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1.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802928

RESUMO

Blood platelets' adenosine receptors (AR) are considered to be a new target for the anti-platelet therapy. This idea is based on in vitro studies which show that signaling mediated by these receptors leads to a decreased platelet response to activating stimuli. In vivo evidence for the antithrombotic activity of AR agonists published to date were limited, however, to the usage of relatively high doses given in bolus. The present study was aimed at verifying if these substances used in lower doses in combination with inhibitors of P2Y12 could serve as components of dual anti-platelet therapy. We have found that a selective A2A agonist 2-hexynyl-5'-N-ethylcarboxamidoadenosine (HE-NECA) improved the anti-thrombotic properties of either cangrelor or prasugrel in the model of ferric chloride-induced experimental thrombosis in mice. Importantly, HE-NECA was effective not only when applied in bolus as other AR agonists in the up-to-date published studies, but also when given chronically. In vitro thrombus formation under flow conditions revealed that HE-NECA enhanced the ability of P2Y12 inhibitors to decrease fibrinogen content in thrombi, possibly resulting in their lower stability. Adenosine receptor agonists possess a certain hypotensive effect and an ability to increase the blood-brain barrier permeability. Therefore, the effects of anti-thrombotic doses of HE-NECA on blood pressure and the blood-brain barrier permeability in mice were tested. HE-NECA applied in bolus caused a significant hypotension in mice, but the effect was much lower when the substance was given in doses corresponding to that obtained by chronic administration. At the same time, no significant effect of HE-NECA was observed on the blood-brain barrier. We conclude that chronic administration of the A2A agonist can be considered a potential component of a dual antithrombotic therapy. However, due to the hypotensive effect of the substances, dosage and administration must be elaborated to minimize the side-effects. The total number of animals used in the experiments was 146.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Adenosina-5'-(N-etilcarboxamida)/análogos & derivados , Antitrombinas/farmacologia , Fibrinogênio/metabolismo , Cloridrato de Prasugrel/farmacologia , Agonistas do Receptor Purinérgico P1/farmacologia , Trombose/metabolismo , Monofosfato de Adenosina/farmacologia , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Adulto , Animais , Pressão Sanguínea/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Cloretos , Diástole/efeitos dos fármacos , Feminino , Compostos Férricos , Humanos , Fluxometria por Laser-Doppler , Masculino , Camundongos Endogâmicos C57BL , Permeabilidade/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Sístole/efeitos dos fármacos
2.
Life Sci ; 269: 119073, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33460666

RESUMO

AIMS: Coagulation is a common event that play a double-edged role in physiological and pathological process. Anti-coagulation methods were applied in joint surgery or scaffolds implantation to encourage new vascular formation and avoid coagulation block. However, whether anti-coagulation drug perform regulatory roles in bone structure is unknown. This study aims to explore a direct thrombin inhibitor, argatroban, effects on bone marrow stromal cells (BMSCs) and decipher the underlying mechanisms. MATERIALS AND METHODS: Argatroban effects on BMSCs were investigated in vivo and in vitro. The drug was applied in periodontal disease model mice and bone loss was evaluated by µCT and histology. BMSCs were treated with different doses argatroban or vehicle. Cellular reactions were analyzed using wound healing assay, qRT-PCR, Alizarin Red S staining and western blotting. KEY FINDINGS: We demonstrated that local injection of argatroban can rescue bone loss in periodontal disease in vivo. To explore the underlying mechanism, we examined that cell proliferation and differentiation capability. Proliferation and migration of BMSCs were both inhibited by applying lower dose of argatroban. Interestingly, without affecting osteoclastogenesis, osteogenic differentiation was significantly induced by argatroban, which were shown by extracellular mineralization and upregulation of early osteoblastic differentiation markers, alkaline phosphatase, Osteocalcin, transcription factors RUNX2 and Osterix. In addition, molecular analysis revealed that argatroban promoted ß-catenin nuclear translocation and led to an increase of osteogenesis through activating canonical Wnt signaling. SIGNIFICANCE: Taken together, our results show the novel application of the anti-coagulation compound argatroban in the commitment of BMSCs-based alveolar bone regeneration and remodeling.


Assuntos
Perda do Osso Alveolar/prevenção & controle , Arginina/análogos & derivados , Células-Tronco Mesenquimais/citologia , Osteogênese , Periodontite/complicações , Ácidos Pipecólicos/farmacologia , Sulfonamidas/farmacologia , Trombina/antagonistas & inibidores , Via de Sinalização Wnt/efeitos dos fármacos , Perda do Osso Alveolar/etiologia , Perda do Osso Alveolar/metabolismo , Perda do Osso Alveolar/patologia , Animais , Antitrombinas/farmacologia , Arginina/farmacologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
3.
Clin Appl Thromb Hemost ; 26: 1076029620954913, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33030036

RESUMO

INTRODUCTION: Sulodexide represents a mixture of fast-moving heparin (FMH) and dermatan sulfate (DS) and has been used for the management of venous diseases such as DVT and related disorders. The purpose of this study is to compare sulodexide and its components with unfractionated heparin (UFH) to determine its suitability for the indications in which UFH is used. MATERIALS AND METHOD: Active pharmaceutical ingredients (API) versions of sulodexide, FMH and DS were obtained from Alfasigma. API versions of UFH were obtained from Medefil Inc. Normal human citrated plasma was obtained from blood bank of the Loyola University Medical Center. Each of the individual agents were supplemented in plasma at a graded concentration of 0.0-10 µg/mL. Clotting assays (PiCT, aPTT, PT and TT), anti-Xa and anti-IIa and thrombin generation studies were carried out. Results were compiled as mean ± SD of 3 individual determination. RESULT: In the clot based (PiCT, aPTT and TT), anti-Xa and IIa assays, both the UFH and FMH produced stronger activities in these assays followed by sulodexide. DS did not show any anticoagulant activity. In the thrombin generation assay, FMH and UFH produced comparable inhibition of thrombin generation as measured by various parameters. Sulodexide was slightly weaker in this assay, whereas DS produced relatively weaker effects. CONCLUSION: In comparison to sulodexide, both UFH and FMH exhibit comparable anticoagulant activity despite differences in their molecular weight. These results suggest that sulodexide can be developed as a parenteral anticoagulant for indications in which UFH is used.


Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Glicosaminoglicanos/farmacologia , Trombina/farmacologia , Anticoagulantes/administração & dosagem , Antitrombinas/administração & dosagem , Antitrombinas/farmacologia , Glicosaminoglicanos/administração & dosagem , Heparina/administração & dosagem , Heparina/farmacologia , Humanos , Itália , Sensibilidade e Especificidade , Trombina/administração & dosagem
4.
Vascul Pharmacol ; 130: 106679, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32387621

RESUMO

BACKGROUND: Treatment with non-vitamin K antagonist oral anticoagulants (NOACs) such as dabigatran (a direct thrombin inhibitor) or rivaroxaban (a direct inhibitor of factor [F] Xa) attenuates atherosclerotic plaque progression in hypercholesterolemic mice. PURPOSE: To evaluate the effect of NOACs application on the expression of coagulation proteins in loco within stenotic aortic valves and in valve interstitial cells (VICs) from patients with severe aortic stenosis (AS). METHODS: Primary cultures of VICs obtained from 90 patients undergoing aortic valve replacement were stimulated with TNF-α (50 ng/mL) and pre-treated with rivaroxaban (1 and 10 ng/mL) or dabigatran (25 and 250 ng/mL). The expression of coagulation proteins was analyzed by immunofluorescence. Cytokine levels were measured by ELISA. RESULTS: FX, FXa, FVII, thrombin and PAR1/2 were present in loco within human aortic stenotic valves. Cultured VICs exhibited constant expression of FX, TF, PAR1/2. Exposure of VICs to TNF-α caused the upregulated expression of TF, PAR1/2 and induced expression of thrombin, FVII and FXa. FX was expressed by 80% of VICs, regardless of stimulation. Cultured VICs were able to synthesize metalloproteinases 1-3, IL-6, IL-32, IL-34, osteopontin and osteocalcin, the levels of which increased under TNF-α stimulation. NOACs added to culture inhibited coagulation factor and PAR1/2 expression. Moreover, NOACs down-regulated VIC-derived proteins responsible for valve calcification and extracellular matrix remodeling. CONCLUSIONS: NOACs at therapeutic concentrations may inhibit the effects of FXa and thrombin at in vitro level. It might be speculated that long-term treatment with rivaroxaban or dabigatran could attenuate the progression of AS in humans.


Assuntos
Antitrombinas/farmacologia , Estenose da Valva Aórtica/tratamento farmacológico , Valva Aórtica/efeitos dos fármacos , Fatores de Coagulação Sanguínea/metabolismo , Dabigatrana/farmacologia , Inibidores do Fator Xa/farmacologia , Mediadores da Inflamação/metabolismo , Rivaroxabana/farmacologia , Idoso , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Estenose da Valva Aórtica/genética , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Fatores de Coagulação Sanguínea/genética , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Transdução de Sinais
5.
Life Sci ; 252: 117665, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32305521

RESUMO

AIMS: Thrombin formation is increased in patients with acute cerebral ischemic stroke, and augments coagulation and inflammation in the brain. Administration of antithrombin (AT) was previously reported to be protective against renal and myocardial ischemic injury. Thus, we hypothesized that treatment with AT would be neuroprotective against cerebral ischemic injury. This study evaluated the effects of AT treatment on ischemic inflammation and brain damage in mice subjected to middle cerebral artery occlusion (MCAO). MAIN METHODS: A mouse model of 4-hour MCAO was used to induce ischemic brain injury. Recombinant AT gamma was administered intravenously immediately after reperfusion at 4 h after MCAO. Infarct volume, neurological deficit, and regional cerebral blood flow (rCBF) were measured at 24 h after MCAO. To evaluate the effect of AT gamma on ischemic inflammation, we measured the number of Iba1-positive cells (marker of macrophage/microglial activation) and levels of proinflammatory cytokines. Further, we investigated the direct anti-inflammatory effects of rAT in the J774.1 cell line. KEY FINDINGS: Treatment with AT gamma (480 U/kg) reduced infarct volume and neurological deficit, and improved rCBF, in MCAO mice. Moreover, AT gamma treatment decreased the number of Iba1-positive cells and levels of proinflammatory cytokines. In vitro, treatment with thrombin significantly increased proinflammatory cytokine levels, which was significantly reduced by pretreatment with AT gamma. SIGNIFICANCE: Treatment with AT showed neuroprotective effects via anticoagulation actions, as well as direct anti-inflammatory effects on macrophage/microglial activation. These data suggest that AT may be a useful new therapeutic option for cerebral ischemia.


Assuntos
Antitrombinas/farmacologia , Isquemia Encefálica/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Antitrombinas/administração & dosagem , Isquemia Encefálica/patologia , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média , Inflamação/tratamento farmacológico , Inflamação/patologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microglia/efeitos dos fármacos , Microglia/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Proteínas Recombinantes , Acidente Vascular Cerebral/patologia
6.
Yonsei Med J ; 61(2): 120-128, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31997620

RESUMO

PURPOSE: Stroke prevention in patients with atrial fibrillation (AF) is influenced by many factors. Using a contemporary registry, we evaluated variables associated with the use of warfarin or direct oral anticoagulants (OACs). MATERIALS AND METHODS: In the prospective multicenter CODE-AF registry, 10529 patients with AF were evaluated. Multivariate analyses were performed to identify variables associated with the use of anticoagulants. RESULTS: The mean age of the patients was 66.9±14.4 years, and 64.9% were men. The mean CHA2DS2-VASc and HAS-BLED scores were 2.6±1.7 and 1.8±1.1, respectively. In patients with high stroke risk (CHA2DS2-VASc ≥2), OACs were used in 83.2%, including direct OAC in 68.8%. The most important factors for non-OAC treatment were end-stage renal disease [odds ratio (OR) 0.27; 95% confidence interval (CI): 0.19-0.40], myocardial infarct (OR 0.53; 95% CI: 0.40-0.72), and major bleeding (OR 0.57; 95% CI: 0.39-0.84). Female sex (OR 1.40; 95% CI: 1.21-1.61), cancer (OR 1.78; 95% CI: 1.38-2.29), and smoking (OR 1.60; 95% CI: 1.15-2.24) were factors favoring direct OAC use over warfarin. Among patients receiving OACs, the rate of combined antiplatelet agents was 7.8%. However, 73.6% of patients did not have any indication for a combination of antiplatelet agents. CONCLUSION: Renal disease and history of valvular heart disease were associated with warfarin use, while cancer and smoking status were associated with direct OAC use in high stroke risk patients. The combination of antiplatelet agents with OAC was prescribed in 73.6% of patients without definite indications recommended by guidelines.


Assuntos
Anticoagulantes/uso terapêutico , Fibrilação Atrial/tratamento farmacológico , Administração Oral , Idoso , Anticoagulantes/administração & dosagem , Anticoagulantes/efeitos adversos , Antitrombinas/farmacologia , Antitrombinas/uso terapêutico , Fibrilação Atrial/complicações , Feminino , Hemorragia/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores da Agregação de Plaquetas/farmacologia , Inibidores da Agregação de Plaquetas/uso terapêutico , Estudos Prospectivos , República da Coreia , Fatores de Risco , Acidente Vascular Cerebral/complicações
7.
Vascul Pharmacol ; 124: 106632, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31759113

RESUMO

Diabetic patients have coagulation abnormalities, in which thrombin plays a key role. Whereas accumulating evidence suggests that it also contributes to the development of vascular dysfunction through the activation of protease-activated receptors (PARs). Here we investigated whether the blockade of thrombin attenuates endothelial dysfunction in diabetic mice. Induction of diabetes by streptozotocin (STZ) increased the expression of PAR1, PAR3, and PAR4 in the aorta. STZ-induced diabetic mice showed impairment of endothelial function, while the administration of dabigatran etexilate, a direct thrombin inhibitor, significantly attenuated endothelial dysfunction in diabetic mice with no alteration of metabolic parameters including blood glucose level. Dabigatran did not affect endothelium-independent vasodilation. Dabigatran decreased the expression of inflammatory molecules (e.g., MCP-1 and ICAM-1) in the aorta of diabetic mice. Thrombin increased the expression of these inflammatory molecules and the phosphorylation of IκBα, and decreased the phosphorylation of eNOSSer1177 in human umbilical endothelial cells (HUVEC). Thrombin significantly impaired the endothelium-dependent vascular response of aortic rings obtained from wild-type mice. Inhibition of NF-κB attenuated thrombin-induced inflammatory molecule expression in HUVEC and ameliorated thrombin-induced endothelial dysfunction in aortic rings. Dabigatran attenuated the development of diabetes-induced endothelial dysfunction. Thrombin signaling may serve as a potential therapeutic target in diabetic condition.


Assuntos
Antitrombinas/farmacologia , Dabigatrana/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Angiopatias Diabéticas/prevenção & controle , Endotélio Vascular/efeitos dos fármacos , Animais , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Angiopatias Diabéticas/metabolismo , Angiopatias Diabéticas/fisiopatologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Mediadores da Inflamação/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Receptores Ativados por Proteinase/metabolismo
8.
Platelets ; 31(3): 360-364, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31161848

RESUMO

Dabigatran, a direct oral thrombin inhibitor, has two therapeutic effects: anticoagulation; and antiplatelet activity. In the clinical field, evaluation of the effect of dabigatran on thrombin-induced platelet aggregation is difficult because of fibrin clot formation and platelet aggregation. The aim of this study was to establish a new platelet aggregation method and to investigate the effects of dabigatran on thrombin-induced platelet aggregation. Platelet aggregation with thrombin was performed with automated light transmission aggregometry (CS2400; Sysmex, Kobe, Japan) in 40 healthy subjects. Thrombin-induced platelet aggregation was performed using thrombin and platelet-rich plasma (PRP), and thrombin-induced fibrin polymerization was inhibited by adding the peptide Gly-Pro-Arg-Pro (GPRP). The effect of dabigatran was then evaluated using the above method. Thrombin at < 0.2 U/mL did not induce platelet aggregation in most normal subjects. Median maximum aggregation percent (MA%) (25th-75th percentile) with 0.5 and 1.0 U/mL of thrombin was 87.0% (79.3-90.8%), and 90.2% (86.5-92.2%), respectively. The anti-platelet effects of dabigatran were then evaluated with these concentrations of thrombin. Dabigatran (final concentration, 2.5-1000 nM) inhibited platelet aggregation by 0.2-1.0 U/mL of thrombin in a concentration-dependent manner in vitro. Dabigatran showed potent inhibitory effects against platelet aggregation induced by 0.5 and 1.0 U/mL thrombin with half maximal inhibitory concentrations of 10.5 and 40.4 nM, respectively. A standard for thrombin-induced platelet aggregation was developed using the CS2400 in healthy subjects, and dabigatran was confirmed to inhibit thrombin-induced platelet aggregation in vitro with PRP.


Assuntos
Antitrombinas/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Dabigatrana/farmacologia , Agregação Plaquetária , Testes de Função Plaquetária , Trombina/metabolismo , Adulto , Biomarcadores , Coagulação Sanguínea/efeitos dos fármacos , Feminino , Humanos , Masculino , Agregação Plaquetária/efeitos dos fármacos , Contagem de Plaquetas , Testes de Função Plaquetária/métodos , Trombina/farmacologia , Adulto Jovem
9.
Blood Coagul Fibrinolysis ; 31(1): 97-100, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31833869

RESUMO

: Anticoagulation in a neonate is a challenge and the availability of anticoagulant options is extremely limited. Here we describe the use of a direct thrombin inhibitor, bivalirudin, in a full-term neonate with symptomatic cerebral sinovenous thrombosis complicated by bilateral thalamic hemorrhagic stroke and intraventricular hemorrhage, who could not be effectively treated with sodium heparin due to heparin resistance (HR) and showed thrombosis regression after start of bivalirudin treatment, without worsening of the hemorrhage. While the use of bivalirudin in neonates has been previously described, the indication of cerebral sinovenous thrombosis and the setting of HR are unique.


Assuntos
Antitrombinas/uso terapêutico , Trombose Intracraniana/tratamento farmacológico , Fragmentos de Peptídeos/uso terapêutico , Antitrombinas/farmacologia , Hirudinas/farmacologia , Humanos , Recém-Nascido , Masculino , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico
10.
Biochemistry (Mosc) ; 84(12): 1521-1528, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31870256

RESUMO

DNA aptamers (oligonucleotides) interacting with thrombin exosite I contain G-quadruplex, two T-T, and one T-G-T loops in their structure. They prevent exosite I binding with fibrinogen and thrombin receptors on platelet surface, thereby suppressing thrombin-stimulated formation of fibrin from fibrinogen and platelet aggregation. Earlier, we synthesized original antithrombin aptamer RE31 (5'-GTGACGTAGGTTGGTGTGGTTGGGGCGTCAC-3') that contained (in addition to G-quadruplex) a hinge region connected to six pairs of complementary bases (duplex region). In this study, we compared properties of RE31 aptamer and its analogues containing varying number of bases in the duplex region and nucleotide insertions in the hinge region. Reduction in the number of nucleotides in the duplex region by 1 to 4 pairs (in comparison with RE31 aptamer) resulted in the decrease of the structural stability of aptamers (manifested as lower melting temperatures) and their ability to inhibit thrombin-stimulated fibrin formation in human blood plasma in tests of thrombin, prothrombin, and activated partial thromboplastin times. However, an increase in the number of bases by 1 to 2 pairs did not cause significant changes in the stability and antithrombin activity of the aptamers. Insertions into the hinge region of RE31 aptamer decreased its antithrombin activity. Investigation of RE31 antithrombotic properties demonstrated that RE31 (i) slowed down thrombin formation in human blood plasma (thrombin generation test), (ii) accelerated lysis of fibrin clot by tissue plasminogen activator in in vitro model, and (iii) suppressed arterial thrombosis in in vivo model. Based on the obtained data, RE31 aptamer can be considered as a potentially effective antithrombotic compound.


Assuntos
Antitrombinas/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Trombina/efeitos dos fármacos , Aptâmeros de Nucleotídeos/química , Sítios de Ligação , Testes de Coagulação Sanguínea , Humanos , Agregação Plaquetária/efeitos dos fármacos , Relação Estrutura-Atividade
11.
Thromb Res ; 183: 159-162, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31678711

RESUMO

INTRODUCTION: Clinical benefit-risk balance of direct oral anticoagulants (DOAC) in atherothrombosis prevention differs between anti-Xa and anti-IIa drugs and their specific effect on platelet functions remains controversial. We hence investigated rivaroxaban and dabigatran effect on platelets in identical experimental conditions. MATERIALS AND METHODS: Blood of fifteen healthy volunteers was spiked with DOAC which plasma concentrations were measured by specific anti-Xa or anti-IIa assays. Light transmission aggregometry measured in platelet-rich plasma used low doses of agonists: 0.5 mM arachidonic acid, 2.5 µM ADP, 0.5 µM epinephrine, 0.8 µg/ml collagen, 7.5 µM TRAP-6 and 0.5 pM tissue factor in the presence of H-Gly-Pro-Arg-Pro-OH to prevent fibrin polymerization. Platelet adhesion on collagen fibres was evaluated in whole blood under flow. Same experiments were reproduced in the presence of aspirin. RESULTS: Median [95% CI] plasma concentrations were of 28 [23-36], 128 [119-144] and 321 [293-361] ng/ml for rivaroxaban and 39 [34-45], 171 [166-193] and 353 [349-382] ng/ml for dabigatran. DOAC did not modify platelet aggregation or adhesion on collagen fibres at any tested concentrations. However, they delayed platelet aggregation secondary to coagulation activation with a more potent effect with dabigatran (p < 0.001). Aspirin did not modify DOAC effect. CONCLUSION: Efficacy of combining DOAC and aspirin in atherothrombosis prevention would not stem from a direct antiplatelet effect of the formers but to their additive inhibitory effect on platelet aggregation secondary to coagulation activation. This effect differs according to DOAC molecules and may also result from the pleiotropic roles of the different coagulation factors targeted by DOAC.


Assuntos
Antitrombinas/efeitos adversos , Dabigatrana/efeitos adversos , Inibidores do Fator Xa/efeitos adversos , Rivaroxabana/efeitos adversos , Antitrombinas/farmacologia , Antitrombinas/uso terapêutico , Dabigatrana/farmacocinética , Dabigatrana/uso terapêutico , Inibidores do Fator Xa/farmacologia , Inibidores do Fator Xa/uso terapêutico , Feminino , Voluntários Saudáveis , Humanos , Masculino , Testes de Função Plaquetária , Rivaroxabana/farmacologia , Rivaroxabana/uso terapêutico
12.
Semin Thromb Hemost ; 45(8): 760-766, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31627216

RESUMO

Heatstroke-induced disseminated intravascular coagulation represents potential targets for specific intensive treatments. However, the effect of antithrombin or thrombomodulin treatment remains uncertain. Using a large nationwide inpatient database in Japan, this study aimed to evaluate whether treatment with antithrombin or thrombomodulin could reduce mortality among patients with heatstroke-induced disseminated intravascular coagulation. Using the Japanese Diagnosis Procedure Combination inpatient database from April 2014 to March 2017, we identified heatstroke patients who developed disseminated intravascular coagulation. We allocated patients who started treatment with antithrombin or thrombomodulin within 2 days after admission to the treatment group and allocated others to the control group. A primary outcome was in-hospital mortality. We used a doubly robust analysis to ensure the robustness of our findings. We also conducted two sensitivity analyses for thrombomodulin versus others and antithrombin versus others. We identified 1,606 eligible patients during the 81-month study period. Of these, 556 (35%) received antithrombin or thrombomodulin. The doubly robust analysis demonstrated that in-hospital mortality was significantly lower among patients in the treatment group than among those in the control group (risk difference -6.5%; 95% confidence interval: -12 to -1.4%). In-hospital mortality was significantly lower in patients with thrombomodulin than in others (risk difference -5.5%; 95% confidence interval: -9.5 to -1.6%). There was no significant difference in in-hospital mortality between patients with antithrombin and others (risk difference -4.2%; 95% confidence interval: -9.3 to 0.9%). Treatment with recombinant human thrombomodulin may be associated with lower in-hospital mortality among patients with heatstroke-induced disseminated intravascular coagulation.


Assuntos
Antitrombinas/uso terapêutico , Coagulação Intravascular Disseminada/tratamento farmacológico , Golpe de Calor/complicações , Trombomodulina/uso terapêutico , Idoso , Antitrombinas/farmacologia , Coagulação Intravascular Disseminada/sangue , Coagulação Intravascular Disseminada/mortalidade , Feminino , Humanos , Masculino , Análise de Sobrevida
15.
Thromb Res ; 182: 101-109, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31476483

RESUMO

BACKGROUND: Depending on test assays and the time of last DOAC intake, direct thrombin inhibitors (DTI) and direct FXa inhibitors (DXI) may or may not affect prothrombin time (PT), international normalized ratio (INR) or activated thromboplastin time (aPTT) but the clinical impact is unknown. METHODS: Using data from the Dresden NOAC Registry, we evaluated the impact of DOAC on first PT, INR or aPTT tests during emergency hospitalizations of DTI/DXI patients and the assay performance across 50 coagulation laboratories. RESULTS: In 724 emergency admissions (77 DTI; 647 DXI), 490 cases (67.7%) had a reported last DOAC intake within 12 h before blood sampling. INR and PT were elevated above the upper limit of normal (ULN) in >65% of all cases and aPTT was elevated in 45%. On the other hand, >30% of all cases had normal values of INR, PR and aPTT despite a DOAC intake within the last 12 h. Assay performance for detecting or ruling out therapeutic DOAC levels was highly variable and, overall, insufficient to guide clinical decisions. DOAC specific testing was performed in <10% of all cases. CONCLUSION: Many DOAC recipients present with elevated PT, INR or aPTT during emergency admissions but false negative values within 12 h of last intake as well as elevated values beyond 24 h after last DOAC intake are common. Both scenarios may result in clinical misinterpretation and, potentially, in patient harm, also because DOAC specific testing is rarely performed in emergency settings.


Assuntos
Antitrombinas/uso terapêutico , Testes de Coagulação Sanguínea , Coagulação Sanguínea/efeitos dos fármacos , Inibidores do Fator Xa/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Antitrombinas/farmacologia , Testes de Coagulação Sanguínea/métodos , Serviço Hospitalar de Emergência , Inibidores do Fator Xa/farmacologia , Feminino , Alemanha , Hospitalização , Humanos , Coeficiente Internacional Normatizado/métodos , Masculino , Tempo de Tromboplastina Parcial/métodos , Estudos Prospectivos , Sistema de Registros , Tempo de Coagulação do Sangue Total/métodos
16.
Sci Adv ; 5(8): eaaw2851, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31457083

RESUMO

Macrocyclic compounds are an attractive modality for drug development, but the limited availability of large, structurally diverse macrocyclic libraries hampers the discovery of leads. Here, we describe the discovery of efficient macrocyclization reactions based on thiol-to-amine ligations using bis-electrophiles, their application to synthesize and screen large libraries of macrocyclic compounds, and the identification of potent small macrocyclic ligands. The thiol-to-amine cyclization reactions showed unexpectedly high yields for a wide substrate range, which obviated product purification and enabled the generation and screening of an 8988 macrocycle library with a comparatively small effort. X-ray structure analysis of an identified thrombin inhibitor (K i = 42 ± 5 nM) revealed a snug fit with the target, validating the strategy of screening large libraries with a high skeletal diversity. The approach provides a route for screening large sub-kilodalton macrocyclic libraries and may be applied to many challenging drug targets.


Assuntos
Aminas/química , Compostos Macrocíclicos/química , Bibliotecas de Moléculas Pequenas , Compostos de Sulfidrila/química , Antitrombinas/química , Antitrombinas/farmacologia , Ciclização , Descoberta de Drogas , Humanos , Ligantes , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/farmacologia , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Inibidores da Tripsina/química , Inibidores da Tripsina/farmacologia
17.
J Pharmacol Exp Ther ; 371(2): 375-384, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31451508

RESUMO

JNJ-64179375 (JNJ-9375) is a recombinant human IgG4 monoclonal antibody engineered to mimic an IgA antibody that was identified in a patient who exhibited markedly prolonged clotting times but without spontaneous bleeding episodes over several years of follow-up. The crystal structure of the JNJ-9375 antigen-binding fragment/thrombin complex showed an almost identical binding mode to that of the patient IgA. In the current study, we characterized the in vitro and in vivo properties of JNJ-9375. Surface plasmon resonance studies demonstrated that JNJ-9375 binds to α-thrombin with high affinity and specificity (K D: 0.8 nM for human thrombin). JNJ-9375 produced concentration-dependent prolongation of in vitro clotting assays in human plasma, including thrombin time (TT), ecarin clotting time, prothrombin time, and activated partial thromboplastin time, with EC2X values of 4.4, 12.4, 172.6, and 202.7 µg/ml, respectively. JNJ-9375 inhibited thrombin-induced platelet aggregation in human plasma with an IC50 value of 52.6 nM (7.8 µg/ml) and produced concentration-dependent prolongation of reaction time tested by thromboelastography. JNJ-9375 pretreatment resulted in dose-dependent reduction in thrombus formation in the rat arteriovenous (AV) shunt model of thrombosis. Robust efficacy was observed at 0.3 mg/kg accompanied by 1.5× of TT. Bleeding was increased at 3 mg/kg in a rat tail transection bleeding model demonstrating a therapeutic index of 10× compared with 1× for apixaban in the same models. Our data suggest that thrombin exosite I inhibition is efficacious against thrombosis in a pretreatment prevention animal model. SIGNIFICANCE STATEMENT: JNJ-9375 is a novel, fully human monoclonal antibody that binds to the exosite I region of thrombin with high affinity and specificity. JNJ-9375 concentration dependently prolonged clotting times and inhibited thrombin-induced platelet aggregation in in vitro assays based on its mechanism of action. In an in vivo rat AV shunt model, JNJ-9375 prevented thrombus formation in a dose-dependent fashion while demonstrating reduced bleeding risk. The present study demonstrated the antithrombotic effects of inhibiting the exosite I region of thrombin when given in a prevention mode in preclinical animal models.


Assuntos
Anticorpos Monoclonais/farmacologia , Antitrombinas/farmacologia , Imunoglobulina G/farmacologia , Inibidores da Agregação de Plaquetas/farmacologia , Proteínas Recombinantes/farmacologia , Animais , Anticorpos Monoclonais/metabolismo , Antitrombinas/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Relação Dose-Resposta a Droga , Humanos , Imunoglobulina G/metabolismo , Macaca fascicularis , Masculino , Camundongos , Inibidores da Agregação de Plaquetas/metabolismo , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo
18.
J Clin Pathol ; 72(12): 817-824, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31366633

RESUMO

AIMS: Bivalent direct thrombin inhibitors (DTIs), hirudin and bivalirudin, bind to the active site and exosite 1 of thrombin irreversibly and reversibly, respectively. The present study aims to assess in vitro effects of hirudin and bivalirudin through clot waveform analysis (CWA) and enzyme kinetics in coagulation assays. METHODS: The pooled normal plasma and its dilutions were spiked with hirudin or bivalirudin. The activated partial thromboplastin time (APTT) assay and the Clauss fibrinogen assay were performed using the CS-5100 (Sysmex). The APTT-CWA data were automatically gained by the CS-5100 programme. RESULTS: In APTT-CWA, the maximum coagulation velocity, acceleration and deceleration were decreased dependently on the drug concentrations, demonstrating evidence for the blockade of thrombin-positive feedback by hirudin or bivalirudin. The Hill plot analysis was applied to the dose-dependent curves in bivalirudin. The Hill coefficients were greater than 1, showing positive anticoagulant cooperativity. Regarding the dose-dependent curves in hirudin, all the parameters dropped to almost zero without making an asymptotic line. In the Clauss fibrinogen assay, the Lineweaver-Burk plots demonstrated that both drugs exhibit mixed inhibition mimicking uncompetitive binding. The Dixon plots in bivalirudin were linear and supported the inhibition type described above. The Dixon plots in hirudin were non-linear and inappropriate to use for determination of the inhibition type. In addition, the inverse function of the clotting time appeared to drop to zero without making an asymptotic line, suggesting complete loss of thrombin activity by irreversible binding. CONCLUSIONS: The results provide insights into anticoagulation with bivalent DTIs.


Assuntos
Antitrombinas/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Hirudinas/farmacologia , Tempo de Tromboplastina Parcial , Fragmentos de Peptídeos/farmacologia , Trombina/antagonistas & inibidores , Biomarcadores/sangue , Relação Dose-Resposta a Droga , Fibrinogênio/metabolismo , Humanos , Cinética , Modelos Biológicos , Proteínas Recombinantes/farmacologia , Trombina/metabolismo
19.
Pediatr Crit Care Med ; 20(11): 1027-1033, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31274779

RESUMO

OBJECTIVES: To make practical and evidence-based recommendations on improving understanding of bleeding and thrombosis with pediatric extracorporeal life support and to make recommendations for research directions. DATA SOURCES: Evaluation of literature and consensus conferences of pediatric critical care and extracorporeal life support experts. STUDY SELECTION: A team of 10 experts with pediatric cardiac and extracorporeal membrane oxygenation experience and expertise met through the Pediatric Cardiac Intensive Care Society to review current knowledge and make recommendations for future research to establish "best practice" for anticoagulation management related to extracorporeal life support. DATA EXTRACTION/SYNTHESIS: The first of a two-part white article focuses on clinical understanding and limitations of medications in use for anticoagulation, including novel medications. For each medication, limitations of current knowledge are addressed and research recommendations are suggested to allow for more definitive clinical guidelines in the future. CONCLUSIONS: No consensus on best practice for anticoagulation exists. Structured scientific evaluation to answer questions regarding anticoagulant medication and bleeding and thrombotic events should occur in multicenter studies using standardized approaches and well-defined endpoints. Outcomes related to need for component change, blood product administration, healthcare outcome, and economic assessment should be incorporated into studies. All centers should report data on patients receiving extracorporeal life support to a registry. The Extracorporeal Life Support Organization registry, designed primarily for quality improvement purposes, remains the primary and most successful data repository to date.


Assuntos
Oxigenação por Membrana Extracorpórea/efeitos adversos , Hemorragia/prevenção & controle , Trombose/prevenção & controle , Anticoagulantes/administração & dosagem , Anticoagulantes/efeitos adversos , Anticoagulantes/farmacologia , Antitrombinas/administração & dosagem , Antitrombinas/efeitos adversos , Antitrombinas/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Criança , Estado Terminal/terapia , Oxigenação por Membrana Extracorpórea/métodos , Oxigenação por Membrana Extracorpórea/tendências , Humanos , Inibidores da Agregação de Plaquetas/administração & dosagem , Inibidores da Agregação de Plaquetas/efeitos adversos , Inibidores da Agregação de Plaquetas/farmacologia
20.
J Med Food ; 22(10): 976-981, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31211650

RESUMO

Fermentation has shown to be an effective technique in bioactive peptides release. That is why in this study antihypertensive, antithrombotic, and antioxidant activity was evaluated during amaranth proteins fermentation with Lactobacillus casei Shirota and Streptococcus thermophilus 54102 in mono and combined culture. During fermentation an increase of free amine groups was observed, and no statistical differences among monocultures were shown, getting higher concentration in combined culture. This was related to antihypertensive and antioxidant activities, where the highest values were also found in the combined process (45% of angiotensin-converting enzyme inhibition, and 168 µmol Trolox equivalents per liter [TE/L] for 2,2-diphenyl-1-picrylhydrazyl, 268 µmol TE/L for 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid, and 381 µmol Fe2E/L for ferric reducing ability of plasma). On the contrary, antithrombotic activity was not related to free amine groups during fermentation, having the highest bioactivity in different moments in each experiment. L. casei Shirota and S. thermophilus 54102 are strains that are able to release bioactive peptides from amaranth protein, although amaranth is not a common matrix for the development of lactic acid bacteria. In addition, in this study it was observed for the first time that lactic acid strains are able to release bioactive peptides from amaranth protein. In addition, this methodology could be part for the development of fermented beverages, different from fermented milk, to diversify matrix to obtain a novel functional food.


Assuntos
Amaranthus/química , Fermentação , Lactobacillales/metabolismo , Peptídeos/farmacologia , Proteínas de Plantas/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Anti-Hipertensivos/farmacologia , Antioxidantes/farmacologia , Antitrombinas/farmacologia , Ácido Láctico/metabolismo , Lactobacillus casei , Sementes/química , Streptococcus thermophilus
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