-induced injury was investigated. Local application of WT-FSAP led to a 70% reduction in the neointima formation, and this effect was dependent on the protease activity of FSAP. MI-FSAP did not inhibit neointima formation in vivo. This is due to a reduced proteolytic activity of MI-FSAP, compared to WT-FSAP, toward platelet-derived growth factor BB, a key mediator of neointima development. The inability of MI-FSAP to inhibit vascular smooth muscle accumulation explains the observed linkage between the MI-polymorphism and increased cardiovascular risk. Hence, FSAP has a protective function in the vasculature, and analysis of MI polymorphism is likely to be clinically relevant in restenosis.


Assuntos
Reestenose Coronária/genética , Polimorfismo Genético , Serina Endopeptidases/genética , Túnica Íntima/metabolismo , Actinas/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Becaplermina , Catálise , Proliferação de Células/efeitos dos fármacos , Reestenose Coronária/prevenção & controle , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Expressão Gênica , Heparina/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-sis , Serina Endopeptidases/metabolismo , Serina Endopeptidases/farmacologia , Inibidores de Serina Proteinase/farmacologia , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/patologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Fator de von Willebrand/metabolismo
15.
Circ J ; 76(11): 2653-61, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22850287

RESUMO

BACKGROUND: Factor VII activating protease (FSAP) is a circulating serine protease strongly expressed in unstable plaques and may serve as a marker of plaque destabilization. The aim of this study was to examine the relation between plasma concentrations of FSAP and clinical instability and outcome in coronary artery disease (CAD). METHODS AND RESULTS: Circulating FSAP concentration and activity, as well as FSAP mRNA expression in monocytes, were measured in 231 sequential patients who underwent coronary angiography because of stable angina pectoris (n=50), unstable angina pectoris (n=43), or acute myocardial infarction (n=87). FSAP activity, but not FSAP antigen concentration, was elevated in patients with CAD compared with a control group. Elevated FSAP activity (≥1.035 plasma equivalent units [PEU]/ml) indicated a significantly increased risk of death or non-fatal myocardial infarction during 1 year of follow-up as compared with patients with low activity of FSAP (odds ratio 1.895 [95% confidence interval 1.093-3.283]; P=0.023). Furthermore, there were no significant changes in the FSAP expression in monocytes from CAD and control subjects in the basal state but there were differences after stimulation with proinflammatory factors. CONCLUSIONS: Plasma FSAP activity was significantly increased in patients with acute coronary syndrome and may be involved in the pathogenesis of these conditions. High levels of FSAP activity were predictive of adverse events during follow-up, suggesting its potential role in risk stratification and clinical management of CAD patients.


Assuntos
Síndrome Coronariana Aguda/sangue , Regulação Enzimológica da Expressão Gênica , Monócitos/enzimologia , Serina Endopeptidases/sangue , Síndrome Coronariana Aguda/complicações , Síndrome Coronariana Aguda/diagnóstico por imagem , Síndrome Coronariana Aguda/mortalidade , Idoso , Angiografia Coronária , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/mortalidade , Estudos Prospectivos , RNA Mensageiro/biossíntese , Fatores de Risco , Fatores de Tempo
16.
Thromb Res ; 220: 24-34, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36265411

RESUMO

Human genetic studies based on the Marburg I polymorphism in the factor VII activating protease (FSAP) encoding gene, analysis of FSAP activity in plasma and biochemical characterization of FSAP substrates indicate a possible causal link between FSAP activity and venous thrombosis. We hypothesized that a direct standardized assay to measure FSAP activity in plasma could provide convincing arguments for or against such a potential link. Using Ac-Pro-DTyr-Lys-Arg-AMC as a highly specific and sensitive substrate, histones as a trigger to activate pro-FSAP and plasma-purified active FSAP as a calibrator, we have developed a fluorogenic kinetic assay that reveals the FSAP generating potential in human plasma in real time. This assay is similar to the thrombin generation assay and allows analysis of lag phase, time to peak and velocity, as well as peak FSAP and the endogenous FSAP potential (EFP) of plasma samples. Carriers of the Marburg I polymorphism showed clearly delayed FSAP generation and lower peak FSAP and EFP level. There were no significant differences in all FSAP activity parameters between plasma from patients with a history of venous thrombosis and controls. When excluding Marburg I carriers, which were evenly distributed between groups, delayed FSAP generation significantly correlated with venous thrombosis in postmenopausal women. The novel FSAP activity assay is robust and easy to perform and will be a useful tool for analyzing plasma FSAP activity, also, in other pathophysiological conditions.


Assuntos
Fator VII , Trombose Venosa , Feminino , Humanos , Peptídeo Hidrolases , Serina Endopeptidases/genética , Trombina , Trombose Venosa/genética
17.
PLoS One ; 17(2): e0263238, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35180240

RESUMO

BACKGROUND: Envenomation by the European adder, Vipera berus berus (Vbb), is a medical emergency. The overall in vivo haemostatic effects of pro- and anticoagulant components in Vbb venom, and the downstream effects of cellular injury and systemic inflammation, are unclear. OBJECTIVES: To longitudinally describe the global coagulation status of dogs after Vbb envenomation and compare to healthy controls. A secondary aim was to investigate differences between dogs treated with and without antivenom. METHODS: Citrated plasma was collected at presentation, 12 hours (h), 24 h, 36 h and 15 days after bite from 28 dogs envenomated by Vbb, and from 28 healthy controls at a single timepoint. Thrombin generation (initiated with and without exogenous phospholipids and tissue factor), thrombin-antithrombin (TAT)-complexes and the procoagulant activity of phosphatidylserine (PS)-expressing extracellular vesicles (EVs), expressed as PS-equivalents, were measured. RESULTS: At presentation the envenomated dogs were hypercoagulable compared to controls, measured as increased thrombin generation, TAT-complexes and PS-equivalents. The hypercoagulability decreased gradually but compared to controls thrombin generation and PS-equivalents were still increased at day 15. The discrepancy in peak thrombin between envenomated dogs and controls was greater when the measurement was phospholipid-dependent, indicating that PS-positive EVs contribute to hypercoagulability. Lag time was shorter in non-antivenom treated dogs, compared to antivenom treated dogs <24 h after envenomation. CONCLUSIONS: Hypercoagulability was measured in dogs up to 15 days after Vbb envenomation. Dogs treated with antivenom may be less hypercoagulable than their non-antivenom treated counterparts. Thrombin generation is a promising diagnostic and monitoring tool for Vbb envenomation.


Assuntos
Antivenenos/uso terapêutico , Doenças do Cão/etiologia , Doenças do Cão/terapia , Fatores Imunológicos/uso terapêutico , Mordeduras de Serpentes/complicações , Trombofilia/etiologia , Trombofilia/veterinária , Viperidae , Animais , Antitrombina III , Estudos de Casos e Controles , Cães , Feminino , Inflamação/sangue , Inflamação/etiologia , Inflamação/terapia , Inflamação/veterinária , Estudos Longitudinais , Masculino , Peptídeo Hidrolases/sangue , Trombina/análise , Trombofilia/sangue , Trombofilia/terapia , Resultado do Tratamento , Venenos de Víboras/imunologia
18.
ACS Chem Biol ; 17(9): 2631-2642, 2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-36070465

RESUMO

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.


Assuntos
Bacteriófagos , Fator VII , Animais , Humanos , Camundongos , Aminoácidos , Anticorpos Monoclonais/metabolismo , Bacteriófagos/metabolismo , Precursores Enzimáticos/metabolismo , Fator VII/metabolismo , Histonas , Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Serina Endopeptidases/metabolismo
19.
Blood ; 113(3): 714-22, 2009 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-18971420

RESUMO

Various virus infections cause dysfunctional hemostasis and in some instances lead to the development of viral hemorrhagic fever syndrome. How do diverse viruses induce the expression of tissue factor on vascular cells? We hypothesize that a direct stimulation of pattern recognition receptors (PRR) by viral nucleic acids may be the key. Double-stranded RNA (dsRNA) is produced by many viruses and is recognized by various PRR, including Toll-like receptor-3 (TLR3). We have investigated whether poly I:C, a model for viral dsRNA, can influence cellular hemostasis. Poly I:C could up-regulate tissue factor and down-regulate thrombomodulin expression on endothelial cells but not on monocytes. The response to poly I:C was diminished upon small interfering RNA (siRNA)-mediated inhibition of TLR3, but not other PRR. In vivo, application of poly I:C induced similar changes in the aortic endothelium of mice as determined by enface microscopy. D-dimer, a circulating marker for enhanced coagulation and fibrinolysis, and tissue fibrin deposition was elevated. All the hemostasis-related responses to poly I:C, but not cytokine secretion, were blunted in TLR3(-/-) mice. Hence, the activation of TLR3 can induce the procoagulant state in the endothelium, and this could be relevant for understanding the mechanisms of viral stimulation of hemostasis.


Assuntos
Células Endoteliais/metabolismo , Hemostasia/fisiologia , Tromboplastina/metabolismo , Receptor 3 Toll-Like/metabolismo , Animais , Coagulação Sanguínea/efeitos dos fármacos , Coagulação Sanguínea/fisiologia , Western Blotting , Células Cultivadas , Citocinas/biossíntese , Células Endoteliais/efeitos dos fármacos , Feminino , Imunofluorescência , Expressão Gênica/efeitos dos fármacos , Humanos , Indutores de Interferon/farmacologia , Masculino , Camundongos , Camundongos Mutantes , Microscopia de Fluorescência , Poli I-C/farmacologia , RNA Interferente Pequeno , Receptores de Reconhecimento de Padrão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trombomodulina/efeitos dos fármacos , Trombomodulina/metabolismo , Tromboplastina/efeitos dos fármacos , Receptor 3 Toll-Like/efeitos dos fármacos , Receptor 3 Toll-Like/genética
20.
Nat Med ; 8(7): 687-93, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12091905

RESUMO

Staphylococcus aureus is a human pathogen that secretes proteins that contribute to bacterial colonization. Here we describe the extracellular adherence protein (Eap) as a novel anti-inflammatory factor that inhibits host leukocyte recruitment. Due to its direct interactions with the host adhesive proteins intercellular adhesion molecule 1 (ICAM-1), fibrinogen or vitronectin, Eap disrupted beta(2)-integrin and urokinase receptor mediated leukocyte adhesion in vitro. Whereas Eap-expressing S. aureus induced a 2 3-fold lower neutrophil recruitment in bacterial peritonitis in mice as compared with an Eap-negative strain, isolated Eap prevented beta(2)-integrin-dependent neutrophil recruitment in a mouse model of acute thioglycollate-induced peritonitis. Thus, the specific interactions with ICAM-1 and extracellular matrix proteins render Eap a potent anti-inflammatory factor, which may serve as a new therapeutic substance to block leukocyte extravasation in patients with hyperinflammatory pathologies.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Aderência Bacteriana , Proteínas de Bactérias/farmacologia , Leucócitos/fisiologia , Staphylococcus aureus/fisiologia , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Endotélio Vascular/microbiologia , Endotélio Vascular/fisiologia , Fibronectinas/fisiologia , Humanos , Molécula 1 de Adesão Intercelular/fisiologia , Leucócitos/microbiologia , Antígeno de Macrófago 1/fisiologia , Camundongos , Camundongos Endogâmicos , Peritonite/microbiologia , Peritonite/prevenção & controle , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/prevenção & controle , Células U937 , Veias Umbilicais
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