RESUMO
Plant viruses cause systemic diseases that severely impair plant growth and development. While the accumulation of viruses in the root system has long been established, little is known as to how viruses affect root architecture. Here, we examined how the emerging tobamovirus, tomato brown rugose fruit virus (ToBRFV), alters root development in tomato. We found that ToBRFV and tobacco mosaic virus both invaded root systems during the first week of infection. ToBRFV infection of tomato plants resulted in a significant decrease in root biomass and elongation and root-to-shoot ratio and a marked suppression of root branching. Mutation in RNA-dependent RNA polymerase 6 increased the susceptibility of tomato plants to ToBRFV, resulting in severe reduction of various root growth parameters including root branching. Viral root symptoms were associated with the accumulation of auxin response factor 10a (SlARF10a) transcript, a homolog of Arabidopsis ARF10, a known suppressor of lateral root development. Interestingly, loss-of-function mutation in SlARF10a moderated the effect of ToBRFV on root branching. In contrast, downregulation of sly-miR160a, which targets SlARF10a, was associated with constitutive suppression root branching independent of viral infection. In addition, overexpression of a microRNA-insensitive mutant of SlARF10a mimicked the effect of ToBRFV on root development, suggesting a specific role for SlARF10a in ToBRFV-mediated suppression of root branching. Taken together, our results provide new insights into the impact of tobamoviruses on root development and the role of ARF10a in the suppression of root branching in tomato.
Assuntos
Solanum lycopersicum , Tobamovirus , Solanum lycopersicum/genética , Tobamovirus/genética , Fator Xa/genética , Ácidos Indolacéticos , Mutação , Doenças das PlantasRESUMO
Background: Mitochondria have been involved in host defense upon viral infections. Factor Xa (FXa), a coagulating factor, may also have influence on mitochondrial functionalities. The aim was to analyze if in human pulmonary microvascular endothelial cells (HPMEC), the SARS-CoV-2 (COVID-19) spike protein subunits, S1 and S2 (S1+S2), could alter mitochondrial metabolism and what is the role of FXA. Methods: HPMEC were incubated with and without recombinants S1+S2 (10 nmol/L each). Results: In control conditions, S1+S2 failed to modify FXa expression. However, in LPS (1 µg/mL)-incubated HPMEC, S1+S2 significantly increased FXa production. LPS tended to reduce mitochondrial membrane potential with respect to control, but in higher and significant degree, it was reduced when S1+S2 were present. LPS did not significantly modify cytochrome c oxidase activity as compared with control. Addition of S1+S2 spike subunits to LPS-incubated HPMEC significantly increased cytochrome c oxidase activity with respect to control. Lactate dehydrogenase activity was also increased by S1+S2 with respect to control and LPS alone. Protein expression level of uncoupled protein-2 (UCP-2) was markedly expressed when S1+S2 were added together to LPS. Rivaroxaban (50 nmol/L), a specific FXa inhibitor, significantly reduced all the above-mentioned alterations induced by S1+S2 including UCP-2 expression. Conclusions: In HPMEC undergoing to preinflammatory condition, COVID-19 S1+S2 spike subunits promoted alterations in mitochondria metabolism suggesting a shift from aerobic towards anaerobic metabolism that was accompanied of high FXa production. Rivaroxaban prevented all the mitochondrial metabolic changes mediated by the present COVID-19 S1 and S2 spike subunits suggesting the involvement of endogenous FXa.
Assuntos
COVID-19 , Inibidores do Fator Xa , Fator Xa , Mitocôndrias , Rivaroxabana , Glicoproteína da Espícula de Coronavírus , Humanos , COVID-19/genética , COVID-19/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Células Endoteliais/metabolismo , Fator Xa/genética , Fator Xa/metabolismo , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Subunidades Proteicas/metabolismo , Rivaroxabana/metabolismo , Rivaroxabana/farmacologia , Rivaroxabana/uso terapêutico , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Tratamento Farmacológico da COVID-19 , Inibidores do Fator Xa/metabolismo , Inibidores do Fator Xa/farmacologia , Inibidores do Fator Xa/uso terapêutico , Antivirais/metabolismo , Antivirais/farmacologia , Antivirais/uso terapêuticoRESUMO
Blood coagulation is an essential physiological process for hemostasis; however, abnormal coagulation can lead to various potentially fatal disorders, generally known as thromboembolic disorders, which are a major cause of mortality in the modern world. Recently, the FDA has approved several anticoagulant drugs for Factor Xa (FXa) which work via the common pathway of the coagulation cascade. A main side effect of these drugs is the potential risk for bleeding in patients. Coagulation Factor IXa (FIXa) has recently emerged as the strategic target to ease these risks as it selectively regulates the intrinsic pathway. These aforementioned coagulation factors are highly similar in structure, functional architecture, and inhibitor binding mode. Therefore, it remains a challenge to design a selective inhibitor which may affect only FIXa. With the availability of a number of X-ray co-crystal structures of these two coagulation factors as protein-ligand complexes, structural alignment, molecular docking, and pharmacophore modeling were employed to derive the relevant criteria for selective inhibition of FIXa over FXa. In this study, six ligands (three potent, two selective, and one inactive) were selected for FIXa inhibition and six potent ligands (four FDA approved drugs) were considered for FXa. The pharmacophore hypotheses provide the distribution patterns for the principal interactions that take place in the binding site. None of the pharmacophoric patterns of the FXa inhibitors matched with any of the patterns of FIXa inhibitors. Based on pharmacophore analysis, a selectivity of a ligand for FIXa over FXa may be defined quantitatively as a docking score of lower than -8.0 kcal/mol in the FIXa-grids and higher than -7.5 kcal/mol in the FXa-grids.
Assuntos
Anticoagulantes/farmacologia , Fator IXa/antagonistas & inibidores , Inibidores do Fator Xa/farmacologia , Fator Xa/metabolismo , Anticoagulantes/química , Cristalografia por Raios X , Fator IXa/genética , Fator IXa/metabolismo , Fator Xa/genética , Inibidores do Fator Xa/química , Humanos , Modelos Moleculares , Estrutura MolecularRESUMO
Bloodfeeding is employed by many parasitic animals and requires specific innovations for efficient feeding. Some of these innovations are molecular features that are related to the inhibition of hemostasis. For example, bloodfeeding insects, bats, and leeches release proteins with anticoagulatory activity through their salivary secretions. The antistasin-like protein family, composed of serine protease inhibitors with one or more antistasin-like domains, is tightly linked to inhibition of hemostasis in leeches. However, this protein family has been recorded also in non-bloodfeeding invertebrates, such as cnidarians, mollusks, polychaetes, and oligochaetes. The present study aims to 1) root the antistasin-like gene tree and delimit the major orthologous groups, 2) identify potential independent origins of salivary proteins secreted by leeches, and 3) identify major changes in domain and/or motif structure within each orthologous group. Five clades containing leech antistasin-like proteins are distinguishable through rigorous phylogenetic analyses based on nine new transcriptomes and a diverse set of comparative data: the trypsin + leukocyte elastase inhibitors clade, the antistasin clade, the therostasin clade, and two additional, unnamed clades. The antistasin-like gene tree supports multiple origins of leech antistasin-like proteins due to the presence of both leech and non-leech sequences in one of the unnamed clades, but a single origin of factor Xa and trypsin + leukocyte elastase inhibitors. This is further supported by three sequence motifs that are exclusive to antistasins, the trypsin + leukocyte elastase inhibitor clade, and the therostasin clade, respectively. We discuss the implications of our findings for the evolution of this diverse family of leech anticoagulants.
Assuntos
Anelídeos/genética , Anelídeos/metabolismo , Evolução Molecular , Hormônios de Invertebrado/genética , Hormônios de Invertebrado/metabolismo , Sanguessugas/genética , Animais , Anticoagulantes/química , Fator Xa/genética , Hemostasia , Filogenia , Proteínas e Peptídeos Salivares/genética , Inibidores de Serina Proteinase/genética , TranscriptomaRESUMO
Humans express seven heparan sulfate (HS) 3-O-sulfotransferases that differ in substrate specificity and tissue expression. Although genetic studies have indicated that 3-O-sulfated HS modulates many biological processes, ligand requirements for proteins engaging with HS modified by 3-O-sulfate (3-OS) have been difficult to determine. In particular, the context in which the 3-OS group needs to be presented for binding is largely unknown. We describe herein a modular synthetic approach that can provide structurally diverse HS oligosaccharides with and without 3-OS. The methodology was employed to prepare 27 hexasaccharides that were printed as a glycan microarray to examine ligand requirements of a wide range of HS-binding proteins. The binding selectivity of antithrombin-III (AT-III) compared well with anti-Factor Xa activity supporting robustness of the array technology. Many of the other examined HS-binding proteins required an IdoA2S-GlcNS3S6S sequon for binding but exhibited variable dependence for the 2-OS and 6-OS moieties, and a GlcA or IdoA2S residue neighboring the central GlcNS3S. The HS oligosaccharides were also examined as inhibitors of cell entry by herpes simplex virus type 1, which, surprisingly, showed a lack of dependence of 3-OS, indicating that, instead of glycoprotein D (gD), they competitively bind to gB and gC. The compounds were also used to examine substrate specificities of heparin lyases, which are enzymes used for depolymerization of HS/heparin for sequence determination and production of therapeutic heparins. It was found that cleavage by lyase II is influenced by 3-OS, while digestion by lyase I is only affected by 2-OS. Lyase III exhibited sensitivity to both 3-OS and 2-OS.
Assuntos
Células Epiteliais/metabolismo , Heparina Liase/metabolismo , Heparitina Sulfato/metabolismo , Herpesvirus Humano 1/metabolismo , Sulfatos/metabolismo , Sulfotransferases/metabolismo , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Antitrombina III/química , Antitrombina III/genética , Antitrombina III/metabolismo , Sítios de Ligação , Ligação Competitiva , Sequência de Carboidratos , Linhagem Celular , Córnea/citologia , Córnea/metabolismo , Células Epiteliais/patologia , Células Epiteliais/virologia , Fator Xa/química , Fator Xa/genética , Fator Xa/metabolismo , Inibidores do Fator Xa/química , Inibidores do Fator Xa/metabolismo , Expressão Gênica , Ácido Glucurônico/química , Ácido Glucurônico/metabolismo , Heparina Liase/química , Heparina Liase/genética , Heparitina Sulfato/química , Herpesvirus Humano 1/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno/genética , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Análise em Microsséries , Ligação Proteica , Proteólise , Bibliotecas de Moléculas Pequenas , Especificidade por Substrato , Sulfatos/química , Sulfotransferases/química , Sulfotransferases/genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Coagulation factor V (FV) plays an anticoagulant role but serves as a procoagulant cofactor in the prothrombinase complex once activated to FVa. At the heart of these opposing effects is the proteolytic removal of its central B-domain, including conserved functional landmarks (basic region, BR; 963-1008 and acidic region 2, AR2; 1493-1537) that enforce the inactive FV procofactor state. Tissue factor pathway inhibitor α (TFPIα) has been associated with FV as well as FV-short, a physiologically relevant isoform with a shortened B-domain missing the BR. However, it is unclear which forms of FV are physiologic ligands for TFPIα. Here, we characterize the binding and regulation of FV and FV-short by TFPIα via its positively charged C-terminus (TFPIα-BR) and examine how bond cleavage in the B-domain influences these interactions. We show that FV-short is constitutively active and functions in prothrombinase like FVa. Unlike FVa, FV-short binds with high affinity (Kd â¼1 nM) to TFPIα-BR, which blocks procoagulant function unless FV-short is cleaved at Arg1545, removing AR2. Importantly, we do not observe FV binding (µM detection limit) to TFPIα. However, cleavage at Arg709 and Arg1018 displaces the FV BR, exposing AR2 and allowing TFPIα to bind via its BR. We conclude that for full-length FV, the detachment of FV BR from AR2 is necessary and sufficient for TFPIα binding and regulation. Our findings pinpoint key forms of FV, including FV-short, that act as physiologic ligands for TFPIα and establish a mechanistic framework for assessing the functional connection between these proteins.
Assuntos
Fator V/química , Fator Va/química , Lipoproteínas/química , Trombina/genética , Coagulação Sanguínea/genética , Fator V/genética , Fator Va/genética , Fator Xa/química , Fator Xa/genética , Humanos , Ligantes , Lipoproteínas/genética , Ligação Proteica/genética , Domínios Proteicos/genética , Proteólise/efeitos dos fármacos , Trombina/química , Tromboplastina/química , Tromboplastina/genéticaRESUMO
The coronavirus disease, COVID-19, caused by the novel coronavirus SARS-CoV-2, which first emerged in Wuhan, China and was made known to the World in December 2019 turned into a pandemic causing more than 126,124 deaths worldwide up to April 16th, 2020. It has 79.5% sequence identity with SARS-CoV-1 and the same strategy for host cell invasion through the ACE-2 surface protein. Since the development of novel drugs is a long-lasting process, researchers look for effective substances among drugs already approved or developed for other purposes. The 3D structure of the SARS-CoV-2 main protease was compared with the 3D structures of seven proteases, which are drug targets, and docking analysis to the SARS-CoV-2 protease structure of thirty four approved and on-trial protease inhibitors was performed. Increased 3D structural similarity between the SARS-CoV-2 main protease, the HCV protease and α-thrombin was found. According to docking analysis the most promising results were found for HCV protease, DPP-4, α-thrombin and coagulation Factor Xa known inhibitors, with several of them exhibiting estimated free binding energy lower than -8.00 kcal/mol and better prediction results than reference compounds. Since some of the compounds are well-tolerated drugs, the promising in silico results may warrant further evaluation for viral anticipation. DPP-4 inhibitors with anti-viral action may be more useful for infected patients with diabetes, while anti-coagulant treatment is proposed in severe SARS-CoV-2 induced pneumonia.
Assuntos
Anticoagulantes/química , Antivirais/química , Betacoronavirus/efeitos dos fármacos , Inibidores da Dipeptidil Peptidase IV/química , Inibidores de Proteases/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Anticoagulantes/farmacologia , Antivirais/farmacologia , Betacoronavirus/química , Betacoronavirus/enzimologia , Betacoronavirus/genética , Sítios de Ligação , COVID-19 , Proteases 3C de Coronavírus , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Fator Xa/química , Fator Xa/genética , Fator Xa/metabolismo , Hepacivirus/química , Hepacivirus/enzimologia , Hepacivirus/genética , Humanos , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , SARS-CoV-2 , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato , Termodinâmica , Trombina/antagonistas & inibidores , Trombina/química , Trombina/genética , Trombina/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Australian elapid snakes are some of the most venomous snakes in the world and are unique among venomous snakes in having mutated forms of the blood clotting factor X in an activated form (FXa) as a key venom component. In human bite victims, an overdose of this activated clotting enzyme results in the systemic consumption of fibrinogen due to the large amounts of endogenous thrombin generated by the conversion of prothrombin to thrombin by venom FXa. Within Australian elapids, such procoagulant venom is currently known from the tiger snake clade (Hoplocephalus, Notechis, Paroplocephalus, and Tropidechis species), brown/taipan (Oxyuranus and Pseudonaja species) clade, and the red-bellied black snake Pseudechis porphyriacus. We used a STA-R Max coagulation analyser and TEG5000 thromboelastographers to test 47 Australian elapid venoms from 19 genera against human plasma in vitro. In addition to activity being confirmed in the two clades above, FXa-driven potent procoagulant activity was found in four additional genera (Cryptophis, Demansia, Hemiaspis, and Suta). Ontogenetic changes in procoagulant function was also identified as a feature of Suta punctata venom. Phylogenetic analysis of FX sequences confirmed that snake venom FXa toxins evolved only once, that the potency of these toxins against human plasma has increased in a stepwise fashion, and that multiple convergent amplifications of procoagulant activity within Australian elapid snakes have occurred. Cofactor dependence tests revealed all procoagulant venoms in our study, except those of the tiger snake clade, to be highly calcium-dependent, whereas phospholipid dependence was less of a feature but still displayed significant variation between venoms. Antivenom testing using CSL Tiger Snake Antivenom showed broad but differential cross-reactivity against procoagulant venoms, with P. porphyriacus and S. punctata extremely well neutralised but with Cryptophis, Demansia, and Hemiaspis less well-neutralised. The relative variation was not in accordance to genetic relatedness of the species used in antivenom production (Notechis scutatus), which underscores a fundamental principle that the rapid evolution characteristic of venoms results in organismal phylogeny being a poor predictor of antivenom efficacy. Our results have direct and immediate implications for the design of clinical management plans in the event of snakebite by such lesser known Australian elapid snake species that have been revealed in this study to be as potent as the better studied, and proven lethal, species.
Assuntos
Antivenenos/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Venenos Elapídicos/antagonistas & inibidores , Elapidae , Inibidores do Fator Xa/farmacologia , Fator Xa/metabolismo , Mordeduras de Serpentes/tratamento farmacológico , Animais , Reações Cruzadas , Venenos Elapídicos/genética , Venenos Elapídicos/imunologia , Venenos Elapídicos/metabolismo , Elapidae/classificação , Elapidae/genética , Elapidae/imunologia , Elapidae/metabolismo , Evolução Molecular , Fator Xa/genética , Fator Xa/imunologia , Fibrinólise/efeitos dos fármacos , Mutação , Filogenia , Mordeduras de Serpentes/imunologia , Mordeduras de Serpentes/metabolismo , TromboelastografiaRESUMO
Pentamidine is bis-oxybenzamidine-based antiprotozoal drug. The parenteral use of pentamidine appears to affect the processes of blood coagulation and/or fibrinolysis resulting in rare but potentially life-threatening blood clot formation. Pentamidine was also found to cause disseminated intravascular coagulation syndrome. To investigate the potential underlying molecular mechanism(s) of pentamidine's effects on coagulation and fibrinolysis, we studied its effects on clotting times in normal and deficient human plasmas. Using normal plasma, pentamidine isethionate doubled the activated partial thromboplastin time at 27.5 µM, doubled the prothrombin time at 45.7 µM, and weakly doubled the thrombin time at 158.17 µM. Using plasmas deficient of factors VIIa, IXa, XIa, or XIIa, the concentrations to double the activated partial thromboplastin time were similar to that obtained using normal plasma. Pentamidine also inhibited plasmin-mediated clot lysis with half-maximal inhibitory concentration (IC50) value of ~3.6 µM. Chromogenic substrate hydrolysis assays indicated that pentamidine inhibits factor Xa and plasmin with IC50 values of 10.4 µM and 8.4 µM, respectively. Interestingly, it did not significantly inhibit thrombin, factor XIa, factor XIIIa, neutrophil elastase, or chymotrypsin at the highest concentrations tested. Michaelis-Menten kinetics and molecular modeling studies revealed that pentamidine inhibits factor Xa and plasmin in a competitive fashion. Overall, this study provides quantitative mechanistic insights into the in vitro effects of pentamidine isethionate on coagulation and fibrinolysis via the disruption of the proteolytic activity of factor Xa and plasmin.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Fibrinólise/efeitos dos fármacos , Pentamidina/farmacologia , Trombose/tratamento farmacológico , Testes de Coagulação Sanguínea , Fator VIIa/genética , Fator XIIa/genética , Fator XIa/genética , Fator Xa/genética , Humanos , Tempo de Tromboplastina Parcial , Tempo de Protrombina , Trombina/química , Trombina/genética , Tempo de Trombina , Trombose/sangue , Trombose/patologiaRESUMO
This paper presents a systemic investigation of ADA development and ADA impact of a human coagulation factor in nonclinical species during drug development and provides insights into potential implications in human if a similar ADA occurs. FXaI16L-induced ADA response was characterized in monkey, mouse, rat, and dog in different studies, and ADA effects on pharmacokinetic and/or pharmacodynamics of FXaI16L were further examined in ADA-negative and ADA-positive animals. After repeated administrations, FXaI16L elicited a dose and exposure day-dependent ADA response which ranged from no response to a transient or persistent response. Increase in exposure day and increase in dose generally enhanced ADA incidence except for a decrease in ADA incidence was observed in monkeys after repeated high-dose administrations. The observable ADA impact on pharmacokinetics was only found in some ADA+ animals and included decrease in clearance and increase in systemic exposure but no increase in half-life. In addition, no or limited effect on pharmacodynamics by ADA was observed. The earliest ADA response was observed after three exposure days, marked elevation of drug exposure was observed in some animals at log titer > 2.0, and the highest antibody titer excited was about 4 (Log10) in all species. A correlation between ADA induction and accumulative exposure after various repeat treatments in different species was found for FXaI16L. In addition, potential immunogenicity risk and mitigation of ADA in clinics are discussed.
Assuntos
Fator Xa/imunologia , Hemofilia A/tratamento farmacológico , Animais , Coagulação Sanguínea/efeitos dos fármacos , Coagulação Sanguínea/imunologia , Modelos Animais de Doenças , Cães , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Fator Xa/administração & dosagem , Fator Xa/genética , Feminino , Meia-Vida , Hemofilia A/sangue , Hemofilia A/diagnóstico , Hemofilia A/imunologia , Humanos , Macaca fascicularis , Masculino , Camundongos , Tempo de Tromboplastina Parcial , Tempo de Protrombina , Ratos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
INTRODUCTION: Andexanet alfa is a recombinant modified factor Xa protein that has been developed to reverse factor Xa inhibitors. Since May 2018, the FDA has approved its utilization in patients treated with apixaban and rivaroxaban in case of life-threatening or uncontrolled bleeding. On 28 of February 2019, the Committee for Medicinal Products for Human Use adopted a positive opinion, recommending the granting of a conditional marketing authorization for andexanet alfa in Europe. Area covered: The authors provide an overview of andexanet alfa development and its pharmacokinetic and pharmacodynamic properties. The results of the clinical phase III trial ANNEXA as well as current limitations related to andexanet alfa are also discussed. Expert opinion: Although phase I and II studies have proven that andexanet alfa can be effective in reversing the effect of factor Xa inhibitors, its efficacy in major bleeding patients has only been shown for apixaban and rivaroxaban, without any comparator group. Well-designed studies comparing the efficacy and safety of andexanet alfa to other reversal strategies are required to confirm preliminary data. The benefit of andexanet alfa in specific settings needs to be investigated and its use in clinical practice needs to be facilitated by the implementation of international guidelines.
Assuntos
Inibidores do Fator Xa/imunologia , Fator Xa/uso terapêutico , Hemorragia/tratamento farmacológico , Proteínas Recombinantes/uso terapêutico , Ensaios Clínicos como Assunto , Fator Xa/genética , Fator Xa/metabolismo , Fator Xa/farmacocinética , Meia-Vida , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacocinéticaRESUMO
Current thought holds that factor Xa (FXa) bound in the prothrombinase complex is resistant to regulation by protein protease inhibitors during prothrombin activation. Here we provide evidence that, contrary to this view, the FXa-specific serpin inhibitor, protein Z-dependent protease inhibitor (ZPI), complexed with its cofactor, protein Z (PZ), functions as a physiologically significant inhibitor of prothrombinase-bound FXa during prothrombin activation. Kinetics studies showed that the rapid rate of inhibition of FXa by the ZPI-PZ complex on procoagulant membrane vesicles (ka(app) â¼107 m-1 s-1) was decreased â¼10-fold when FXa was bound to FVa in prothrombinase and a further â¼3-4-fold when plasma levels of S195A prothrombin were present (ka(app) 2 × 105 m-1 s-1). Nevertheless, the ZPI-PZ complex produced a major inhibition of thrombin generation during prothrombinase-catalyzed activation of prothrombin under physiologically relevant conditions. The importance of ZPI-PZ complex anticoagulant regulation of FXa both before and after incorporation into prothrombinase was supported by thrombin generation assays in plasma. These showed enhanced thrombin generation when the inhibitor was neutralized with a PZ-specific antibody and decreased thrombin generation when exogenous ZPI-PZ complex was added whether prothrombin was activated directly by FXa or through extrinsic or intrinsic pathway activators. Moreover, the PZ antibody enhanced thrombin generation both in the absence and presence of activated protein C (APC) anticoagulant activity. Taken together, these results suggest an important anticoagulant role for the ZPI-PZ complex in regulating both free FXa generated in the initiation phase of coagulation as well as prothrombinase-bound FXa in the propagation phase that complement prothrombinase regulation by APC.
Assuntos
Coagulação Sanguínea , Fator V/química , Fator Xa/química , Protrombina/química , Serpinas/química , Trombina/química , Substituição de Aminoácidos , Anticorpos/química , Fator V/genética , Fator V/metabolismo , Fator Xa/genética , Fator Xa/metabolismo , Humanos , Cinética , Mutação de Sentido Incorreto , Proteína C/química , Proteína C/metabolismo , Protrombina/genética , Protrombina/metabolismo , Serpinas/genética , Serpinas/metabolismo , Trombina/genética , Trombina/metabolismoRESUMO
Venous thromboembolism (VTE) is a leading cause of morbidity and mortality in elderly people. Increased expression of tumor suppressor protein 53 (p53) has been implicated in vascular senescence. Here, we examined the importance of endothelial p53 for venous thrombosis and whether endothelial senescence and p53 overexpression are involved in the exponential increase of VTE with age. Mice with conditional, endothelial-specific deletion of p53 (End.p53-KO) and their wild-type littermates (End.p53-WT) underwent subtotal inferior vena cava (IVC) ligation to induce venous thrombosis. IVC ligation in aged (12-month-old) End.p53-WT mice resulted in higher rates of thrombus formation and greater mean thrombus size vs adult (12-week-old) End.p53-WT mice, whereas aged End.p53-KO mice were protected from vein thrombosis. Analysis of primary endothelial cells from aged mice or human vein endothelial cells after induction of replicative senescence revealed significantly increased early growth response gene-1 (Egr1) and heparanase expression, and plasma factor Xa levels were elevated in aged End.p53-WT, but not in End.p53-KO mice. Increased endothelial Egr1 and heparanase expression also was observed after doxorubicin-induced p53 overexpression, whereas p53 inhibition using pifithrin-α reduced tissue factor (TF) expression. Importantly, inhibition of heparanase activity using TF pathway inhibitor-2 (TFPI2) peptides prevented the enhanced venous thrombus formation in aged mice and restored it to the thrombotic phenotype of adult mice. Our findings suggest that p53 accumulation and heparanase overexpression in senescent endothelial cells are critically involved in mediating the increased risk of venous thrombosis with age and that heparanase antagonization may be explored as strategy to ameliorate the prothrombotic endothelial phenotype with age.
Assuntos
Envelhecimento/metabolismo , Células Endoteliais/metabolismo , Proteína Supressora de Tumor p53/biossíntese , Trombose Venosa/metabolismo , Envelhecimento/genética , Envelhecimento/patologia , Animais , Proteína 1 de Resposta de Crescimento Precoce/biossíntese , Proteína 1 de Resposta de Crescimento Precoce/genética , Células Endoteliais/patologia , Fator Xa/biossíntese , Fator Xa/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Heparina Liase/genética , Camundongos , Camundongos Knockout , Proteína Supressora de Tumor p53/genética , Trombose Venosa/genéticaRESUMO
Unfractionated heparin (UFH), the standard anticoagulant for cardiopulmonary bypass (CPB) surgery, carries a risk of post-operative bleeding and is potentially harmful in patients with heparin-induced thrombocytopenia-associated antibodies. To improve the activity of an alternative anticoagulant, the RNA aptamer 11F7t, we solved X-ray crystal structures of the aptamer bound to factor Xa (FXa). The finding that 11F7t did not bind the catalytic site suggested that it could complement small-molecule FXa inhibitors. We demonstrate that combinations of 11F7t and catalytic-site FXa inhibitors enhance anticoagulation in purified reaction mixtures and plasma. Aptamer-drug combinations prevented clot formation as effectively as UFH in human blood circulated in an extracorporeal oxygenator circuit that mimicked CPB, while avoiding side effects of UFH. An antidote could promptly neutralize the anticoagulant effects of both FXa inhibitors. Our results suggest that drugs and aptamers with shared targets can be combined to exert more specific and potent effects than either agent alone.
Assuntos
Anticoagulantes/administração & dosagem , Inibidores do Fator Xa/administração & dosagem , Fator Xa/química , Hemorragia Pós-Operatória/tratamento farmacológico , Anticoagulantes/química , Aptâmeros de Nucleotídeos/administração & dosagem , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Ponte Cardiopulmonar/efeitos adversos , Cristalografia por Raios X , Combinação de Medicamentos , Fator Xa/genética , Inibidores do Fator Xa/química , Heparina/efeitos adversos , Humanos , Hemorragia Pós-Operatória/genética , Hemorragia Pós-Operatória/patologia , Conformação Proteica/efeitos dos fármacosRESUMO
The absence of an adequate reversal strategy to prevent and stop potential life-threatening bleeding complications is a major drawback to the clinical use of the direct oral inhibitors of blood coagulation factor Xa. Here we show that specific modifications of the substrate-binding aromatic S4 subpocket within the factor Xa active site disrupt high-affinity engagement of the direct factor Xa inhibitors. These modifications either entail amino-acid substitution of S4 subsite residues Tyr99 and/or Phe174 (chymotrypsinogen numbering), or extension of the 99-loop that borders the S4 subsite. The latter modifications led to the engineering of a factor Xa variant that is able to support coagulation in human plasma spiked with (supra-)physiological concentrations of direct factor Xa inhibitors. As such, this factor Xa variant has the potential to be employed to bypass the direct factor Xa inhibitor-mediated anticoagulation in patients that require restoration of blood coagulation.A major drawback in the clinical use of the oral anticoagulants that directly inhibit factor Xa in order to prevent blood clot formation is the potential for life threatening bleeding events. Here the authors describe factor Xa variants that are refractory to inhibition by these anticoagulants and could serve as rescue agents in treated patients.
Assuntos
Inibidores do Fator Xa/química , Inibidores do Fator Xa/farmacologia , Fator Xa/genética , Fator Xa/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Testes de Coagulação Sanguínea , Fator Xa/química , Humanos , Cinética , Engenharia de ProteínasRESUMO
Antithrombin mainly inhibits factor Xa and thrombin. The reactive center loop (RCL) is crucial for its interactions with its protease targets and is fully inserted into the A-sheet after its cleavage, causing translocation of the covalently linked protease to the opposite end of the A-sheet. Antithrombin variants with altered RCL hinge residues behave as substrates rather than inhibitors, resulting in stoichiometries of inhibition greater than one. Other antithrombin residues have been suggested to interfere with RCL insertion or the stability of the antithrombin-protease complex, but available crystal structures or mutagenesis studies have failed to identify such residues. Here, we characterized two mutations, S365L and I207T, present in individuals with type II antithrombin deficiency and identified a new antithrombin functional domain. S365L did not form stable complexes with thrombin or factor Xa, and the I207T/I207A variants inhibited both proteases with elevated stoichiometries of inhibition. Close proximity of Ile-207 and Ser-365 to the inserted RCL suggested that the preferred reaction of these mutants as protease substrates reflects an effect on the rate of the RCL insertion and protease translocation. However, both residues lie within the final docking site for the protease in the antithrombin-protease complex, supporting the idea that the enhanced substrate reactions may result from an increased dissociation of the final complexes. Our findings demonstrate that the distal end of the antithrombin A-sheet is crucial for the last steps of protease inhibition either by affecting the rate of RCL insertion or through critical interactions with proteases at the end of the A-sheet.
Assuntos
Proteínas Antitrombina/química , Transtornos Herdados da Coagulação Sanguínea , Fator Xa/química , Simulação de Acoplamento Molecular , Trombina/química , Substituição de Aminoácidos , Proteínas Antitrombina/genética , Proteínas Antitrombina/metabolismo , Domínio Catalítico , Fator Xa/genética , Fator Xa/metabolismo , Feminino , Humanos , Masculino , Mutação de Sentido Incorreto , Domínios Proteicos , Estrutura Secundária de Proteína , Trombina/genética , Trombina/metabolismoRESUMO
Tissue factor pathway inhibitor (TFPI), the main inhibitor of initiation of coagulation, exerts an important anticoagulant role through the factor Xa (FXa)-dependent inhibition of tissue factor/factor VIIa. Protein S is a TFPI cofactor, enhancing the efficiency of FXa inhibition. TFPI can also inhibit prothrombinase assembly by directly interacting with coagulation factor V (FV), which has been activated by FXa. Because full-length TFPI associates with FV in plasma, we hypothesized that FV may influence TFPI inhibitory function. Using pure component FXa inhibition assays, we found that although FV alone did not influence TFPI-mediated FXa inhibition, it further enhanced TFPI in the presence of protein S, resulting in an â¼8-fold reduction in Ki compared with TFPI alone. A FV variant (R709Q/R1018Q/R1545Q, FVΔIIa) that cannot be cleaved/activated by thrombin or FXa also enhanced TFPI-mediated inhibition of FXa â¼12-fold in the presence of protein S. In contrast, neither activated FV nor recombinant B-domain-deleted FV could enhance TFPI-mediated inhibition of FXa in the presence of protein S, suggesting a functional contribution of the B domain. Using TFPI and protein S variants, we show further that the enhancement of TFPI-mediated FXa inhibition by protein S and FV depends on a direct protein S/TFPI interaction and that the TFPI C-terminal tail is not essential for this enhancement. In FXa-catalyzed prothrombin activation assays, both FV and FVΔIIa (but not activated FV) enhanced TFPI function in the presence of protein S. These results demonstrate a new anticoagulant (cofactor) function of FV that targets the early phase of coagulation before prothrombinase assembly.
Assuntos
Anticoagulantes/metabolismo , Coagulação Sanguínea/fisiologia , Fator V/metabolismo , Substituição de Aminoácidos , Fator V/genética , Fator Xa/genética , Fator Xa/metabolismo , Humanos , Lipoproteínas/genética , Lipoproteínas/metabolismo , Mutação de Sentido Incorreto , Domínios Proteicos , Proteína S/genética , Proteína S/metabolismo , Protrombina/genética , Protrombina/metabolismoRESUMO
Cleavage activation of the hemagglutinin (HA) protein by host proteases is a crucial step in the infection process of influenza A viruses (IAV). However, IAV exists in eighteen different HA subtypes in nature and their cleavage sites vary considerably. There is uncertainty regarding which specific proteases activate a given HA in the human respiratory tract. Understanding the relationship between different HA subtypes and human-specific proteases will be valuable in assessing the pandemic potential of circulating viruses. Here we utilized fluorogenic peptides mimicking the HA cleavage motif of representative IAV strains causing disease in humans or of zoonotic/pandemic potential and tested them with a range of proteases known to be present in the human respiratory tract. Our results show that peptides from the H1, H2 and H3 subtypes are cleaved efficiently by a wide range of proteases including trypsin, matriptase, human airway tryptase (HAT), kallikrein-related peptidases 5 (KLK5) and 12 (KLK12) and plasmin. Regarding IAVs currently of concern for human adaptation, cleavage site peptides from H10 viruses showed very limited cleavage by respiratory tract proteases. Peptide mimics from H6 viruses showed broader cleavage by respiratory tract proteases, while H5, H7 and H9 subtypes showed variable cleavage; particularly matriptase appeared to be a key protease capable of activating IAVs. We also tested HA substrate specificity of Factor Xa, a protease required for HA cleavage in chicken embryos and relevant for influenza virus production in eggs. Overall our data provide novel tool allowing the assessment of human adaptation of IAV HA subtypes.
Assuntos
Hemaglutininas/metabolismo , Vírus da Influenza A/metabolismo , Peptídeos/metabolismo , Fator Xa/genética , Fator Xa/metabolismo , Fibrinolisina/genética , Fibrinolisina/metabolismo , Hemaglutininas/genética , Humanos , Vírus da Influenza A Subtipo H9N2/metabolismo , Vírus da Influenza A/genética , Calicreínas/genética , Calicreínas/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Peptídeos/genética , Proteólise , Sistema Respiratório/metabolismo , Tripsina/genética , Tripsina/metabolismoRESUMO
OBJECTIVE: The role of hypercoagulability in the pathogenesis of diabetic nephropathy (DN) remains elusive. We recently reported the increased infiltration of macrophages expressing tissue factor in diabetic kidney glomeruli; tissue factor activates coagulation factor X (FX) to FXa, which in turn stimulates protease-activated receptor 2 (PAR2) and causes inflammation. APPROACH AND RESULTS: Here, we demonstrated that diabetes mellitus increased renal FX mRNA, urinary FXa activity, and FX expression in glomerular macrophages. Administration of an oral FXa inhibitor, edoxaban, ameliorated DN with concomitant reductions in the expression of PARs (Par1 and Par2) and of proinflammatory and profibrotic genes. Diabetes mellitus induced PAR2, and lack of Par2 ameliorated DN. FXa or PAR2 agonist increased inflammatory cytokines in endothelial cells and podocytes in vitro. CONCLUSIONS: We conclude that enhanced FXa and PAR2 exacerbate DN and that both are promising targets for preventing DN. Alleviating inflammation is probably more important than inhibiting coagulation per se when treating kidney diseases using anticoagulants.
Assuntos
Anti-Inflamatórios/farmacologia , Nefropatias Diabéticas/prevenção & controle , Inibidores do Fator Xa/farmacologia , Fator Xa/efeitos dos fármacos , Glomérulos Renais/efeitos dos fármacos , Piridinas/farmacologia , Receptor PAR-2/metabolismo , Tiazóis/farmacologia , Animais , Coagulação Sanguínea/efeitos dos fármacos , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fator Xa/genética , Fator Xa/metabolismo , Genótipo , Humanos , Mediadores da Inflamação/metabolismo , Insulina/genética , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/genética , Fenótipo , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Receptor PAR-2/antagonistas & inibidores , Receptor PAR-2/deficiência , Receptor PAR-2/genética , Transdução de Sinais/efeitos dos fármacos , Regulação para CimaRESUMO
There is a clinical need to develop safe and rapid therapeutic strategies to control bleeding arising from a host of emergent situations. Over the past several years our laboratory has developed novel zymogen-like FXa variants and tested their safety and efficacy using hemophilia as a model system. The variants have a spectrum of properties resulting from an amino acid change at the N-terminus of the heavy chain that alters a critical conformational change. These properties, which include resistance to plasma protease inhibitors, low activity in the absence of FVa, and rescue of low activity upon incorporation in prothrombinase, yield remarkably effective pro-hemostatic agents. The FVa-dependent restoration of activity is a key aspect to their efficacy and also contributes to localizing the variants to the site of vascular injury. While pre-clinical data support their use in the setting of hemophilia, they have the potential to act as rapid pro-hemostatic agents for the treatment of a range of bleeding conditions. This review will discuss the biochemical properties of these FXa zymogen-like variants and their in vivo characterization.