RESUMO
The proper performance and interpretation of factor inhibitor assays is a critical role for the hemostasis laboratory. Both false-positive and false-negative inhibitor assays may be reported, leading to serious patient mismanagement. Knowledge and recognition of common causes of both false-positive and negative-results can aid in the identification of these potential pitfalls. Safeguards to reporting accurate factor inhibitor assays include initial characterization of the sample, using the Nijmegen modification, properly performing and interpreting an incubated mixing test in conjunction, and performing two dilutions for each dependent dilution in the factor inhibitor assay.
Assuntos
Inibidores dos Fatores de Coagulação Sanguínea/uso terapêutico , Fatores de Coagulação Sanguínea/antagonistas & inibidores , Testes de Coagulação Sanguínea/métodos , Testes de Coagulação Sanguínea/normas , Reações Falso-Negativas , Reações Falso-Positivas , Humanos , Garantia da Qualidade dos Cuidados de Saúde/métodos , Garantia da Qualidade dos Cuidados de Saúde/normas , Reprodutibilidade dos TestesRESUMO
Viper bites cause high morbidity and mortality especially in tropical and subtropical regions, affecting a large number of the rural population in these areas. Even though anti-venoms are available, in most cases they fail to tackle viper venom-induced local manifestations that persist even after anti-venom administration. Several studies have been reported the use of plant products and approved drugs along side anti-venom therapy for efficient management of local tissue damage. In this regard, the present study focuses on the protective efficacy of Cassia auriculata L. (Leguminosae) against Echis carinatus venom (ECV) induced toxicity. C. auriculata is a traditional medicinal plant, much valued in alternative medicine for its wide usage in ayurveda, naturopathy, and herbal therapy. Further, it has been used widely by traditional healers for treatment of snake and scorpion bites in the Western Ghats of Karnataka, India. In the present study, C. auriculata leaf methanol extract (CAME) significantly inhibited enzymatic activities of ECV proteases (96 ± 1 %; P = 0.001), PLA2 (45 ± 5 %; P = 0.01) and hyaluronidases (100 %; P = 0.0003) in vitro and hemorrhage, edema and myotoxicity in vivo. Further, CAME effectively reduced the lethal potency of ECV and increased the survival time of mice by ~6 times (17 vs 3 h). These inhibitory potentials of CAME towards hydrolytic enzymes, mortal and morbid symptoms of ECV toxins clearly substantiates the use by traditional healers of C. auriculata as a folk medicinal remedy for snakebite.
Assuntos
Antivenenos/uso terapêutico , Cassia/química , Fitoterapia , Venenos de Víboras/antagonistas & inibidores , Animais , Antivenenos/química , Fatores de Coagulação Sanguínea/antagonistas & inibidores , Edema/induzido quimicamente , Edema/prevenção & controle , Hemorragia/induzido quimicamente , Hemorragia/prevenção & controle , Humanos , Hialuronoglucosaminidase/antagonistas & inibidores , Masculino , Metanol , Camundongos , Fenóis/isolamento & purificação , Compostos Fitoquímicos/química , Extratos Vegetais/química , Folhas de Planta/química , Venenos de Víboras/toxicidadeRESUMO
BACKGROUND: Recombinant human activated protein C (APC) improves survival of patients with severe sepsis; this beneficial effect is especially apparent in patients with pneumococcal pneumonia. The aim of this study was to determine the effect of APC treatment initiated after induction of pneumococcal pneumonia on pulmonary coagulation, inflammation, and survival, with or without concurrent antibiotic therapy. METHODS: Mice were infected intranasally with viable Streptococcus pneumoniae and were treated intraperitoneally after 24 h of infection with vehicle, recombinant mouse (rm) APC (125 µg), ceftriaxone (500 µg), or rm-APC plus ceftriaxone. Treatment with rm-APC or vehicle was repeated every 8 h for a maximum of 96 h. Animals were either killed 48 h after infection or were monitored in a survival study (with an extra dose of ceftriaxone given after 72 h). RESULTS: Rm-APC treatment inhibited pulmonary activation of coagulation, as reflected by lower levels of thrombin-antithrombin complexes and D-dimer. Rm-APC did not affect the pulmonary levels of 55 inflammatory mediators in the context of antibiotic therapy. Rm-APC added to ceftriaxone markedly improved survival, compared with ceftriaxone treatment alone. CONCLUSIONS: Rm-APC inhibits pulmonary activation of coagulation and, when added to antibiotic therapy, improves survival in murine pneumococcal pneumonia.
Assuntos
Transtornos de Proteínas de Coagulação/tratamento farmacológico , Transtornos de Proteínas de Coagulação/microbiologia , Pneumonia Pneumocócica/tratamento farmacológico , Proteína C/uso terapêutico , Animais , Antibacterianos/uso terapêutico , Fatores de Coagulação Sanguínea/antagonistas & inibidores , Ceftriaxona/administração & dosagem , Ceftriaxona/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Quimioterapia Combinada , Humanos , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia Pneumocócica/complicações , Proteína C/administração & dosagem , Proteína C/farmacologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêuticoRESUMO
Prothrombin complex concentrates (PCC) have been used as bypassing agents for the treatment of haemophilia A patients with inhibitor as well as for replacement therapy in congenital and acquired deficiencies of vitamin-K-dependent clotting factors. The efficacy of PCC is variable, however, especially during long-term and high-dose use, and all currently available products of this nature contain heparin. We have examined the haemostatic properties of PCC using reconstituted whole blood made by mixing coagulation-factor-deficient plasma and washed blood cells. In rotation thromboelastometry (ROTEM), the recommended therapeutic dose of Proplex ST corrected the abnormal patterns. At higher concentrations, however, the ROTEM patterns regressed. In addition, specific assays of coagulation factors appeared unreliable in the presence of 2.5 U/ml Proplex ST; the abnormalities were corrected when protamine sulfate was added. The findings suggest that the presence of heparin in PCC might have a greater effect on global haemostasis. Careful attention to the anticoagulant effect as well as thrombogenicity of PCC is required. Monitoring therapy using such as ROTEM analysis could be highly informative.
Assuntos
Fatores de Coagulação Sanguínea/antagonistas & inibidores , Testes de Coagulação Sanguínea/normas , Hemostasia , Heparina/farmacologia , Fatores de Coagulação Sanguínea/análise , Testes de Coagulação Sanguínea/instrumentação , Testes de Coagulação Sanguínea/métodos , Monitoramento de Medicamentos/normas , Humanos , CinéticaRESUMO
Thrombin and factor Xa, two important procoagulant enzymes, have been prime targets for regulation of clotting through the direct and indirect mechanism of inhibition. Our efforts on exploiting the indirect mechanism led us to study a carboxylic acid-based scaffold, which displayed major acceleration in the inhibition of these enzymes [J. Med. Chem.2005, 48, 1269, 5360]. This work advances the study to chemo-enzymatically prepared oligomers of 4-hydroxycinnamic acids, DHPs, which display interesting anticoagulant properties. Oligomers, ranging in size from tetramers to pentadecamers, were prepared through peroxidase-catalyzed oxidative coupling of caffeic, ferulic, and sinapic acids, and sulfated using triethylamine-sulfur trioxide complex. Chromatographic, spectroscopic, and elemental studies suggest that the DHPs are heterogeneous, polydisperse preparations composed of inter-monomer linkages similar to those found in natural lignins. Measurement of activated thromboplastin and prothrombin time indicates that both the sulfated and unsulfated derivatives of the DHPs display anticoagulant activity, which is dramatically higher than that of the reference polyacrylic acids. More interestingly, this activity approaches that of low-molecular-weight heparin with the sulfated derivative showing approximately 2- to 3-fold greater potency than the unsulfated parent. Studies on the inhibition of factor Xa and thrombin indicate that the oligomers exert their anticoagulant effect through both direct and indirect inhibition mechanisms. This dual inhibition property of 4-hydroxycinnamic acid-based DHP oligomers is the first example in inhibitors of coagulation. This work puts forward a novel, non-heparin structure, which may be exploited for the design of potent, dual action inhibitors of coagulation through combinatorial virtual screening on a library of DHP oligomers.
Assuntos
Fatores de Coagulação Sanguínea/antagonistas & inibidores , Ácidos Cumáricos/síntese química , Ácidos Cumáricos/farmacologia , Inibidores de Proteases/síntese química , Anticoagulantes/síntese química , Anticoagulantes/química , Anticoagulantes/farmacologia , Testes de Coagulação Sanguínea , Técnicas de Química Combinatória , Ácidos Cumáricos/química , Dimerização , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Relação Estrutura-AtividadeRESUMO
Haemophilia A and B are inherited bleeding disorders whose diagnosis and management is generally well established and best provided by specialists in a comprehensive care setting. Patients may be put at unnecessary risk if appropriate expertise is not sought for the management of accidents and surgery. The delivery of a high quality comprehensive service to patients with bleeding disorders depends upon defined standards and a network of haemophilia centres in the UK with similar models in other countries. In developing countries, despite a shortage or absence of treatment products, development of local expertise results in an improved outlook and reduction in mortality. Optimal care for severe haemophilia includes accurate diagnosis, early and adequate factor replacement for bleeding episodes and the provision of prophylaxis from an early age to prevent joint bleeding and the consequent arthropathy. Haemophilia treatment is expensive resulting in considerable inequity in provision of care across the world. Despite decades of experience, optimal treatment levels are not robustly defined. Transfusion-transmitted infections continue to have a significant impact on patient management. The development of inhibitory antibodies seriously complicates the management both in morbidity and cost. While gene therapy has not yet produced the hoped-for cure, new technologies will produce improved products.
Assuntos
Hemofilia A/terapia , Adolescente , Adulto , Fatores de Coagulação Sanguínea/antagonistas & inibidores , Fatores de Coagulação Sanguínea/uso terapêutico , Criança , Assistência Integral à Saúde/métodos , Análise Custo-Benefício , Organização do Financiamento , Infecções por HIV/transmissão , Hemofilia A/economia , Hemofilia A/prevenção & controle , Hemofilia B/economia , Hemofilia B/prevenção & controle , Hemofilia B/terapia , Hepatite C/complicações , Humanos , Equipe de Assistência ao Paciente , Qualidade da Assistência à Saúde , Sepse/transmissãoRESUMO
Plasminogen activator inhibitor-1 (PAI-1) is a key negative regulator of the fibrinolytic system. In animal studies, inhibition of PAI-1 activity prevents arterial and venous thrombosis, indicating that PAI-1 inhibitors may be used as a new class of antithrombotics. In this study, we characterize a small molecule PAI-1 inhibitor, ZK4044, which was identified by high throughput screening and chemically optimized. In a chromogenic substrate-based urokinse (uPA)/PAI-1 assay and a tissue-type plasminogen activator (tPA)-mediated clot lysis assay, ZK4044 inhibited human PAI-1 activity with IC50 values of 644+/-255 and 100+/-90 nM, respectively. ZK4044 had no detectable inhibitory activity toward other serpins such as antithrombin III, alpha1-antitrypsin and alpha2-antiplasmin, indicating that ZK4044 is a specific PAI-1 inhibitor. ZK4044 was shown to bind directly to PAI-1 and prevent the binding of PAI-1 to tPA in a dose-dependent manner in surface plasmon resonance Biacore-based experiments. ZK4044 also prevented PAI-1/tPA complex formation, as analyzed by SDS/PAGE. ZK4044 had little effect on elastase-mediated cleavage of active PAI-1, indicating that the primary mode of action of ZK4044 is most likely to directly block the PAI-1/tPA interaction rather than to convert active PAI-1 to latent PAI-1. In the chromogenic substrate-based uPA/PAI-1 assay, ZK4044 was approximately 2-fold less potent against a mutant PAI-1 (14B-1), which contains four mutations at N150H, K154T, Q319L and M354I, compared with wild-type PAI-1, suggesting that the ZK4044 binding site on the surface of PAI-1 is close to these mutant residues. Together, our data show that ZK4044 represents a new class of small molecule PAI-1 inhibitors with anti-thrombotic potential.
Assuntos
Compostos de Anilina/farmacologia , Benzoatos/farmacologia , Fatores de Coagulação Sanguínea/antagonistas & inibidores , Fibrinolíticos/farmacologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Compostos de Anilina/química , Animais , Benzoatos/química , Avaliação Pré-Clínica de Medicamentos , Fibrinolíticos/química , Humanos , Técnicas In Vitro , Cinética , Mutação , Inibidor 1 de Ativador de Plasminogênio/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serpinas/efeitos dos fármacos , Serpinas/metabolismo , Ressonância de Plasmônio de Superfície , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
Although the central role of thrombin in arterial thrombosis is well established, the efficacy of vitamin K-dependent factor depletion by warfarin at preventing this process has not been established. To assess the efficacy of warfarin in the prevention of arterial thrombosis, two intensities of anticoagulation were compared in a well-characterized porcine model of carotid angioplasty. For 10 days prior to angioplasty, pigs received either high-dose warfarin (n = 9), low-dose warfarin plus aspirin (n = 9), or control tablets (n = 10). Injured arteries were assessed for (111)In-platelet ( x 10(6) cm(-2)) and (125)I-fibrin(ogen) (molecules x 10(12) cm(-2)) deposition and the incidence of macroscopic thrombus. Platelet (30 +/- 7 vs. 332 +/- 137; P = 0.001) and fibrinogen (156 +/- 17 vs. 365 +/- 90; P < 0.05) deposition were significantly reduced in animals receiving high-intensity warfarin whereas low-intensity warfarin/ASA (520 +/- 240 and 1193 +/- 638) was similar to control (P =NS). At the time of angioplasty, the PT-INR and vitamin K-dependent factors varied over a broad range. The greatest reduction of platelet and fibrinogen deposition occurred as the PT-INR increased from 1.0 to 2.2. Increasing the PT-INR beyond 3.0 resulted in little, if any, incremental reduction of either platelet or fibrinogen deposition. Macroscopic thrombus was abolished at PT-INR > 2.2. Despite a broad range of vitamin K factor activities, no single factor was predictive of either platelet or fibrinogen deposition. Warfarin at PT-INR > 2.2 effectively eliminates thrombosis following deep arterial injury. Arterial thrombosis correlates poorly with any single vitamin K-dependent factor but rather appears to be a function of the entire extrinsic coagulation pathway as measured by the PT-INR.
Assuntos
Artérias/lesões , Fatores de Coagulação Sanguínea/antagonistas & inibidores , Trombose/prevenção & controle , Varfarina/farmacologia , Angioplastia/efeitos adversos , Animais , Aspirina/uso terapêutico , Fatores de Coagulação Sanguínea/metabolismo , Plaquetas/patologia , Relação Dose-Resposta a Droga , Fibrinogênio/metabolismo , Coeficiente Internacional Normatizado , Adesividade Plaquetária , Suínos , Trombose/tratamento farmacológico , Trombose/etiologia , Vitamina K , Varfarina/administração & dosagemRESUMO
Heparin and coumarin derivatives have long been used for the prophylaxis and treatment of venous thromboembolism (VTE). Although they have demonstrated efficacy and safety, they act at multiple targets within the coagulation cascade and their efficacy is influenced by many patient variables. Because of the need to improve the benefit-to-risk ratio of antithrombotic drugs, newer agents that target single coagulation factors have been developed. These include selective factor Xa inhibitors, direct thrombin inhibitors (DTIs), and inhibitors of factor IXa and the factor VII-tissue factor complex. Three DTIs-hirudin, bivalirudin, and argatroban-have been approved for clinical use. Fondaparinux, a novel pentasaccharide and the first selective factor Xa inhibitor to be approved by the U.S. Food and Drug Administration and the European Agency for the Evaluation of Medicinal Products, is indicated for the prevention of VTE after major orthopedic surgery. Fondaparinux has predictable pharmacokinetics, is almost 100% bioavailable, and has a half-life that allows once-daily dosing in all indications. In addition, the routine monitoring of standard indicators of hemostasis is not required. The availability of these agents and the continued development of investigational selective coagulation inhibitors have the potential to improve efficacy and decrease adverse events in patients at risk for VTE.
Assuntos
Anticoagulantes/uso terapêutico , Antitrombinas/uso terapêutico , Fatores de Coagulação Sanguínea/antagonistas & inibidores , Trombose/tratamento farmacológico , Anticoagulantes/farmacologia , Antitrombinas/farmacologia , Sítios de Ligação/efeitos dos fármacos , Coagulação Sanguínea/efeitos dos fármacos , Fator Xa/fisiologia , Inibidores do Fator Xa , Hemostasia , Humanos , Trombina/antagonistas & inibidores , Trombina/fisiologiaRESUMO
In this study we compared the antithrombotic and anticoagulant properties of sodium and calcium derivatives of pentosan polysulfate (Na-PPS, Ca-PPS), unfractionated heparin (UFH), and low-molecular-weight heparin (Fraxiparin). The antithrombotic effects of these agents have been investigated in an experimental thrombosis model in which rat mesenteric venules with a diameter of 20-30 microm were injured by well-defined argon laser lesions. Furthermore, the in vivo and in vitro anticoagulant activities [activated partial thromboplastin time (aPTT), Heptest] of these agents have been studied. Thrombus formation was significantly inhibited after s.c. injection of Na-PPS and Ca-PPS in doses >10 mg/kg. The duration of the antithrombotic effect lasted 8 h for Na-PPS and 12 h for Ca-PPS. After oral administration of Na-PPS, an antithrombotic effect was not observed. Oral application of Ca-PPS in doses >20 mg/kg significantly inhibited thrombus formation. Na-PPS and Ca-PPS markedly prolonged clotting time in aPTT and Heptest in concentrations ranging from 0.01 to 0.2 mg/ml rat PTT. Two h after s.c. administration of these agents in a dose of 10 mg/kg, the aPTT increased threefold and the Heptest 2.5-fold compared with controls. After oral application of 50 mg/kg Na-PPS and Ca-PPS, no effect on the coagulation test could be measured. Intravenous injection of UFH prolonged the Heptest after 1 min and the aPTT after 30 min. In ex vivo studies of aPTT and Heptest performed in rat plasma between 2 and 24 h after s.c. injection of 0.2 mg/kg Fraxiparin, no inhibition of any coagulation test was measured. The antithrombotic effect of 0.2 mg/kg Fraxiparin after s.c. injection was significant. Intravenous injection of 20 U/kg UFH significantly inhibited thrombus formation. The smallest antithrombotic effect was after i.v. injection of UFH.
Assuntos
Fibrinolíticos/uso terapêutico , Heparinoides/uso terapêutico , Trombose Venosa/tratamento farmacológico , Animais , Fatores de Coagulação Sanguínea/antagonistas & inibidores , Modelos Animais de Doenças , Heparina/uso terapêutico , Heparinoides/química , Lasers , Masculino , Nadroparina/uso terapêutico , Poliéster Sulfúrico de Pentosana/uso terapêutico , Ratos , Ratos Wistar , Trombose/metabolismoRESUMO
An anticoagulant activity was identified and isolated from the leaves of a West African plant, Aspilia africana by gel filtration on Sephadex G-100. The anticoagulant factor had an apparent molecular weight of approximately 60,000 d. Upon incubation with plasma, it prolonged the partial thromboplastin time, prothrombin time, thrombin and reptilase time. The factor decreased the fibrinogen content of plasma as well as the activity of coagulation factors V, VIII and IX but not factor VII, X or XI activities. After incubation with fibrinogen, the thrombin clotting time was prolonged and the quantity of clottable fibrinogen reduced. The action on fibrinogen was characterized by sequential lytic breakdown of the A-alpha-chain and B-beta-chain, the gamma-chain being lysed last, after prolonged incubation. Benzamidine, Epsilon aminocaproic acid or soybean trypsin inhibitor did not impede lysis.