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1.
Biochem Med (Zagreb) ; 30(1): 010702, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31839722

RESUMO

Introduction: Clinical application of rivaroxaban and apixaban does not require therapeutic monitoring. Commercial anti-activated factor X (anti-FXa) inhibition methods for all anti-FXa drugs are based on the same principle, so there are attempts to evaluate potential clinical application of heparin-calibrated anti-FXa assay as an alternative method for direct FXa inhibitors. We aimed to evaluate relationship between anti-FXa methods calibrated with low molecular weight heparin (LMWH) and with drug specific calibrators, and to determine whether commercial LMWH anti-FXa assay can be used to exclude the presence of clinically relevant concentrations of rivaroxaban and apixaban. Materials and methods: Low molecular weight heparin calibrated reagent (Siemens Healthineers, Marburg, Germany) was used for anti-FXa activity measurement. Innovance heparin (Siemens Healthineers, Marburg, Germany) calibrated with rivaroxaban and apixaban calibrators (Hyphen BioMed, Neuville-sur-Oise, France) was used for quantitative determination of FXa inhibitors. Results: Analysis showed good agreement between LMWH calibrated and rivaroxaban calibrated activity (κ = 0.76) and very good agreement with apixaban calibrated anti-Xa activity (κ = 0.82), respectively. Low molecular weight heparin anti-FXa activity cut-off values of 0.05 IU/mL and 0.1 IU/mL are suitable for excluding the presence of clinically relevant concentrations (< 30 ng/mL) of rivaroxaban and apixaban, respectively. Concentrations above 300 ng/mL exceeded upper measurement range for LMWH anti-FXa assay and cannot be determined by this method. Conclusion: Low molecular weight heparin anti-FXa assay can be used in emergency clinical conditions for ruling out the presence of clinically relevant concentrations of rivaroxaban and apixaban. However, use of LMWH anti-FXa assay is not appropriate for their quantitative determination as an interchangeable method.


Assuntos
Anticoagulantes/química , Testes de Coagulação Sanguínea/métodos , Heparina de Baixo Peso Molecular/química , Pirazóis/química , Piridonas/química , Rivaroxabana/química , Anticoagulantes/metabolismo , Área Sob a Curva , Testes de Coagulação Sanguínea/normas , Calibragem , Compostos Cromogênicos/química , Fator Xa/química , Fator Xa/metabolismo , Inibidores do Fator Xa/química , Inibidores do Fator Xa/metabolismo , Heparina de Baixo Peso Molecular/metabolismo , Humanos , Pirazóis/metabolismo , Piridonas/metabolismo , Curva ROC
2.
J Chem Theory Comput ; 15(11): 6504-6512, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31584802

RESUMO

We present an explicit solvent alchemical free-energy method for optimizing the partial charges of a ligand to maximize the binding affinity with a receptor. This methodology can be applied to known ligand-protein complexes to determine an optimized set of ligand partial atomic changes. Three protein-ligand complexes have been optimized in this work: FXa, P38, and the androgen receptor. The sets of optimized charges can be used to identify design principles for chemical changes to the ligands which improve the binding affinity for all three systems. In this work, beneficial chemical mutations are generated from these principles and the resulting molecules tested using free-energy perturbation calculations. We show that three quarters of our chemical changes are predicted to improve the binding affinity, with an average improvement for the beneficial mutations of approximately 1 kcal/mol. In the cases where experimental data are available, the agreement between prediction and experiment is also good. The results demonstrate that charge optimization in explicit solvent is a useful tool for predicting beneficial chemical changes such as pyridinations, fluorinations, and oxygen to sulfur mutations.


Assuntos
Fator Xa/química , Ligantes , Receptores Androgênicos/química , Proteínas Quinases p38 Ativadas por Mitógeno/química , Sítios de Ligação , Fator Xa/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Receptores Androgênicos/metabolismo , Eletricidade Estática , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
3.
Toxicol Lett ; 316: 171-182, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31442586

RESUMO

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 , Tromboelastografia
4.
J Ethnopharmacol ; 243: 112099, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31326559

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Cardiovascular diseases are the major cause of mortality and morbidity, causing over 17.9 million deaths a year worldwide. Currently used therapy is often having side effects and expensive, dietary interventions and alternative medicines are required. Clerodendrum colebrookianum has been used to treat cardiac hypertension but anticoagulant potency was not evaluated. AIM OF THE STUDY: To characterize an active anticoagulant fraction (AAFCC) and a 30 kDa fibrin(ogen)olytic serine protease (clerofibrase) isolated from aqueous leave extract of C. colebrookianum. MATERIALS AND METHODS: AAFCC/clerofibrase was subjected to extensive biochemical and pharmacological characterization including LC-MS/MS, amino acid compositional and GC-MS analyses. Interaction between clerofibrase with fibrinogen was studied by spectrofluorometric analysis. In vitro thrombolytic, antiplatelet and cytotoxicity assay were performed. In vivo toxicity, anticoagulant, defibrinogen and antithrombotic activities were determined on Swiss albino mice. RESULTS: The in vitro anticoagulant activity of AAFCC was found to be superior to heparin and clerofibrase and comparable to Nattokinase and warfarin. The proteomics and amino acid composition analyses suggest that clerofibrase is a previously uncharacterized novel plant protease capable of degrading the -αß chains of fibrinogen/fibrin. AAFCC/clerofibrase exerts their anticoagulant action via fibrinogenolytic activity and partially by antiplatelet activity albeit they have no effect on thrombin and FXa inhibition. The spectrofluorometric analysis revealed the binding of clerofibrase to fibrinogen but not to thrombin and FXa. The phytochemical constituents and bioactive components of AAFCC were characterized by biochemical, and GC-MS analyses. The AAFCC and clerofibrase inhibited collagen/ADP-induced mammalian platelet aggregation, showed in vitro thrombolytic activity, and non-cytotoxic to mammalian cells. The AAFCC showed and dose-dependent in vivo plasma defibrinogenating and anticoagulant activities and inhibited k-carrageen-induced thrombus formation in the tails of mice. CONCLUSION: The potent in vivo anticoagulant and antithrombotic effects of AAFCC suggests its pharmacological significance as herbal anticoagulant drug for the prevention and/or treatment of hyperfibrinogenemia- and thrombosis associated cardiovascular disorders.


Assuntos
Anticoagulantes/uso terapêutico , Clerodendrum , Fibrinolíticos/uso terapêutico , Extratos Vegetais/uso terapêutico , Animais , Anticoagulantes/farmacologia , Anticoagulantes/toxicidade , Sistema Enzimático do Citocromo P-450/metabolismo , Fator Xa/metabolismo , Feminino , Fibrina/metabolismo , Fibrinogênio/metabolismo , Fibrinolíticos/farmacologia , Fibrinolíticos/toxicidade , Células HEK293 , Humanos , Hipertensão/tratamento farmacológico , Masculino , Medicina Tradicional , Camundongos , Extratos Vegetais/farmacologia , Extratos Vegetais/toxicidade , Folhas de Planta , Plantas Medicinais , Serina Endopeptidases , Trombina/metabolismo , Trombose/tratamento farmacológico
5.
Clin Appl Thromb Hemost ; 25: 1076029619847524, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31088146

RESUMO

The currently available oral anti-Xa agents are claimed to produce their anticoagulant and antithrombotic effects solely by the inhibition of factor Xa. This study profiled various anti-Xa drugs in routinely used laboratory assays to demonstrate that their effects are not solely related to the anti-Xa activities. Apixaban, betrixaban, edoxaban, and rivaroxaban were obtained commercially. Native and citrated whole blood was used for the activated clotting time (ACT) and thromboelastography (TEG). Citrated plasma was used for monitoring the prothrombin time (PT), activated partial thromboplastin time (aPTT), Heptest, and prothrombinase-induced clotting time (PiCT) tests. An amidolytic method was used for the determination of anti-Xa effects. Thrombin-induced fibrinokinetics was monitored optically. Thrombin generation studies were carried out using the calibrated automated thrombogram. All of the anti-Xa agents produced concentration- and assay-dependent effects. In the ACT at 2.5 µg/mL and TEG at 1.0 µg/mL, edoxaban exhibited the strongest anticoagulation effect. In the PiCT, PT, and aPTT assay at 1 µg/mL, edoxaban showed stronger effects than other agents. The half maximal inhibitory concentration of these agents for the inhibition of factor Xa ranged from 340 to >1000 ng/mL. In the thrombin generation inhibition assay, apixaban showed the strongest activity. In the fibrinokinetics, different anti-Xa agents produced varying degrees of inhibition. These results demonstrate that the measured anti-Xa activity alone does not fully reflect the overall biologic spectrum of these agents.


Assuntos
Benzamidas , Inibidores do Fator Xa , Fator Xa/metabolismo , Tempo de Protrombina , Pirazóis , Piridinas , Piridonas , Rivaroxabana , Benzamidas/farmacocinética , Benzamidas/farmacologia , Inibidores do Fator Xa/farmacocinética , Inibidores do Fator Xa/farmacologia , Humanos , Tempo de Tromboplastina Parcial , Pirazóis/farmacocinética , Pirazóis/farmacologia , Piridinas/farmacocinética , Piridinas/farmacologia , Piridonas/farmacocinética , Piridonas/farmacologia , Rivaroxabana/farmacocinética , Rivaroxabana/farmacologia , Tiazóis/farmacocinética , Tiazóis/farmacologia , Tromboelastografia
6.
Blood ; 134(8): 699-708, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31133602

RESUMO

Ixolaris is a potent tick salivary anticoagulant that binds coagulation factor Xa (FXa) and zymogen FX, with formation of a quaternary tissue factor (TF)/FVIIa/ FX(a)/Ixolaris inhibitory complex. Ixolaris blocks TF-induced coagulation and PAR2 signaling and prevents thrombosis, tumor growth, and immune activation. We present a high-resolution structure and dynamics of Ixolaris and describe the structural basis for recognition of FX. Ixolaris consists of 2 Kunitz domains (K1 and K2) in which K2 is strikingly dynamic and encompasses several residues involved in FX binding. This indicates that the backbone plasticity of K2 is critical for Ixolaris biological activity. Notably, a nuclear magnetic resonance-derived model reveals a mechanism for an electrostatically guided, high-affinity interaction between Ixolaris and FX heparin-binding (pro)exosite, resulting in an allosteric switch in the catalytic site. This is the first report revealing the structure-function relationship of an anticoagulant targeting a zymogen serving as a scaffold for TF inhibition.


Assuntos
Inibidores do Fator Xa/química , Inibidores do Fator Xa/farmacologia , Fator Xa/metabolismo , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/farmacologia , Animais , Fator Xa/química , Humanos , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Domínios Proteicos , Carrapatos/química
7.
Chem Pharm Bull (Tokyo) ; 67(5): 426-432, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31061367

RESUMO

Quantitative structure-activity relationship (QSAR) techniques, especially those that possess three-dimensional attributes, such as the comparative molecular field analysis (CoMFA), are frequently used in modern-day drug design and other related research domains. However, the requirement for accurate alignment of compounds in CoMFA increases the difficulties encountered in its use. This has led to the development of several techniques-such as VolSurf, Grid-independent descriptors (GRIND), and Anchor-GRIND-which do not require such an alignment. We propose a technique to construct the prediction model that uses molecular interaction field grid potentials as inputs to convolutional neural network. The proposed model has been found to demonstrate higher accuracy compared to the conventional descriptor-based QSAR models as well as Anchor-GRIND techniques. In addition, the method is target independent, and is capable of providing useful information regarding the importance of individual atoms constituting the compounds contained in the chemical dataset used in the proposed analysis. In view of these advantages, the proposed technique is expected to find wide applications in future drug-design operations.


Assuntos
Relação Quantitativa Estrutura-Atividade , Algoritmos , Aprendizado Profundo , Fator Xa/química , Fator Xa/metabolismo , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica
8.
Toxicol In Vitro ; 58: 195-206, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30930232

RESUMO

Envenomations by Asian pitvipers can induce multiple clinical complications resulting from coagulopathic and neuropathic effects. While intense research has been undertaken for some species, functional coagulopathic effects have been neglected. As these species' venoms affect the blood coagulation cascade we investigated their effects upon the human clotting cascade using venoms of species from the Azemiops, Calloselasma, Deinagkistrodon and Hypnale genera. Calloselasma rhodostoma, Deinagkistrodon acutus, and Hypnale hypnale produced net anticoagulant effects through pseudo-procoagulant clotting of fibrinogen, resulting in weak, unstable, transient fibrin clots. Tropidolaemus wagleri was only weakly pseudo-procoagulant, clotting fibrinogen with only a negligible net anticoagulant effect. Azemiops feae and Tropidolaemus subannulatus did not affect clotting. This is the first study to examine in a phylogenetic context the coagulotoxic effects of related genera of basal Asiatic pit-vipers. The results reveal substantial variation between sister genera, providing crucial information about clinical effects and implications for antivenom cross-reactivity.


Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Venenos de Víboras/toxicidade , Viperidae , Animais , Fator Xa/metabolismo , Humanos , Filogenia , Plasminogênio/metabolismo , Protrombina/metabolismo , Tromboelastografia
9.
Toxicol In Vitro ; 58: 97-109, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30910521

RESUMO

Snakebite is a neglected tropical disease that disproportionately affects the poor. Antivenom is the only specific and effective treatment for snakebite, but its distribution is severely limited by several factors, including the prohibitive cost of some products. Papua New Guinea (PNG) is a snakebite hotspot but the high costs of Australian antivenoms (thousands of dollars per treatment) makes it unaffordable in PNG. A more economical taipan antivenom has recently been developed at the Instituto Clodomiro Picado (ICP) in Costa Rica for PNG and is currently undergoing clinical trials for the treatment of envenomations by coastal taipans (Oxyuranus scutellatus). In addition to potentially having the capacity to neutralise the effects of envenomations of non-PNG taipans, this antivenom may have the capacity to neutralise coagulotoxins in venom from closely related brown snakes (Pseudonaja spp.) also found in PNG. Consequently, we investigated the cross-reactivity of taipan antivenom across the venoms of all Oxyuranus and Pseudonaja species. In addition, to ascertain differences in venom biochemistry that influence variation in antivenom efficacy, we tested for relative cofactor dependence. We found that the new ICP taipan antivenom exhibited high selectivity for Oxyuranus venoms and only low to moderate cross-reactivity with any Pseudonaja venoms. Consistent with this genus level distinction in antivenom efficacy were fundamental differences in the venom biochemistry. Not only were the Pseudonaja venoms significantly more procoagulant, but they were also much less dependent upon the cofactors calcium and phospholipid. There was a strong correlation between antivenom efficacy, clotting time and cofactor dependence. This study sheds light on the structure-function relationships of the procoagulant toxins within these venoms and may have important clinical implications including for the design of next-generation antivenoms.


Assuntos
Antivenenos/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Venenos Elapídicos/toxicidade , Animais , Elapidae , Fator Xa/metabolismo , Feminino , Humanos , Masculino
10.
J Biol Chem ; 294(19): 7644-7657, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30918026

RESUMO

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) ∼107 m-1 s-1) was decreased ∼10-fold when FXa was bound to FVa in prothrombinase and a further ∼3-4-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/metabolismo
11.
Burns ; 45(4): 818-824, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30827851

RESUMO

INTRODUCTION: Patients recovering from burn injury are at high risk of developing deep venous thrombosis (DVT). While 30-mg twice-daily enoxaparin is accepted as the standard prophylactic dose, recent evidence in injured patients suggests this dosing strategy may result in sub-optimal pharmacologic DVT prophylaxis. We hypothesized that standard enoxaparin dosing would result in inadequate DVT prophylaxis in burn patients. METHODS: A retrospective review of an ABA-verified Burn center's registry from January 2012 - December 2016 identified patients with peak plasma anti-Xa levels to monitor the efficacy of pharmacologic DVT prophylaxis. Patients ≥18 years old were included if they received at least 3 doses of enoxaparin and had appropriately timed peak anti-Xa levels. We analyzed data including patient demographics, body weight, body mass index (BMI) and total body surface area burn (TBSA). Diagnosis of DVT was collected. RESULTS: During the study period, 393 patients were screened with a plasma anti-Xa levels. Of the 157 patients that met inclusion criteria, 81 (51.6%) achieved target peak plasma anti-Xa levels (0.2-0.4 IU/mL) on standard 30-mg twice-daily prophylactic enoxaparin and 76 (48.4%) had sub-prophylactic levels. Sub-prophylactic patients were more likely to be male, have increased body weight and elevated BMI. 49 of the 76 sub-prophylactic patients received a dose-adjustment in order to reach target anti-Xa levels; 37 patients required 40mg twice-daily, 10 required 50mg twice-daily and 2 required 60mg twice-daily. The overall DVT rate was 3.8%. CONCLUSIONS: The current recommended prophylactic dose of 30-mg twice-daily enoxaparin is inadequate in many burn patients. Alternate dosing strategies should be considered to increase the number of burn patients achieving target prophylactic anti-Xa levels. Determining whether prophylactic enoxaparin dose adjustment decreases DVT rates in burn injured patients should be evaluated in future prospective trials.


Assuntos
Anticoagulantes/administração & dosagem , Queimaduras/terapia , Enoxaparina/administração & dosagem , Fator Xa/metabolismo , Trombose Venosa/prevenção & controle , Adulto , Idoso , Testes de Coagulação Sanguínea , Índice de Massa Corporal , Peso Corporal , Queimaduras/sangue , Quimioprevenção , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores Sexuais , Trombose Venosa/sangue , Adulto Jovem
12.
J Biochem Mol Toxicol ; 33(5): e22287, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30719803

RESUMO

Activated factor X has a central role in the coagulation activation and also contributes to chronic inflammation and tissue fibrosis. In this study, rivaroxaban, a direct factor X inhibitor, attenuates liver fibrosis induced by carbon tetrachloride (CCl4 ). Male rats were randomly allocated into three groups: a control group, CCl 4 fibrotic group, and CCl 4 +rivaroxaban (5 mg/kg) group. Liver fibrosis was induced by subcutaneous injection of CCl 4 twice a week for 6 weeks. Rivaroxaban significantly restored the biochemical parameter including inflammatory and fibrosis markers with histopathological evidence using routine and Masson trichrome staining. It reduced also the expression of tissue factor, fibrin, transforming growth factor and α-smooth muscle actin in the liver tissues. This concludes that rivaroxaban attenuates liver injury caused by CCl 4 , at least in part by inhibiting coagulation and proinflammatory activation. In conclusion, rivaroxaban may be used for the management of liver fibrosis.


Assuntos
Intoxicação por Tetracloreto de Carbono/metabolismo , Inibidores do Fator Xa/farmacologia , Fator Xa/metabolismo , Cirrose Hepática/metabolismo , Rivaroxabana/farmacologia , Animais , Tetracloreto de Carbono/toxicidade , Intoxicação por Tetracloreto de Carbono/patologia , Intoxicação por Tetracloreto de Carbono/prevenção & controle , Fator Xa/farmacologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Cirrose Hepática/prevenção & controle , Masculino , Ratos , Ratos Wistar
13.
Int J Hematol ; 109(4): 390-401, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30756344

RESUMO

Factor VIIa/tissue factor (FVIIa/TF) initiates blood coagulation by promoting FXa generation (extrinsic-Xa). Subsequent generation of intrinsic FXa (intrinsic-Xa) amplifies thrombin formation. Previous studies suggested that FVIIa/TF activates FVIII rapidly in immediate coagulation reactions, and FVIIa/TF/FXa activates FVIII prior to thrombin-dependent feedback. We investigated FVIII/FVIIa/TF/FXa relationships in early coagulation mechanisms. Total FXa generated by FVIIa/TF and FVIIa/TF-activated FVIII (FVIIIaVIIa/TF) was 22.6 ± 1.7 nM (1 min); total FXa with FVIIa-inhibitor was 3.4 ± 0.7 nM, whereas FXa generated by FVIIa/TF or FVIII/TF was 10.4 ± 1.1 or 0.74 ± 0.14 nM, respectively. Little Xa was generated by FVIII alone, suggesting that intrinsic-Xa mechanisms were mediated by FVIIIaVIIa/TF and FVIII/TF in the initiation phase. Intrinsic-Xa was delayed somewhat by von Willebrand factor (VWF). FVIII activation by FXa with FVIIa/TF was comparable to activation with Glu-Gly-Arg-inactivated-FVIIa/TF. TF counteracted the inhibitory effects of VWF on FXa-induced FVIII activation mediated by Arg372 cleavage. The FVIII-C2 domain bound to cytoplasmic domain-deleted TF (TF1-243), and VWF blocked this binding by > 80%, indicating an overlap between VWF- and TF1-243-binding site(s) on C2. Overall, these data suggest that FVIII-associated intrinsic-Xa, governed by both FVIIa/TF-induced and FXa-induced FVIII activation mediated by FVIII-TF interactions, together with FVIIa-dependent extrinsic-Xa mechanisms, may be central to the initiation phase of coagulation.


Assuntos
Coagulação Sanguínea , Fator VIIIa/metabolismo , Fator VIIa/metabolismo , Fator Xa/metabolismo , Tromboplastina/metabolismo , Humanos
14.
Bioorg Med Chem ; 27(7): 1320-1326, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30792102

RESUMO

Kirenol is one of the biologically active diterpenoids from Siegesbeckia pubescens. In terms of the high content and typical structure, many ent-diterpenoids separated from S. pubescens were presumed to be biologically related to kirenol. Among them, epoxy-pimarane diterpenoids are belonging to a special family of naturally occurring compounds that attracted our attentions on their putative biosynthesis pathway and biological activities. Here, we designed and synthesized two known 14,16-epoxy-pimarane diterpenoids (2 and 3) and five 8,15-epoxy-pimarane diterpenoids (4-8) from kirenol. Their absolute structures were determined by 1D and 2D NMR data and the absolute configurations of 4 were confirmed by X-ray crystallographic data. Their inhibition effects on factor Xa (FXa) were evaluated to assess the potentiality of epoxy-pimarane diterpenoids as FXa inhibitor agents.


Assuntos
/farmacologia , Diterpenos/química , Diterpenos/farmacologia , Compostos de Epóxi/farmacologia , Inibidores do Fator Xa/síntese química , Inibidores do Fator Xa/farmacologia , Fator Xa/metabolismo , /síntese química , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Compostos de Epóxi/síntese química , Compostos de Epóxi/química , Inibidores do Fator Xa/química , Humanos , Estrutura Molecular , Relação Estrutura-Atividade
15.
Thromb Res ; 175: 13-20, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30677622

RESUMO

Alterations in the endothelial permeability occur in response to the activation of coagulation mechanisms in order to control clot formation. The activation of the protease activated receptors (PAR) can induce signals that regulate such cellular responses. PAR2 is a target for the coagulation factor Xa (fXa) and tissue factor-factor VIIa (TF-fVIIa) complex. By measuring the permeability of dextran blue across endothelial monolayer, we examined the mechanisms linking coagulation and endothelial permeability. Activation of PAR2 using the agonist peptide (PAR2-AP) resulted in increased permeability across the monolayer and was comparable to that obtained with VEGF at 60 min. Incubation of cells with activated factor Xa (fXa) resulted in an initial decrease in permeability by 30 min, but then significantly increased at 60 min. These responses required fXa activity, and were abrogated by incubation of the cells with a PAR2-blocking antibody (SAM11). Activation of PAR2 alone, or inhibition of PAR1, abrogated the initial reduction in permeability. Additionally, inclusion of Rivaroxaban (0.6 µg/ml) significantly inhibited the response to fXa. Finally, incubation of the endothelial monolayers up to 2 h with TF-containing microvesicles derived from MDA-MB-231 cells, in the presence or absence of fVIIa, did not influence the permeability across the monolayers. In conclusion, fXa but not TF-fVIIa is a noteworthy mediator of endothelial permeability. The rapid initial decrease in permeability requires PAR2 and PAR1 which may act to constrain bleeding. The longer-term response is mediated by PAR2 with increased permeability, presumably to enhance clot formation at the site of damage.


Assuntos
Endotélio/metabolismo , Fator VIIa/metabolismo , Fator Xa/metabolismo , Receptor PAR-2/metabolismo , Linhagem Celular Tumoral , Humanos , Permeabilidade
16.
Toxicol Lett ; 302: 1-6, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30502385

RESUMO

Due to their potent coagulotoxicity, Australian elapid venoms are unique relative to non-Australian members of the Elapidae snake family. The majority of Australian elapids possess potent procoagulant venom, while only a few species have been identified as possessing anticoagulant venoms. The majority of research to-date has concentrated on large species with range distributions overlapping major city centres, such as brown snakes (Pseudonaja spp.) and taipans (Oxyuranus spp.). We investigated the venom from the poorly studied genus Denisonia and documented anticoagulant activities that were differentially potent on amphibian, avian, and human plasmas. Both species were potently anticoagulant upon amphibian plasma, consistent with these snakes preying upon frogs as their primary food source. While D. devisi was only relatively weakly active on avian and human plasma, D. maculata was potently anticoagulant to amphibian, avian, and human plasma. The mechanism of anticoagulant action was determined to be the inhibition of prothrombin activation by Factor Xa by blocking the formation of the prothrombinase complex. Fractionation of D. maculata venom followed by MS sequencing revealed that the toxins responsible were Group I phospholipase A2. As no antivenom is produced for this species or its near relatives, we examined the ability of Seqirus Australian snake polyvalent antivenom to neutralise the anticoagulant effects, with this antivenom shown to be effective. These results contribute to the body of knowledge regarding adaptive evolution of venom, revealing a unique taxon-specific anticoagulant effect for D. devisi venom. These results also reveal the potential effects and mechanisms behind envenomation by the potently acting D. maculata venom on human plasma, while the discovery of the efficacy of an available antivenom provides information crucial to the design of snakebite management strategies.


Assuntos
Antivenenos/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Venenos Elapídicos/metabolismo , Elapidae/metabolismo , Fator V/antagonistas & inibidores , Inibidores do Fator Xa/farmacologia , Mordeduras de Serpentes/tratamento farmacológico , Animais , Antivenenos/metabolismo , Bufo marinus/sangue , Galinhas/sangue , Relação Dose-Resposta a Droga , Fator V/metabolismo , Fator Xa/metabolismo , Inibidores do Fator Xa/metabolismo , Humanos , Mordeduras de Serpentes/sangue , Especificidade da Espécie
17.
Cardiol Rev ; 27(2): 108-111, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30362966

RESUMO

The direct oral anticoagulants (DOACs) have gained popularity recently among both patients and providers for their comparable or better efficacy and safety profiles compared with warfarin and the lack of need for routine monitoring of anticoagulant effect. One obstacle for the more widespread use of the DOACs in clinical practice has been the lack of a reversal agent. Most DOACs act by directly binding to and inhibiting the effects of factor Xa. Andexanet alfa (Andexxa, Portola Pharmaceuticals, San Francisco, CA) is a modified form of factor Xa that acts as a decoy binding entity for DOACs, thereby allowing endogenous factor Xa to perform its normal clotting functions. Andexanet has proven efficacious in clinical trials for reversing the anticoagulant effects of apixaban, edoxaban, and rivaroxaban, although its impact on clinical outcomes has not been adequately studied. Andexanet has a boxed warning for thromboembolic risks, ischemic risks, cardiac arrest, and sudden death, with these adverse events occurring in up to 18% of patients in clinical trials. However, the occurrence of these adverse events needs to be considered in relation to the fragile nature of patients who receive this agent. Because the duration of the DOACs is much less than that of warfarin, it is unclear how many patients would actually need andexanet in clinical practice, because cessation of the DOAC may be all that is needed to effectively manage bleeding. Nonetheless, having andexanet available in cases of DOAC-associated severe or life-threatening bleeding represents a therapeutic advance and should provide an added level of comfort with the clinical use of DOACs.


Assuntos
Fator Xa/efeitos dos fármacos , Hemorragia/prevenção & controle , Proteínas Recombinantes/administração & dosagem , Tromboembolia/tratamento farmacológico , Administração Oral , Fator Xa/administração & dosagem , Fator Xa/metabolismo , Inibidores do Fator Xa/efeitos adversos , Hemorragia/sangue , Hemorragia/induzido quimicamente , Humanos , Tromboembolia/sangue , Resultado do Tratamento
18.
Curr Med Chem ; 26(17): 3175-3200, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-29376487

RESUMO

Factor Xa (FXa) plays a key role in haemostasis, it is a central part of the blood coagulation cascade which catalyzes the production of thrombin and leads to clot formation and wound closure. Therefore, FXa is an attractive target for the development of new anticoagulant agents. In this review, we will first describe the molecular features of this fundamental protein in order to understand its mechanism of action, an essential background for the design of novel inhibitors by means of synthetic organic chemistry or using peptides obtained from recombinant methodologies. Then, we will review the current state of the synthesis of novel direct FXa inhibitors along with their mechanisms of action. Finally, approved reversal agents that aid in maintaining blood haemostasis by using these commercial drugs will also be discussed.


Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Fator Xa/metabolismo , Peptídeos/farmacologia , Anticoagulantes/síntese química , Anticoagulantes/química , Hemostasia/efeitos dos fármacos , Humanos , Peptídeos/síntese química , Peptídeos/química
19.
J Biol Chem ; 294(7): 2422-2435, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30578302

RESUMO

The proteolytic conversion of factor V to factor Va is central for amplified flux through the blood coagulation cascade. Heterodimeric factor Va is produced by cleavage at three sites in the middle of factor V by thrombin, yielding an N terminus-derived heavy chain and a C terminus-derived light chain. Here, we show that light chain formation resulting from the C-terminal cleavage is the rate-limiting step in the formation of fully cleaved Va. This rate-limiting step also corresponded to and was sufficient for the ability of cleaved factor V to bind Xa and assemble into the prothrombinase complex. Meizothrombin, the proteinase intermediate in thrombin formation, cleaves factor V more slowly than does thrombin, resulting in a pronounced defect in the formation of the light chain. A ∼100-fold reduced rate of meizothrombin-mediated light chain formation by meizothrombin corresponded to equally slow production of active cofactor and an impaired ability to amplify flux through the coagulation cascade initiated in plasma. We show that this defect arises from the occlusion of anion-binding exosite 2 in the catalytic domain by the covalently retained propiece in meizothrombin. Our findings provide structural insights into the prominent role played by exosite 2 in the rate-limiting step of factor V activation. They also bear on how factor V is converted into a cofactor capable of assembling into prothrombinase.


Assuntos
Precursores Enzimáticos/química , Fator Va/química , Proteólise , Trombina/química , Precursores Enzimáticos/metabolismo , Fator Va/metabolismo , Fator Xa/química , Fator Xa/metabolismo , Humanos , Ligação Proteica , Domínios Proteicos , Trombina/metabolismo
20.
Toxins (Basel) ; 10(12)2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30518149

RESUMO

Snakebite is a global tropical disease that has long had huge implications for human health and well-being. Despite its long-standing medical importance, it has been the most neglected of tropical diseases. Reflective of this is that many aspects of the pathology have been underinvestigated. Snakebite by species in the Elapidae family is typically characterised by neurotoxic effects that result in flaccid paralysis. Thus, while clinically significant disturbances to the coagulation cascade have been reported, the bulk of the research to date has focused upon neurotoxins. In order to fill the knowledge gap regarding the coagulotoxic effects of elapid snake venoms, we screened 30 African and Asian venoms across eight genera using in vitro anticoagulant assays to determine the relative inhibition of the coagulation function of thrombin and the inhibition of the formation of the prothrombinase complex through competitive binding to a nonenzymatic site on Factor Xa (FXa), thereby preventing FXa from binding to Factor Va (FVa). It was revealed that African spitting cobras were the only species that were potent inhibitors of either clotting factor, but with Factor Xa inhibited at 12 times the levels of thrombin inhibition. This is consistent with at least one death on record due to hemorrhage following African spitting cobra envenomation. To determine the efficacy of antivenom in neutralising the anticoagulant venom effects, for the African spitting cobras we repeated the same 8-point dilution series with the addition of antivenom and observed the shift in the area under the curve, which revealed that the antivenom performed extremely poorly against the coagulotoxic venom effects of all species. However, additional tests with the phospholipase A2 inhibitor LY315920 (trade name: varespladib) demonstrated a powerful neutralisation action against the coagulotoxic actions of the African spitting cobra venoms. Our research has important implications for the clinical treatment of cobra snakebites and also sheds light on the molecular mechanisms involved in coagulotoxicity within Naja. As the most coagulotoxic species are also those that produce characteristic extreme local tissue damage, future research should investigate potential synergistic actions between anticoagulant toxins and cytotoxins.


Assuntos
Acetatos/farmacologia , Anticoagulantes/farmacologia , Venenos Elapídicos/farmacologia , Indóis/farmacologia , Inibidores de Fosfolipase A2/farmacologia , Animais , Antivenenos/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Elapidae , Fator Va/metabolismo , Fator Xa/metabolismo , Fibrinogênio/metabolismo , Humanos , Mordeduras de Serpentes/tratamento farmacológico
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