Purification, Characterization and Evaluation of the Anticoagulant Effect of an Uncompetitive Trypsin Inhibitor obtained from Bauhinia pulchella (Benth) Seeds.

INTRODUCTION: Trypsin inhibitors (TIs) have the ability to competitively or non-competitively bind to trypsin and inhibit its action. These inhibitors are commonly found in plants and are used in protease inhibition studies involved in biochemical pathways of pharmacological interest. OBJECTIVES: This work aimed to purify a trypsin inhibitor from Bauhinia pulchella seeds (BpuTI), describing its kinetic mechanism and anticoagulant effect. METHODS: Affinity chromatography, protein assay, and SDS-PAGE were used to purify the inhibitor. Mass spectrometry, inhibition assays, and enzyme kinetics were used to characterize the inhibitor. In vitro assays were performed to verify its ability to prolong blood clotting time. RESULTS: Affinity chromatography on a Trypsin-Sepharose 4B column gave a yield of 43.1. BpuTI has an apparent molecular mass of 20 kDa with glycosylation (1.15%). Protein identification was determined by MS/MS, and BpuTI showed similarity to several Kunitz-type trypsin inhibitors. BpuTI inhibited bovine trypsin as an uncompetitive inhibitor with IC50 (3 x 10-6 M) and Ki (1.05 x 10-6 M). Additionally, BpuTI showed high stability to temperature and pH variations, maintaining its activity up to 100ºC and in extreme pH ranges. However, the inhibitor was susceptible to reducing agents, such as DTT, which completely abolished its activity. BpuTI showed an anticoagulant effect in vitro at a concentration of 33 µM, prolonging clotting time by 2.6 times. CONCLUSION: Our results suggest that BpuTI can be a biological tool to be used in blood clotting studies.

Anticoagulants and their effects on the hematological and biochemical parameters of yellow-spotted amazon river turtle / Anticoagulantes e seus efeitos em parâmetros hematológicos e bioquímicos de tracajá

Knowledge of suitable methods and reagents for assessing the health condition of specimens of a given species is essential. The present study evaluated the efficacy of the heparin anticoagulants 5,000 I.U. mL-1, Na2EDTA, and K3EDTA on the blood parameters of yellow-spotted amazon river turtle Podocnemis unifilis, employing different solutions for red blood cells count. The use of the various anticoagulants evaluated after 10 hours of storage efficiently inhibited coagulation in blood samples from P. unifilis. An increased number of erythrocytes was observed with the use of K3EDTA 5% when compared with heparin. Statistically significant changes in the erythrocyte number were observed with the use of the different solutions. Solutions which featured sodium citrate and formaldehyde in their composition, allowed erythrocytes counting up to 120 hours after blood collection, without a change in values. The use of the heparin anticoagulants 5,000 I.U. mL-1, Na2EDTA 3%, Na2EDTA 5%, K3EDTA 3% was recommended in the hematological analysis of P. unifilis. Also recommended was the use of the formaldehyde-citrate solution containing 1.9 g of sodium citrate and 1.0 mL of formaldehyde (in 50 mL of distilled water) to perform red blood cells counts in yellow-spotted amazon river turtle.

O conhecimento sobre métodos e reagentes apropriados para as avaliações da condição de saúde de exemplares de determinada espécie é fundamental. Este estudo avaliou a eficácia dos anticoagulantes heparina 5.000 U.I. mL-1, Na2EDTA (3% e 5%), e K3EDTA (3 e 5%) em parâmetros sanguíneos de tracajá (Podocnemis unifilis), bem como diferentes soluções para contagem de eritrócitos totais. A coagulação foi eficientemente inibida nas amostras de sangue de P. unifilis com o uso dos diferentes anticoagulantes avaliados após 10 horas de armazenamento. Maior número de eritrócitos com o uso de K3EDTA 5% foi observado quando comparado com a coleta de sangue realizada com heparina. Diferenças, estatisticamente significativas, entre as contagens de eritrócitos com o uso das diferentes soluções de reagentes avaliadas foram verificadas. As soluções contendo citrato de sódio e formol na composição possibilitaram contagens de eritrócitos até 120 horas após a coleta, sem alteração em seus valores. Recomenda-se o uso dos anticoagulantes heparina 5000 U.I. mL-¹, Na2EDTA 3%, Na2EDTA 5%, K3EDTA 3% nas análises hematológicas de tracajá. Assim como o uso da solução de formol-citrato contendo 1,9 g de citrato de sódio e 1,0 mL de formol (em 50 mL de água destilada) para realização das contagens de eritrócitos totais em tracajá.

Towards Development of an Anti-Vampire Bat Vaccine for Rabies Management: Inoculation of Vampire Bat Saliva Induces Immune-Mediated Resistance.

The common vampire bat (Desmodus rotundus) is a hematophagous species responsible for paralytic rabies and bite damage that affects livestock, humans and wildlife from Mexico to Argentina. Current measures to control vampires, based upon coumarin-derived poisons, are not used extensively due in part to the high cost of application, risks for bats that share roosts with vampires and residual environmental contamination. Observations that vampire bat bites may induce resistance in livestock against vampire bat salivary anticoagulants encourage research into novel vaccine-based alternatives particularly focused upon increasing livestock resistance to vampire salivary components. We evaluated the action of vampire bat saliva-Freund's incomplete adjuvant administered to sheep with anticoagulant responses induced by repeated vampire bites in a control group and examined characteristics of vampire bat salivary secretion. We observed that injections induced a response against vampire bat salivary anticoagulants stronger than by repeated vampire bat bites. Based upon these preliminary findings, we hypothesize the utility of developing a control technique based on induction of an immunologically mediated resistance against vampire bat anticoagulants and rabies virus via dual delivery of appropriate host and pathogen antigens. Fundamental characteristics of host biology favor alternative strategies than simple culling by poisons for practical, economical, and ecologically relevant management of vampire populations within a One Health context.

Isolation, fractionation and anticoagulant activity of a sulfated galactan extracted from the green algae Penicillus capitatus / Isolamento, fracionamento e atividade anticoagulante da galactana sulfatada extraída da alga verde Penicillus capitatus

Marine algae are natural sources of macromolecules known as sulfated polysaccharides. This class of compounds has attracted the interest of Pharmaceutical Sciences due to its pharmacological anticoagulant, antiplatelet and antithrombotic properties. Therefore, this study evaluated the anticoagulant potential of sulfated polysaccharides extracted from the algae Penicillus capitatus. The extracted sulfated polysaccharides were purified, partially characterized and their anticoagulant activity was evaluated. The extraction process followed by ethanol precipitation resulted in five fractions. Among the analyzed fractions, F44 contained highest concentration of sulfated polysaccharides. After the purified fraction F23, F44 displayed in vitro anticoagulant activity in a time testing for activated partial thromboplastin time and pro-thrombin time. The preferential mechanism effect was based on interactions between thrombin and factor Xa. Additional studies on structure pharmacological are required to test the viability of the use of sulfated polysaccharides as therapeutic agents.(AU)

As algas marinhas são fontes naturais de macromoléculas conhecidas como polissacarídeos sulfatados. Esta classe de compostos atraiu o interesse das Ciências Farmacêuticas devido às suas propriedades farmacológicas como anticoagulante, antiplaquetária e antitrombótica. Portanto, este estudo tem como objetivo avaliar o potencial anticoagulante de polissacarídeos sulfatados extraídos de algas de Penicillus capitatus. Os polissacarídeos sulfatados extraídos foram purificados, parcialmente caracterizados e sua atividade anticoagulante foi avaliada. O processo de extração seguido pela precipitação com etanol resultou em cinco frações. Entre as frações analisadas, F44 foi a maior concentração de polissacarídeos sulfatados. Após a purificação, as frações F23 e F44 mostraram atividade anticoagulante in vitro em um teste de tempo de tromboplastina parcialmente ativada e tempo de protrombina. Seu mecanismo preferencial é baseado nas interações entre trombina e fator Xa. Estudos adicionais sobre a estrutura farmacológica são necessários para testar a viabilidade do uso como agente terapêutico.(AU)

Isolation, fractionation and anticoagulant activity of a sulfated galactan extracted from the green algae Penicillus capitatus / Isolamento, fracionamento e atividade anticoagulante da galactana sulfatada extraída da alga verde Penicillus capitatus

Marine algae are natural sources of macromolecules known as sulfated polysaccharides. This class of compounds has attracted the interest of Pharmaceutical Sciences due to its pharmacological anticoagulant, antiplatelet and antithrombotic properties. Therefore, this study evaluated the anticoagulant potential of sulfated polysaccharides extracted from the algae Penicillus capitatus. The extracted sulfated polysaccharides were purified, partially characterized and their anticoagulant activity was evaluated. The extraction process followed by ethanol precipitation resulted in five fractions. Among the analyzed fractions, F44 contained highest concentration of sulfated polysaccharides. After the purified fraction F23, F44 displayed in vitro anticoagulant activity in a time testing for activated partial thromboplastin time and pro-thrombin time. The preferential mechanism effect was based on interactions between thrombin and factor Xa. Additional studies on structure pharmacological are required to test the viability of the use of sulfated polysaccharides as therapeutic agents.

As algas marinhas são fontes naturais de macromoléculas conhecidas como polissacarídeos sulfatados. Esta classe de compostos atraiu o interesse das Ciências Farmacêuticas devido às suas propriedades farmacológicas como anticoagulante, antiplaquetária e antitrombótica. Portanto, este estudo tem como objetivo avaliar o potencial anticoagulante de polissacarídeos sulfatados extraídos de algas de Penicillus capitatus. Os polissacarídeos sulfatados extraídos foram purificados, parcialmente caracterizados e sua atividade anticoagulante foi avaliada. O processo de extração seguido pela precipitação com etanol resultou em cinco frações. Entre as frações analisadas, F44 foi a maior concentração de polissacarídeos sulfatados. Após a purificação, as frações F23 e F44 mostraram atividade anticoagulante in vitro em um teste de tempo de tromboplastina parcialmente ativada e tempo de protrombina. Seu mecanismo preferencial é baseado nas interações entre trombina e fator Xa. Estudos adicionais sobre a estrutura farmacológica são necessários para testar a viabilidade do uso como agente terapêutico.

Role of crotoxin in coagulation: novel insights into anticoagulant mechanisms and impairment of inflammation-induced coagulation

Snake venom phospholipases A2 (svPLA2) are biologically active toxins, capable of triggering and modulating a wide range of biological functions. Among the svPLA2s, crotoxin (CTX) has been in the spotlight of bioprospecting research due to its role in modulating immune response and hemostasis. In the present study, novel anticoagulant mechanisms of CTX, and the modulation of inflammation-induced coagulation were investigated. Methods: CTX anticoagulant activity was evaluated using platelet poor plasma (PPP) and whole blood (WB), and also using isolated coagulation factors and complexes. The toxin modulation of procoagulant and pro-inflammatory effects was evaluated using the expression of tissue factor (TF) and cytokines in lipopolysaccharide (LPS)-treated peripheral blood mononuclear cells (PBMC) and in WB. Results: The results showed that CTX impaired clot formation in both PPP and WB, and was responsible for the inhibition of both intrinsic (TF/factor VIIa) and extrinsic (factor IXa/factor VIIIa) tenase complexes, but not for factor Xa and thrombin alone. In addition, the PLA2 mitigated the prothrombinase complex by modulating the coagulation phospholipid role in the complex. In regards to the inflammation-coagulation cross talk, the toxin was capable of reducing the production of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, and was followed by decreased levels of TF and procoagulant activity from LPS-treated PBMC either isolated or in WB. Conclusion: The results obtained in the present study recognize the toxin as a novel medicinal candidate to be applied in inflammatory diseases with coagulation disorders.(AU)

Quantitative Assessment of Poorly Soluble Anticoagulant Rivaroxaban by Microemulsion Electrokinetic Chromatography.

Microemulsion electrokinetic chromatography (MEEKC) is an electrophoretic methodology based on the separation of compounds by a microemulsionated electrolyte. There are few options for the evaluation of the stability and content of the oral anticoagulant rivaroxaban (RIV) in pharmaceutical formulations. RIV has low water solubility and undergoes ionization only under restricted pH conditions (pH < 1 or pH > 13), thus, hindering the application of free zone capillary electrophoresis as an analytical method. Therefore, the work aimed at developing and validating a stability-indicating MEEKC method for the analysis of RIV in pharmaceutical formulations. Separation was performed in a fused-silica capillary applying a voltage of 30 kV. The microemulsion system consisted of 13 mM tetraborate, pH 9.75 + 1.2% SDS + 1.0% ethyl acetate + 2.4% butanol. The linearity range was 25-150 µg mL-1, with r = 0.9982. Drug degradations were performed in acid and basic media (HCl 1 M and NaOH 0.1 M, respectively), oxidation with 3%H2O2, 60°C temperature and exposure to UV-C radiation. No interferences with RIV or internal standard peaks were detected. Method robustness was accessed through Plackett-Burman experimental design, after evaluation of model validity. Trueness values between 100.49 and 100.68% were obtained with repeatability. The method developed was found appropriate for quality control of RIV tablets, as a consistent analytical technique that is considered less damaging to the environment due to its low consumption of organic reagents.

Índice predictivo de la anticoagulación con antagonistas de la vitamina K / Predictive index of the anticoagulant with antagonists of the vitamin K

Introducción: se recomienda la predicción del riesgo de sangrado durante la anticoagulación terapéutica. Objetivo: diseñar un modelo predictivo del control de la anticoagulación en pacientes tratados con warfarina sódica. Método: se realizó un estudio explicativo y prospectivo en 50 pacientes tratados con warfarina sódica en el Hospital General Docente Dr Agostinho Neto durante el período 2015-2016. Estos se agruparon en un grupo control (n=25, aquellos con adecuada anticoagulación, INR entre 2.0 - 3.0) y un grupo estudio (n=25, con inadecuada anticoagulación, INR < 2.0). Resultados: Los factores más predictivos del efecto anticoagulante insatisfactorio fueron: vivir solo y el uso de fármacos que interfieren con más constancia e intensidad en el efecto del fármaco. Conclusiones: se diseñó un modelo predictivo del control de la anticoagulación con warfarina, ajustado a los servicios del Hospital Dr Agostinho Neto(AU)

Introduction: it recommends the prediction of the risk of bleeding during the therapeutic anticoagulation. Aim: design a predictive model of the control of the anticoagulant in patients treated with warfarina sódica. Method: it realised an explanatory and prospective study in 50 patients treated with warfarina sódica in the Educational General Hospital "Dr. Agostinho Neto" during the period 2015-2016. These grouped in a group control (n=25, those with suitable anticoagulation, INR between 2.0-3.0) and a group study (n=25, with unsuitable anticoagulation, INR < 2.0). Results: The most predictive factors of the effect unsatisfactory anticoagulant were: live alone and the use of drugs that interfere with more proof and intensity in the effect of the drug. Conclusions: it designed a predictive model of the control of the anticoagulation with warfarina, adjusted to the services of the Hospital Dr Agostinho Neto(AU)

Perfil hematológico de cães em diferentes concentrações de anticoagulante e bioquímica sérica em tempos diferentes de armazenamento / Hematologic profile of dogs in different concentrations of anticoagulant and serum biochemistry at different storage times

O objetivo deste estudo foi verificar as alterações hematológicas e bioquímicas em amostras sanguíneas de cães, em diferentes concentrações de anticoagulante EDTA-Na2 e diferentes tempos de armazenamento em temperatura entre 4 e 8°C por até 48 horas. Para isso, foram coletados 12mL de sangue por venopunção jugular ou cefálica de 30 cães (Canis lupus familiaris), procedentes do atendimento clínico realizados no Hospital Veterinário da UFERSA - Mossoró-RN. As amostras foram distribuídas em 4 tubos de ensaio, sendo 3 tubos contendo 50uL de EDTA-Na2 para a análise hematológica, preenchidos com 1mL, 3mL e 4mL, e o quarto tubo, para análise bioquímica, sem anticoagulante, foi acondicionado 4mL da amostra. As análises foram realizadas durante a primeira hora, 24 e 48 horas após a coleta. Posteriormente às análises, observou-se que as variáveis hematológicas se mostraram estáveis ao longo do tempo e independente da concentração de anticoagulante, sem diferenças significativas. Os resultados revelaram que a relação sangue/anticoagulante não interfere nos parâmetros do hemograma quando mantidas em condições adequadas de armazenamento e processadas em analisador hematológico Scil Vet abc™. Quanto às análises bioquímicas, submetidas às mesmas condições de armazenamento, também se mostraram estáveis por até 48 horas. Desse modo, conclui-se que as amostras sanguíneas podem ser armazenadas entre 4°C e 8°C por até 48 horas.(AU)

The objective of this study was to verify hematological and biochemical alterations in blood samples from dogs, in different concentrations of anticoagulant EDTA-Na2 and different storage times in temperature between 4 and 8°C for up to 48 hours. For this, 12mL of blood were collected by jugular or cephalic venipuncture from 30 dogs (Canis lupus familiaris), from the clinical care performed at the Veterinary Hospital of UFERSA - Mossoró-RN. The samples were distributed in 4 test tubes, 3 tubes containing 50uL of EDTA-Na2 for the hematological analysis, filled with 1mL, 3mL and 4mL, and the fourth tube, for biochemical analysis, without anticoagulant, was conditioned 4 mL of the sample. The analyzes were performed during the first hour, 24 and 48 hours after collection. Subsequent to the analyzes, it was observed that the hematological variables were stable over time and independent of the anticoagulant concentration, without significant differences. The results showed that the blood / anticoagulant ratio did not interfere in the hemogram when stored under adequate conditions of storage and processed in a hematology analyzer Scil Vet abc ™. As for the biochemical analyzes, submitted to the same storage conditions, they were also stable for up to 48 hours. Thus, it is concluded that blood samples can be stored between 4°C and 8°C for up to 48 hours.(AU)

Thromboelastometric evaluation of horses submitted to experimental thrombosis and jugular thrombectomy / Avaliação tromboelastométrica de equinos submetidos a trombose e trombectomia jugular experimental

Jugular thrombosis in horses occurs commonly in iatrogenic situations, secondary to endotoxemic clinical condition and disseminated vascular coagulation, potentially leading to death. Thus, hemostatic evaluation becomes necessary and extremely important for monitoring the risks of systemic hypercoagulability and for the efficiency of allopathic and surgical treatment. This paper describes the hemostatic behavior in experimental jugular thrombosis of ten healthy equines, subsequently submitted to two thrombectomy techniques and receiving heparin sodium as anti-rethrombosis therapy. These animals were evaluated for 20 days by thromboelastometry (TEM), platelet count, hematocrit and fibrinogen, at four moments: pre-induction to phlebitis (D0-MPF); three days after thrombophlebitis induction (D3-MFM); 6 days after, - moment of thrombophlebitis - (D9-MT); and 54 (D16) and 126 (D19) hours after thrombectomies (PTM). Thrombectomy was performed via a Vollmar Ring (group 1, n=5) and Fogarty catheter (group 2, n=5). All the animals received heparin (150 UI/kg, SC) every 12 hours, for ten days after the respective thrombectomies. Through the blood samples were evaluated TEM, activated partial thromboplastin time (aPTT) and prothrombin time (PT), dosing of fibrinogen, hematocrit and platelet count at the abovementioned moments. For comparison between groups and moments the t test was applied at 5% significance level. No significant difference was verified between treatment groups at any of the moments. There were reductions in clotting time (CT) and clot formation time (CFT), with increase in maximum lysis (ML) until the moment D9-MT. [...] (AU)

A trombose jugular nos equinos ocorre comumente em situações iatrogênicas, secundárias a quadros endotoxêmicos e a coagulação vascular disseminada, podendo levar ao óbito. Por isso, avaliação hemostática se faz necessária e de extrema importância para monitorar os riscos de hipercoagulabilidade sistêmica e também a eficiência do tratamento alopático e cirúrgico. Este trabalho descreve o comportamento hemostático na trombose jugular experimental de dez equinos hígidos, submetidos posteriormente a duas técnicas de trombectomia e recebendo heparina sódica como terapia anti retrombosante. Estes animais foram avaliados durante 20 dias por tromboelastometria (TEM), contagem de plaquetas, hematócrito e fibrinogênio, em quatro momentos: pré-indução à flebite (D0-MPF); três dias após a indução da tromboflebite (D3-MFM); 6 dias após, - momento de tromboflebite - (D9-MT); e 54 (D16) e 126 (D19) horas após as trombectomias (MPT). A trombectomia foi realizada com Anel de Vollmar (grupo 1, n=5) e cateter de Fogarty (grupo 2, n=5). Todos os animais receberam heparina (150 UI/Kg, SC) a cada 12 horas, durante dez dias após as respectivas trombectomias. Através de amostras de sangue, foram avaliadas a TEM, o tempo de tromboplastia parcial ativada (TTPa) e tempo de protrombina (TP), a dosagem de fibrinogênio, hematócrito e contagem de plaquetas nos momentos descritos acima. Para a comparação entre os grupos e momentos foi aplicado teste t, com nível de significância de 5%. Não foi verificada diferença significativa entre os grupos de tratamento em nenhum dos momentos. Houve redução do tempo de coagulação (CT) e do tempo de formação do coágulo (CFT), com aumento da lise máxima (LM) até o momento D9-MT. A avaliação com o reagente intem apresentou prolongamento do CT e do CFT e redução do ângulo α e da LM a partir do D16 e D19. Da mesma forma, o TTPa apresentou diferenças significativas entre os momentos pré (D0, 3 e 9) e pós (D16 e 19) tratamento cirúrgico e anticoagulante.[...] (AU)

Thromboelastometric evaluation of horses submitted to experimental thrombosis and jugular thrombectomy / Avaliação tromboelastométrica de equinos submetidos a trombose e trombectomia jugular experimental

Jugular thrombosis in horses occurs commonly in iatrogenic situations, secondary to endotoxemic clinical condition and disseminated vascular coagulation, potentially leading to death. Thus, hemostatic evaluation becomes necessary and extremely important for monitoring the risks of systemic hypercoagulability and for the efficiency of allopathic and surgical treatment. This paper describes the hemostatic behavior in experimental jugular thrombosis of ten healthy equines, subsequently submitted to two thrombectomy techniques and receiving heparin sodium as anti-rethrombosis therapy. These animals were evaluated for 20 days by thromboelastometry (TEM), platelet count, hematocrit and fibrinogen, at four moments: pre-induction to phlebitis (D0-MPF); three days after thrombophlebitis induction (D3-MFM); 6 days after, - moment of thrombophlebitis - (D9-MT); and 54 (D16) and 126 (D19) hours after thrombectomies (PTM). Thrombectomy was performed via a Vollmar Ring (group 1, n=5) and Fogarty catheter (group 2, n=5). All the animals received heparin (150 UI/kg, SC) every 12 hours, for ten days after the respective thrombectomies. Through the blood samples were evaluated TEM, activated partial thromboplastin time (aPTT) and prothrombin time (PT), dosing of fibrinogen, hematocrit and platelet count at the abovementioned moments. For comparison between groups and moments the t test was applied at 5% significance level. No significant difference was verified between treatment groups at any of the moments. There were reductions in clotting time (CT) and clot formation time (CFT), with increase in maximum lysis (ML) until the moment D9-MT. Evaluation through INTEM® reagent presented prolongations of CT and CFT with reduction of α angle and ML starting from D16 and D19. Similarly, aPTT presented significant differences between moments pre- (D0, 3 and 9) and post- (D16 and 19) anticoagulant and surgical treatment. The platelet numbers were diminished at moments D16 and D19. In evaluation with EXTEM® reagent, prolongation of CT and CFT occurred only between the moments D0 vs. D3 and vs. D9. O PT did not present significant differences. The results obtained demonstrate that experimental jugular thrombophlebitis leads to local clinical alterations, with impairment of tissue and of the extrinsic coagulation pathway (EXTEM® ), but without evidence of systemic hypercoagulability status, since there was no increase of the alpha angle or maximum clot firmness (MCF). Furthermore, TEM was shown useful and more sensitive than conventional coagulation tests (PT, aPTT and fibrinogen) for the monitoring of anticoagulant therapy, as demonstrated in other works.(AU)

A trombose jugular nos equinos ocorre comumente em situações iatrogênicas, secundárias a quadros endotoxêmicos e a coagulação vascular disseminada, podendo levar ao óbito. Por isso, avaliação hemostática se faz necessária e de extrema importância para monitorar os riscos de hipercoagulabilidade sistêmica e também a eficiência do tratamento alopático e cirúrgico. Este trabalho descreve o comportamento hemostático na trombose jugular experimental de dez equinos hígidos, submetidos posteriormente a duas técnicas de trombectomia e recebendo heparina sódica como terapia anti retrombosante. Estes animais foram avaliados durante 20 dias por tromboelastometria (TEM), contagem de plaquetas, hematócrito e fibrinogênio, em quatro momentos: pré-indução à flebite (D0-MPF); três dias após a indução da tromboflebite (D3-MFM); 6 dias após, - momento de tromboflebite - (D9-MT); e 54 (D16) e 126 (D19) horas após as trombectomias (MPT). A trombectomia foi realizada com Anel de Vollmar (grupo 1, n=5) e cateter de Fogarty (grupo 2, n=5). Todos os animais receberam heparina (150 UI/Kg, SC) a cada 12 horas, durante dez dias após as respectivas trombectomias. Através de amostras de sangue, foram avaliadas a TEM, o tempo de tromboplastia parcial ativada (TTPa) e tempo de protrombina (TP), a dosagem de fibrinogênio, hematócrito e contagem de plaquetas nos momentos descritos acima. Para a comparação entre os grupos e momentos foi aplicado teste t, com nível de significância de 5%. Não foi verificada diferença significativa entre os grupos de tratamento em nenhum dos momentos. Houve redução do tempo de coagulação (CT) e do tempo de formação do coágulo (CFT), com aumento da lise máxima (LM) até o momento D9-MT. A avaliação com o reagente intem apresentou prolongamento do CT e do CFT e redução do ângulo α e da LM a partir do D16 e D19. Da mesma forma, o TTPa apresentou diferenças significativas entre os momentos pré (D0, 3 e 9) e pós (D16 e 19) tratamento cirúrgico e anticoagulante. Houve diminuição do número de plaquetas nos momentos D16 e D19. Na avaliação com reagente extem ocorreu apenas o prolongamento do CT e CFT entre os momentos D0 e o D3 e D9. O TP não apresentou diferenças significativas. Os resultados obtidos demonstram que a tromboflebite jugular experimental leva a alterações clínicas locais, com comprometimento tecidual e da via extrínseca da coagulação (extem), porém sem evidências de um estado sistêmico de hipercoagulabilidade, pois não houve aumento do ângulo alfa e da firmeza máxima do coágulo (MCF). Além disso, a TEM se mostrou útil e mais sensível que os testes convencionais de coagulação (TP, TTPa e fibrinogênio) para o acompanhamento da terapia anticoagulante, conforme demonstrado em outros trabalhos.(AU)

Comparação de Custo da enoxaparina e dos novos anticoagulantes orais dabigatran e rivaroxaban na profilaxia do tromboembolismo venoso / Cost comparison between enoxaparin and new oral anticoagulants dabigatran and rivaroxaban in venous thromboembolism prophylaxis

Venous thromboembolism, an important complication that is easily prevented during surgery, is among the few clinicai entities wherein the use of prophylaxis has been found to be effective, such as in the case of antibiotic prophylaxis for the prevention of surgical site infections. The objectives of prophylaxis are to reduce the incidence of deep vein thrombosis, minimize the risk of long-term complications such as chronic venous insufficiency and pulmonary hypertension, and prevent death caused by pulmonary embolism. In this study, we aimed to provide current information to plastic surgeons on the therapeutic options for prophylaxis of venous thromboembolism as well as to compare treatment costs of the drugs enoxaparin, dabigatran, and rivaroxaban. Method: For comparison, the prophylaxis regimen was started 6 hours after the surgery and continued for 10 days. The end user price was obtained from an internet search that included leading pharmacy chains. Results: The patient costs of the new oral anticoagulants dabigatran and rivaroxaban were lower than that of enoxaparin. Conclusions: The cost of venous thromboembolism prophylaxis consisting of the new oral anticoagulants dabigatran and rivaroxaban is lower than that of low molecular weight heparin.

O tromboembolismo venoso é uma complicação importante altamente evitável em cirurgia, sendo uma das poucas entidades clínica em medicina passíveis de profilaxia comprovadamente eficiente, assim como ocorre na profilaxia antibiótica da infecções cirúrgicas. A profilaxia tem por objetivo diminuir a incidência de trombose venosa profunda minimizar os riscos das complicações a longo prazo da insuficiência venosa crônica e hipertensão pulmonar, bem como prevenir a morte consequente de embolia pulmonar. Este estudo tem por objetivo atualizar os cirurgiões plásticos quanto às medicações que podem ser adotadas na profilaxia do tromboembolismo venoso bem como comparar o custo dessas medicações (enoxaparina dabigatran e rivaroxaban). Método: Para efeito de comparação foi adotado um esquema de profilaxia iniciado 6 horas após o fim da cirurgia e mantido por 1O dias. A pequisa foi realizada na internet entre as principais redes farmacêuticas, levando em conta custo para o consumidor. Resultados: Os novos anticoagulantes orais dabigatran e rivaroxaban apresentaram custo menor para o paciente. Conclusões: Os novos anticoagulantes orais dabigatran e rivaroxaban possuem custo menor na profilaxia do tromboembolismo venoso em comparação à heparina de baixo peso molecular.

Freshwater plants synthesize sulfated polysaccharides: heterogalactans from Water Hyacinth (Eicchornia crassipes).

Sulfated polysaccharides (SP) are found mainly in seaweeds and animals. To date, they have only been found in six plants and all inhabit saline environments. Furthermore, there are no reports of SP in freshwater or terrestrial plants. As such, this study investigated the presence of SP in freshwaters Eichhornia crassipes, Egeria densa, Egeria naja, Cabomba caroliniana, Hydrocotyle bonariensis and Nymphaea ampla. Chemical analysis identified sulfate in N. ampla, H. bonariensis and, more specifically, E. crassipes. In addition, chemical analysis, FT-IR spectroscopy, histological analysis, scanning electron microscopy (SEM) and energy-dispersive X-ray analysis (EDXA), as well as agarose gel electrophoresis detected SP in all parts of E. crassipes, primarily in the root (epidermis and vascular bundle). Galactose, glucose and arabinose are the main monosaccharides found in the sulfated polysaccharides from E. crassipes. In activated partial thromboplastin time (APTT) test, to evaluate the intrinsic coagulation pathway, SP from the root and rhizome prolonged the coagulation time to double the baseline value, with 0.1 mg/mL and 0.15 mg/mL, respectively. However, SP from the leaf and petiole showed no anticoagulant activity. Eichornia SP demonstrated promising anticoagulant potential and have been selected for further studies on bioguided fractionation; isolation and characterization of pure polysaccharides from this species. Additionally in vivo experiments are needed and are already underway.

New perspectives and recommendations for anticoagulant therapy post orthopedic surgery

Anticoagulant therapy is essential for the prevention of risks associated with the formation of thrombus in patients after surgery, especially in orthopedics. Recently, new oral anticoagulants were introduced in the therapeutic arsenal. This fact is important, because the current drug of choice in clinical practice is enoxaparin, a low molecular weight heparin. As all injecting drugs, enoxaparin may reduce patients' adherence to treatment by dissatisfaction with and resistance to the administration. This article reviews the available literature on the overall utility of these innovative medicines, approaching the pharmacology, the compared efficacy in relation to current agents, and the potential targets for new agents, as well as points to new trends in research and development. The article also contributes with a practical guide for use and recommendations to health professionals, especially focusing on the reversibility of hemorrhagic events, and discusses the importance of convenience/satisfaction of use, the cost of treatment, and the risk-benefit profile for patients.

A terapia anticoagulante é fundamental para a prevenção de riscos associados à formação de trombos em pacientes pós-cirúrgicos, principalmente em ortopedia. Recentemente, novos anticoagulantes orais foram introduzidos no arsenal terapêutico. Tal fato é importantíssimo, visto que o atual medicamento de primeira escolha na prática clínica é a enoxaparina, uma heparina de baixo peso molecular. Por ser de uso injetável, a enoxaparina pode diminuir a adesão do paciente ao tratamento, devido à insatisfação e à resistência quanto à via de administração. Este artigo revisa a literatura disponível sobre a utilidade total desses medicamentos inovadores ao abordar a farmacologia, a eficácia em comparação com os agentes atuais e os alvos potenciais para novos agentes, bem como aponta as novas tendências em pesquisa e desenvolvimento. O artigo também contribui com um guia prático de uso e recomendações aos profissionais de saúde, com um enfoque especial sobre a reversibilidade de eventos hemorrágicos e, finalmente, discute a importância da conveniência/satisfação de uso, o custo de tratamento e o perfil risco-benefício para o paciente.

Novel and highly sensitive mixed-polymeric electrokinetic chromatography system for determination of contaminants and impurities of heparin samples.

A mixed-polymeric electrokinetic chromatography system has been developed for the simultaneous determination of a contaminant like oversulfated condroitin sulfate (OSCS) and impurities expressed as dermatan (Der) in heparin (Hep) samples. The EKC system consisted of 0.5% w/v polymeric ß-CD, 0.4% w/v tetronic(®) 1107 and 400 mM tris-phosphate buffer at pH 3.5. The optimized electrophoretic conditions included the use of an uncoated-silica capillary of 50 cm of total length and 75 µm id, an applied voltage of -7 kV, a temperature of 30°C and 200 nm UV-detection. The highly sensitive method developed showed low values of LOD, 0.07% w/w (0.07 µg/mL) (OSCS) and 0.1% w/w (0.1 µg/mL) (Der), and values of LOQ 0.2% w/w (0.2 µg/mL) (OSCS) and 0.3% w/w (0.3 µg/mL) (Der) with a concentration level of Hep sample as low as 0.1 mg/mL. Additional parameters of validation such as specificity, linearity, accuracy, and robustness were evaluated according to international guidelines. Owing to its simplicity, high sensitivity, and reliability, the proposed method can be an advantageous alternative to the traditional methodologies for the analysis of Hep in raw material and specially in finished products because of the low amounts of Hep sample required.

Identification and partial purification of an anticoagulant factor from the venom of the Iranian snake Agkistrodon halys

An anticoagulant factor was purified from the venom of the Iranian snake Agkistrodon halys by gel filtration on Sephadex G-50 and ion-exchange chromatography on DEAE-Sepharose. In the final stage of purification, the percentage recovery of purified anticoagulant factor was found to be 83 percent. The purified anticoagulant factor revealed a single protein band in SDS-polyacrylamide electrophoresis under reducing conditions and its molecular weight was about 22 kDa. The purified peptide did not show any effect on casein, BApNA or plasma.(AU)

Avaliação de anticoagulantes naturais e de fatores da coagulação em pacientes com distúrbios congênitos de glicosilação (DCG) tipo I / An evaluation of natural anticoagulants and coagulation factors in patients with congenital disorders of glycosylation type I

Defeitos na incorporação de N-glicanos nas proteínas humanas ocasionam um grupo de doenças multissistêmicas denominadas coletivamente distúrbios congênitos de glicosilação (DCG). Os DCG manifestam-se na infância com sintomas neurológicos que incluem principalmente atraso psicomotor, ataxia, hipotonia e episódios de acidente vascular cerebral. Várias proteínas do sistema hemostático somente tornam-se biologicamente ativas após a glicosilação. O objetivo deste estudo foi avaliar os anticoagulantes naturais (proteína S livre, proteína C e antitrombina) e os fatores da coagulação (VIII, IX e XI) em pacientes com DCG tipo I. Foram avaliados 11 pacientes com diagnóstico positivo para DCG tipo I (três do gênero masculino e oito do gênero feminino), idade média de 5,6 anos; e oito pacientes com diagnóstico negativo para DCG(quatro do gênero masculino e quatro do gênero feminino), idade média de 4,5 anos (grupo-controle). O diagnóstico de DCG tipo I foi realizado pela identificação do padrão de hipoglicosilação da transferrina plasmática. Na avaliação dos anticoagulantes naturais pode-se observar redução dos valores de PS livre e PC e uma redução marcante de AT, quando comparados com o grupo controle. Em relação aos fatores de coagulação não houve diferença significativa para os fatores VIII e IX e houve redução marcante do fator XI. Os resultados do presente estudo sugerem que a deficiência combinada de anticoagulantes naturais é responsável pelo estado pró-trombótico observado em pacientes com DCG. Sugerimos também que a análise dos parâmetros hemostáticos seja realizada para pacientes com DCG quando apresentarem sintomas clínicos de alteração do sistema hemostático e antes de procedimentos invasivos.

Defects in the biosynthesis of N-linked human protein glycosylation leads to a group of multisystem disorders collectively called congenital disorders of glycosylation (CDG). CDG present in infancy with neurologic symptoms that include psychomotor retardation, ataxia, hypotonia and stroke-like episodes. Many haemostatic system proteins only present biological activity after glycosylation. The aim of this study was to evaluate coagulation inhibitors (free protein S, protein C and antithrombin) and coagulation factors (VIII, IX and XI) in CDG type I patients. Eleven patients with CDG type I (three males and eight females) with a mean age of 5.6 years old, and eight patients without CDG (four males and four females) with a mean age of 4.5 years old (control group) were evaluated. The diagnoses of CDG type I were confirmed by isoelectric focusing of serum transferrin. When coagulation inhibitors were evaluated, decreased activity of free protein S and protein C, and a pronounced reduction of antithrombin were observed compared to the control group. There was no significant difference for coagulation factors VIII and IX but a markedly decrease in factor XI. The present results suggest that a combined deficiency of coagulation inhibitors is responsible for the pro-thrombotic state observed in CDG patients. We recommend that a haemostatic analysis should be performed in CDG patients with clinical haemostatic manifestations before invasive procedures are performed.

Warfarin use has no effect on blood pressure of patients with Diabetes and hypertension / Uso da warfarina não tem efeitos sobre a pressão arterial em pacientes com diabetes e hipertensão arterial sistêmica

Introduction: Warfarin causes arterial calcification, arterial stiffness and systolic hypertension in animals. Early evidence in humans indicates that a similar effect may occur in patients with diabetes mellitus (DM) and/or hypertension. Objective: To evaluate whether warfarin use causes elevated blood pressure and pulse pressure in patients with both DM and hypertension. Methods: Cross-sectional study of 159 subjects with both DM and hypertension who received warfarin for at least 2 years and 159 age-matched control subjects with DM and hypertension never exposed to warfarin. The primary focus of analysis was the difference in systolic blood pressure (SBP), diastolic blood pressure (DBP) and pulse pressure (PP) between the two groups. Results: Average age was 73±10 years in both groups. Patients in the warfarin group had received it for an average of 5.5±3.1 years. Subjects in the warfarin group had higher rates of coronary disease and heart failure. SBP and PP were lower in the warfarin group (SBP 130±14 mmHg vs. 134±12 mmHg, P=0.003; PP 58±12 mmHg vs. 62±11 mmHg, P=0.004), while DBP was not different (72±8 vs. 72±7 mmHg, P=0.64). Warfarin patients received more antihypertensive drugsand were seen more often than controls. Multiple regression analyses adjusting for relevant variables did not disclose an association between warfarin useand higher BP; on the contrary, exposure to warfarin was associated with lower SBP and PP on the multivariable models. Conclusion: Use of warfarin in conventional doses for an average of 5.5 years was not associated with increased BP in this cross-sectional study of patients with DM and hypertension.

Introdução: Em animais, a warfarina provoca calcificação arterial, rigidez arterial e hipertensão arterial (HA) sistólica. Dados preliminares em humanos sugerem que o mesmo efeito pode acontecer em pacientes com diabetes mellitus (DM) e/ou HA. Objetivo: Determinar se o uso da warfarina em pacientescom DM e HA resulta em elevação da pressão arterial ou pressão de pulso. Métodos: Estudo transversal de 159 pacientes com DM e HA que haviam sidotratados com warfarina por pelo menos 2 anos, e 159 controles pareados por idade, com DM e HA, mas que nunca haviam usado warfarina. O enfoqueprincipal na análise foi a diferença na pressão arterial sistólica (PAS), diastólica (PAD) e pressão de pulso (PP) entre os dois grupos. Resultados: A média de idade foi 73±10 anos em ambos os grupos. Os pacientes no grupo da warfarina haviam usado a droga por 5.5±3.1 anos. Pacientes no grupo da warfarina tinham uma prevalência maior de doença coronariana e insuficiência cardíaca. A PAS e PP foram mais baixas no grupo warfarina (PAS 130±14 mmHgvs. 134±12 mmHg, P=0.003; PP 58±12 mmHg vs. 62±11 mmHg, P=0.004), mas a PAD não diferiu entre os grupos (72±8 vs. 72±7 mmHg, P=0.64).Pacientes do grupo warfarina usaram mais drogas antihipertensivas e foram avaliados clinicamente com maior freqüência do que os controles. Regressão múltipla ajustada para fatores de relevância clínica não demonstrou nenhuma associação entre o uso da warfarina e elevação da pressão arterial. Pelo contrário, nos modelos de regressão múltipla, a exposição à warfarina associou-se a valores mais baixos de PAS e PP. Conclusão: O uso da warfarina em doses convencionais, por 5.5 anos, não associou-se a um aumento da pressão arterial neste estudo tranversal de pacientes com DM e hipertensão.