Morphologic and molecular study on the lens anterior capsule in systemic sclerosis / Relato sobre estudo morfológico e molecular da cápsula anterior do cristalino na esclerose sistêmica

ABSTRACT This study aimed to analyze the anterior lens capsule specimens from both eyes of a patient with systemic sclerosis and compare them to the eyes of a control patient. No significant differences between systemic sclerosis and control eyes were observed in the results from the hematoxylin-eosin and picrosirius staining. In the samples obtained from both systemic sclerosis and control eyes, there were expressions of caspase, a molecule expressed in cell death by apoptosis. Heparanase was overexpressed in the systemic sclerosis sample compared to the control sample. Therefore, the anterior lens capsule of the patient with systemic sclerosis is probably affected by the disease since it showed marked expression of heparanase 1.(AU)

RESUMO Analisamos as amostras das cápsulas anteriores do cristalino de uma paciente com esclerose sistêmica e comparamos com as de um paciente controle. Não foram observadas diferenças significativas entre esclerose sistêmica e controle nos resultados da coloração com hematoxilina-eosina e picrosirius. Nas amostras obtidas da esclerose sistêmica e do controle, obtivemos expressão de caspase, uma molécula expressa na morte celular por apoptose. A heparinase foi expressa de forma mais marcante na amostra de esclerose sistêmica quando comparada ao controle. Portanto, a cápsula anterior do cristalino da paciente com esclerose sistêmica provavelmente foi afetada pela doença, uma vez que mostrou expressão aumentada de heparinase 1.(AU)

Crude Heparin Preparations Unveil the Presence of Structurally Diverse Oversulfated Contaminants.

Nowadays, pharmaceutical heparin is purified from porcine and bovine intestinal mucosa. In the past decade there has been an ongoing concern about the safety of heparin, since in 2008, adverse effects associated with the presence of an oversulfated chondroitin sulfate (OSCS) were observed in preparations of pharmaceutical porcine heparin, which led to the death of patients, causing a global public health crisis. However, it has not been clarified whether OSCS has been added to the purified heparin preparation, or whether it has already been introduced during the production of the raw heparin. Using a combination of different analytical methods, we investigate both crude and final heparin products and we are able to demonstrate that the sulfated contaminants are intentionally introduced in the initial steps of heparin preparation. Furthermore, the results show that the oversulfated compounds are not structurally homogeneous. In addition, we show that these contaminants are able to bind to cells in using well known heparin binding sites. Together, the data highlights the importance of heparin quality control even at the initial stages of its production.

Is the Expression of the Components of the Carotid Matrix of Rats Influenced by Estrogen, Progestin and Tibolone?

OBJECTIVE: To analyze the effects of estrogen alone or in combination with progestogens and tibolone (TIB) on the expression of the extracellular matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9), of perlecan, and of heparanase (HPSE) of the vascular walls of the carotid arteries. METHODS: A total of 30 250-day-old ovariectomized Wistar rats were orally treated for 5 weeks with: a) 1 mg/kg of estradiol benzoate (EB); b) EB + 0.2 mg/kg of medroxyprogesterone acetate (MPA); c) EB + 0.2mg/kg of norethisterone acetate (NETA); d) EB + 2 mg/kg of dydrogesterone (DI); e) 1 mg/kg of TIB; f) placebo (CTR). Following treatment, the expression of mRNA for MMP-2, MMP-9, and HPSE was analyzed by real-time polymerase chain-reaction (PCR), and the expression of MMP-2, of MMP-9, of tissue inhibitor of metalloproteinase 2 (TIMP-2), and of perlecan was quantified by immunohistochemistry in the carotid arteries. RESULTS: The groups showed significant differences on mRNA HPSE expression (p = 0.048), which was higher in the EB, EB + MPA, and TIB groups. There was no statistically significant difference in mRNA MMP-2 or MMP-9 expression. The immunohistochemical expression of MMP-2, of TIMP-2, of MMP-9, of HPSE, and of perlecan showed no differences between groups. CONCLUSION: Estradiol alone or associated with MPA and TIB treatment can increase mRNA HSPE expression of the walls of the carotid arteries in ovariectomized rats.

OBJETIVO: Analisar os efeitos do estrogênio isolado ou em combinação com progestogênios e tibolona (TIB) na expressão das metaloproteinases 2 e 9 da matriz extracelular (MMP-2 e MMP-9), da perlecan e da heparanase (HPSE) das paredes vasculares das artérias carótidas. MéTODOS: Trinta ratas Wistar ovariectomizadas com 250 dias de idade foram tratadas oralmente por 5 semanas com: a) 1 mg/kg de benzoato de estradiol (EB); b) EB + 0,2 mg/kg de acetato de medroxiprogesterona (MPA); c) EB + 0,2mg/kg de acetato de noretisterona (NETA); d) EB + 2 mg/kg de didrogesterona (DI); e) 1 mg/kg de TIB; f) placebo (CTR). Após o tratamento, a expressão de mRNA para MMP-2, MMP-9, e HPSE foi analisada por reação em cadeia da polimerase (RCP) em tempo real, e a expressão de MMP-2, MMP-9, inibidor tecidual de metaloproteinase 2 (TIMP-2), e de perlecan foi quantificado por imunohistoquímica em artérias carótidas. RESULTADOS: Os grupos apresentaram diferenças significativas na expressão do mRNA HPSE (p = 0,048), sendo maiores nos grupos EB, EB + MPA e TIB. Não houve diferença estatisticamente significativa nas expressões de mRNA MMP-2 ou MMP-9. A expressão imunohistoquímica de MMP-2, TIMP-2, MMP-9, HPSE e perlecan não mostrou diferenças entre os grupos. CONCLUSãO: O estradiol isolado ou associado ao tratamento com MPA e TIB pode aumentar a expressão de mRNA HSPE nas paredes das artérias carótidas em ratas ovariectomizadas.

Is the expression of the components of the carotid matrix of rats influenced by estrogen, progestin and tibolone? / A expressão dos componentes da matriz carotídea de ratos é influenciada pelo estrogênio, progestagênio e tibolona?

Abstract Objective To analyze the effects of estrogen alone or in combination with progestogens and tibolone (TIB) on the expression of the extracellular matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9), of perlecan, and of heparanase (HPSE) of the vascular walls of the carotid arteries. Methods A total of 30 250-day-old ovariectomized Wistar rats were orally treated for 5 weeks with: a) 1 mg/kg of estradiol benzoate (EB); b) EB + 0.2 mg/kg of medroxyprogesterone acetate (MPA); c) EB + 0.2mg/kg of norethisterone acetate (NETA); d) EB + 2 mg/kg of dydrogesterone (DI); e) 1 mg/kg of TIB; f) placebo (CTR). Following treatment, the expression of mRNA for MMP-2, MMP-9, and HPSE was analyzed by realtime polymerase chain-reaction (PCR), and the expression of MMP-2, of MMP-9, of tissue inhibitor of metalloproteinase 2 (TIMP-2), and of perlecan was quantified by immunohistochemistry in the carotid arteries. Results The groups showed significant differences on mRNA HPSE expression (p = 0.048), which was higher in the EB, EB + MPA, and TIB groups. There was no statistically significant difference in mRNA MMP-2 or MMP-9 expression. The immunohistochemical expression of MMP-2, of TIMP-2, of MMP-9, of HPSE, and of perlecan showed no differences between groups. Conclusion Estradiol alone or associated with MPA and TIB treatment can increase mRNA HSPE expression of the walls of the carotid arteries in ovariectomized rats.

Resumo Objetivo Analisar os efeitos do estrogênio isolado ou em combinação com progestogênios e tibolona (TIB) na expressão das metaloproteinases 2 e 9 da matriz extracelular (MMP-2 e MMP-9), da perlecan e da heparanase (HPSE) das paredes vasculares das artérias carótidas. Métodos Trinta ratas Wistar ovariectomizadas com 250 dias de idade foram tratadas oralmente por 5 semanas com: a) 1 mg/kg de benzoato de estradiol (EB); b) EB + 0,2 mg/kg de acetato de medroxiprogesterona (MPA); c) EB + 0,2mg/kg de acetato de noretisterona (NETA); d) EB + 2 mg/kg de didrogesterona (DI); e) 1 mg/kg de TIB; f) placebo (CTR). Após o tratamento, a expressão de mRNA para MMP-2, MMP- 9, e HPSE foi analisada por reação em cadeia da polimerase (RCP) em tempo real, e a expressão de MMP-2, MMP-9, inibidor tecidual de metaloproteinase 2 (TIMP-2), e de perlecan foi quantificado por imunohistoquímica em artérias carótidas. Resultados Os grupos apresentaram diferenças significativas na expressão do mRNA HPSE (p = 0,048), sendo maiores nos grupos EB, EB + MPA e TIB. Não houve diferença estatisticamente significativa nas expressões de mRNA MMP-2 ou MMP-9. A expressão imunohistoquímica de MMP-2, TIMP-2, MMP-9, HPSE e perlecan não mostrou diferenças entre os grupos. Conclusão O estradiol isolado ou associado ao tratamento com MPA e TIB pode aumentar a expressão de mRNA HSPE nas paredes das artérias carótidas em ratas ovariectomizadas.

Tromboprofilaxis en el paciente médico hospitalizado por medicina interna Un estudio de corte transversal / Thromboprophylaxis in the hospitalized medical patient by internal medicine A cross-sectional study

Introducción: el tromboembolismo venoso es una de las principales causas de morbimortalidad prevenible seguida de una hospitalización. Las heparinas han demostrado ser eficaces para su prevención, sin embargo se ha documentado la subutilización de estos fármacos, por lo que implementar medidas que garanticen la formulación adecuada es fundamental. En nuestra institución se han instaurado estrategias para mejorar la formulación de estos fármacos con resultados iniciales favorables, pero se desconoce el efecto a más largo plazo. Métodos: estudio descriptivo, retrospectivo de corte transversal. Se evaluaron pacientes mayores de 18 años, hospitalizados por medicina interna entre junio y noviembre de 2012. Se estimó una muestra representativa de 102 pacientes. Se identificó la formulación de la tromboprofilaxis al segundo día de hospitalización, se determinó si fue adecuada según las guías institucionales y los errores en la prescripción de la misma. Se compararon los resultados con dos mediciones previas realizadas en la institución. Resultados: de los 102 pacientes evaluados, la tromboprofilaxis fue adecuada en 63 (61,8%) e inadecuada en 39 (38.2%). Las causas más frecuentes de error fueron: formulación en pacientes de bajo riesgo 18 (46.1%) y error por omisión en 12 (30.7%) pacientes. La formulación en pacientes con indicación y sin contraindicación disminuyó de 92-82% y en pacientes sin indicación aumentó de 50-56.2%, con relación a una medida previa realizada después de la difusión de guías institucionales. Conclusiones: la tromboprofilaxis en pacientes hospitalizados por medicina interna en nuestra institución se ordena en un alto porcentaje, sin embargo debe ser mejorada. El principal error es la formulación en pacientes con riesgo bajo. La implementación de estrategias para mejorar la tromboprofilaxis logró una mejoría inicial, pero tiende a disminuir con el tiempo. Se requiere un trabajo continuado de múltiples medidas que garanticen su impacto favorable a largo plazo. (Acta Med Colomb 2015; 40: 227-233).

Introduction: venous thromboembolism is one of the major causes of preventable morbidity and mortality following a hospitalization. Heparins have proven effective for prevention; however, under utilization of these drugs has been documented, so that the implementation of measures to ensure the proper formulation is essential. Strategies to improve the formulation of these drugs have been established in our institution with favorable initial results, but the longer-term effect is unknown. Methods: descriptive, retrospective cross-sectional study. Patients over 18 years hospitalized for internal medicine between June and November 2012 were evaluated. A representative sample of 102 patients was estimated. Thromboprophylaxis formulation was identified on the second day of hospitalization and it was determined if its prescription was appropriate in accordance with the institutional guidelines as well as errors in its prescription. The results were compared with two previous measurements realized in the institution. Results: Of the 102 patients evaluated, thromboprophylaxis was adequate in 63 (61.8%) and inadequate in 39 (38.2%). The most frequent causes of error were: prescription in low-risk patients 18 (46.1%) and error of omission in 12 (30.7%) patients. The formulation in patients with an indication and without contraindication decreased from 92% to 82% and in patients with no indication increased from 50% to 56.2%, compared to a previous measurement made after the dissemination of institutional guidelines. Conclusions: thromboprophylaxis in hospitalized patients in internal medicine at our institution is organized in a high percentage, but must be improved. The main error is the formulation in patients with low risk. The implementation of strategies to improve thromboprophylaxis achieved initial improvement, but tends to decrease over time. Continued work of multiple measures to ensure their favorable long-term impact is required. (Acta Med Colomb 2015; 40: 227-233).

Rivaroxaban frente a heparinas de bajo peso molecular para la prevención de la tromboembolia venosa posterior al reemplazo total de cadera o rodilla: revisión sistemática de la literatura y metaanálisisis / Rivaroxaban versus low weight heparins for venous thromboembolism prophylaxis after major hip and knee arthroplasty: systematic review and metaanalysis

Objetivo: evaluar la eficacia y seguridad de la tromboprofilaxis con rivaroxaban comparada con las heparinas de bajo peso molecular en cirugía mayor de cadera o rodilla. Métodos: revisión sistemática en Central Cochrane Database, MEDLINE, EMBASE, LILACS, CINAHL y artículos referenciados. Solo se consideraron ensayos clínicos controlados. La búsqueda se realizó hasta octubre 31 de 2011.Dos revisores evaluaron de forma independiente la calidad metodológica de los artículos y extrajeron los datos. Resultados: se estudiaron 15.638 pacientes (nueve estudios) programados para reemplazo total de cadera o rodilla. Comparada con la enoxaparina, la tromboprofilaxis con rivaroxaban se asoció a menor incidencia de trombosis venosa profunda, tromboembolia pulmonar y muerte por cualquier causa posterior a cirugía ortopédica mayor tanto de cadera (5.475, riesgo relativo [RR]: 0,37; intervalo de confianza del 95% [IC 95%]: 0,29-0,48]) como de rodilla (2.878, RR: 0,65; IC 95%: 0,50-0,84) sin diferencias en los eventos hemorrágicos (sangrado mayor en cirugía de cadera: 7.684, RR: 1,79; IC 95%: 0,78-4,11; y de rodilla: 5.700, RR: 1,59; IC 95%: 0,77-3,27). Conclusión: el rivaroxaban usado para tromboprofilaxis de pacientes sometidos a cirugía ortopédica mayor es más eficaz que la enoxaparina y al menos tan seguro como ella.

Objective: To assess the efficacy and safety of rivaroxaban thromboprophylaxis versus low weight heparins in major hip and knee arthroplasty. Methods: Systematic review in Central Cochrane Database, MEDLINE, EMBASE, LILACS, CINAHL, and referenced articles. Only randomized clinical trials were considered. The search was made until October 31, 2011. Two independent reviewers assessed the quality of articles and extracted the information. Results: 15.638 patients who underwent major orthopedic surgery were studied (nine trials). Compared with enoxaparin, thromboprophylaxis with rivaroxaban was associated with lower incidence of deep vein thrombosis, pulmonary embolism and death from any cause after major hip surgery (5.351, relative risk [RR]: 0.37; confidence interval 95% [IC 95%]: 0.29-0.48) and knee surgery (2.878, RR: 0.65; IC 95%: 0.50-0.84) without differences in bleeding events (major bleeding events in hip surgery: 7.684, RR: 1.79; IC 95%: 0.78-4.11) and knee surgery: 5.700, RR: 1.59; IC 95%: 0.77-3.27). Conclusion: Rivaroxaban thromboprophylaxis in major hip and knee arthroplasty is more effective than enoxaparin and as safe as the latter.

Profilaxis de la enfermedad tromboembólica venosa / Prophylaxis of deep venous thromboembolic disease

Introducción: la incidencia de la enfermedad tromboembólica venosa en la población en general, es alta y aumenta hasta en 100 veces en pacientes hospitalizados, de ahí que se considere la primera causa de muerte prevenible intrahospitalaria. Tiene un alto costo económico y secuelas, la mayoría de estas muertes son súbitas o en las primeras 2 h antes de que el tratamiento pueda ser instaurado de modo efectivo, por lo cual existen suficientes motivos para justificar la implementación de la tromboprofilaxis. Objetivo: brindar una revisión actualizada de las recomendaciones actuales para la tromboprofilaxis. Fuente de datos: se realizó una búsqueda de información en Medline (2000-2012) y la base de datos de ensayos clínicos de la Biblioteca Cochrane. Las palabras clave para la búsqueda se relacionaron con el tromboembolismo pulmonar, trombosis venosa y tromboflebitis, la prevención de esas enfermedades y ensayos clínicos controlados y aleatorizados.Resultados: actualmente existen numerosos métodos mecánicos y farmacológicos para prevenir esta enfermedad y los más recomendado son las heparinas de bajo peso molecular y en algunas situaciones específicas los nuevos anticoagulantes orales, entre ellos, el Rivaroxaban, Dabigatran y Apixaban. Conclusiones: la aplicación de estrategias de profilaxis basadas en la mejor evidencia científica disponible beneficia a los pacientes que ingresan en servicios hospitalarios, por lo que en estos centros se debe desarrollar una estrategia que aborde la prevención de las complicaciones tromboembólicas con una política de tromboprofilaxis, sobre todo para los grupos de alto riesgo(AU)

Introduction: the incidence of venous thromboembolic disease is high in the general population and increases up to 100 times in hospitalized patients; hence, it is considered the first cause of preventable death at hospital. It bears high economic cost and sequelae, most of deaths are sudden or occur in the first two hours before the treatment could be effectively applied. There are enough reasons for the implementation of thromboprophylaxis. Objective: to provide an updated review of the current recommendations on thromboprophylaxis. Source of data: information was collected from Medline(200-2012) and the Cochrane Library clinical trial database. The key words were pulmonary thromboembolism, venous thrombosis and thromboflebitis, prevention of these diseases and controlled and randomized clinical trials. Results: there is a number of mechanical and pharmacological methods to prevent the disease and the most recommended are low molecular weight heparins and, in some specific situations, the new oral anticoagulants such a Rivaroxaban, Dabigatan and Apixaban. Conclusions: the implementation of prophylactic strategies based on the best scientific evidence available will benefit the patients who are admitted to the hospitals; therefore, these centers must develop a line of strategy to address the prevention of thromboembolic complications based on a thromboprophylactic policy aimed at high risk groups(AU)

Heparinas de baixo peso molecular para profilaxia e tratamento de trombose venosa profunda na gravidez / Low molecular weight heparins for prophylaxis and treatment of deep venous thrombosis in pregnancy

A trombose venosa profunda (TVP) caracteriza-se por formação aguda de trombos no sistema venoso profundo. A grávida apresenta um risco seis vezes maior de ocorrência de TVP. Heparinas não fracionada (HNF) e de baixo peso molecular (HBPM) têm sido prescritas para tromboprofilaxia durante a gravidez.

Avaliação comparativa da atividade biológica de heparinas não fracionadas pelas metodologias de inibição da coagulação do plasma ovino (ICPO) e de tempo de tromboplastina parcial ativada (TTPA) / Comparative study on the biological activity of non-fractionated heparin by using sheep plasma coagulation inhibition assay (SPCIA) and activated partial thromboplastin time (APTT) methodologies

O objetivo deste trabalho foi realizar o estudo comparativo entre os métodos ICPO e TTPA, para avaliar a eficácia da implantação do TTPA como método para a avaliação da segurança e eficácia de heparinas não fracionadas em produtos farmacêuticos. Foram avaliados, comparativamente, cinco lotes de diferentes fabricantes de heparinas não fracionadas (polissacarídeo sulfatado usado como droga anticoagulante), de origem suína ou bovina, testadas com base no 5º Padrão Internacional de Heparina. Esses produtos foram provenientes de coletas efetuadas pelas autoridades sanitárias para análise no Instituto Nacional de Controle de Qualidade em Saúde (INCQS). As amostras foram analisadas quanto à pureza e potência anticoagulante, por meio de duas metodologias: inibição da coagulação do plasma ovino (ICPO) e tempo de trombo plastina parcial ativada (TTPA). Houve boa concordância entre as duas metodologias, sendo que a técnica TTPA apresentou ser mais simples, rápida e objetiva, quando da utilização do coagulômetro para a medição do tempo de formação de coágulos, em detrimento da leitura subjetiva dos graus de coagulação no ensaio de ICPO. A implantação e a execução do TTPA em paralelo à utilização do ICPO garantirão o aumento de sensibilidade técnica na avaliação da segurança e eficácia de heparinas não fracionadas.

Glycosaminoglycan backbone is not required for the modulation of hemostasis: effect of different heparin derivatives and non-glycosaminoglycan analogs.

Heparin and its derivatives are known to regulate a variety of pathophysiological events related to vascular biology. In the present manuscript we examine a variety of heparinomimetics biochemically (electrophoretic behavior and enzymatic degradation) and pharmacologically (in vitro anticoagulant activity and in vivo hemorrhagic and antithrombotic tests) as well as their interactions with cells from the vessel wall using a time resolved fluorometric method and confocal microscopy. Data were determined for unfractionated heparin (UFH), enoxaparin, synthetic heparin pentasaccharide, C3 heparin derived oligosaccharides and phosphosulfomannan (PI-88). While being structurally distinct from UFH, all compounds exhibited anticoagulant, antithrombotic and hemorrhagic activities. In addition, besides the pentasaccharide, they all stimulated the synthesis of an antithrombotic heparan sulfate present at the cell surface and secreted by endothelial cells. Also, like UFH, they interacted with both endothelial and smooth muscle cells and dislodged UFH from its binding sites in a dose dependent manner but, with distinct saturable curves showing that the binding of polymeric structures to extracellular matrix (ECM) proteins does not depend on a glycosaminoglycan backbone. The data also suggest a common pathway, which does not depend on the presence of the conventionally accepted antithrombin binding pentasaccharide, for ECM dependent activity of the heparinomimetic stimulated synthesis of antithrombotic heparan sulfate. Notably, although of similar molecular weight as well as polymeric backbone, the synthetic heparin pentasaccharide showed significant hemorrhagic action and negligible antithrombotic activity in a venous thrombosis model, contrasting with C3, that displayed negligible hemorrhagic effect and potent antithrombotic action. These results provide evidence that structurally unrelated polymers can elicit similar hemostatic activities and show that polymeric sequence is not always crucial for certain activities. The results also suggest that non-GAG structures may provide an alternative route for the pharmaceutical control of hemostasis.

Heparin from bovine intestinal mucosa: glycans with multiple sulfation patterns and anticoagulant effects.

Pharmaceutical grade heparins from porcine intestine and bovine lung consist mainly of repeating tri-sulfated units, of the disaccharide →4-α-IdoA2S-1→4-α-GlcNS6S-1→. Heparin preparations from bovine intestine, in contrast, are more heterogeneous. Nuclear magnetic resonance (NMR) and disaccharide analysis after heparinase digestions show that heparin from bovine intestine contains α-glucosamine with significant substitutive variations: 64% are 6-O-sulfated and N -sulfated, as in porcine intestinal heparin while 36% are 6-desulfated. Desulfated α-iduronic acid units are contained in slightly lower proportions in bovine than in porcine heparin. NMR data also indicate N-, 3- and 6-trisulfated α-glucosamine (lower proportions) and α-GlcNS-1→4-α-GlcA and α-IdoA2S-1→4-α-GlcNAc (higher amounts) in bovine than in porcine heparin. Porcine and bovine heparins can be fractionated by anion exchange chromatography into three fractions containing different substitutions on the α-glucosamine units. Each individual fraction shows close disaccharide composition and anticoagulant activity, regardless of their origin (bovine or porcine intestine). However, these two heparins differ markedly in the proportions of the three fractions. Interestingly, fractions with the typical heparin disaccharides of porcine intestine are present in bovine intestinal heparin. These fractions contain high in vitro anticoagulant activity, reduced antithrombotic effect and high bleeding tendency. These observations indicate that the prediction of haemostatic effects of heparin preparations cannot rely exclusively on structural analysis and anticoagulant assays in vitro . Minor structural components may account for variations on in vivo effects. In conclusion, we suggest that pharmaceutical grade bovine intestinal heparin, even after purification procedures, is not an equivalent drug to porcine intestinal heparin.

Análisis y caracterización de la farmacoterapéutica de las heparinas de bajo peso molecular prescritas en pacientes hospitalizados en el Hospital Clínica Bíblica, Costa Rica, durante el periodo de marzo a agosto del 2010 / Analysis and characterization of the pharmacotherapy of low molecular weight heparins prescribed in hospitalized patients at the Hospital Clinica Biblica, Costa Rica, from March to August 2010

Justificación y objetivo: Analizar la farmacoterapéutica empleada con las heparinas de bajo peso molecular, HBPM, prescritas en pacientes hospitalizados en el Hospital Clínica Bíblica, hospital privado en Costa Rica, con base en los lineamientos establecidos por el Colegio Americano de Cirujanos Torácicos, 2008. Material y métodos: En el presente estudio se incluyeron pacientes internados en el período marzo-agosto 2010 que fueron tratados con HBPM. De esta población se analizaron 250 pacientes elegidos de forma aleatoria según la metodología recomendada. Se recopilaron todos los documentos e información necesaria de cada paciente para el correspondiente análisis. Resultados: Un 43 por ciento del total de pacientes hospitalizados utilizaron HBPM, 707 pacientes. En un 91 por ciento de los casos, las HBPM fueron utilizadas con un fin profiláctico. Solamente un 2 por ciento de los pacientes que utilizaron HBPM de manera profiláctica no necesitaban de la misma. En un 90 por ciento de los casos, la dosis utilizada fue correcta. En un 18 por ciento de los casos se requería un ajuste de dosis. Un 80 por ciento de los casos presentó algún tipo de interacción farmacológica de relevancia clínica. Un 4 por ciento de los pacientes presentaron algún tipo de hemorragia, en donde un 2 por ciento de los casos este efecto estaba ligado al uso de HBPM. El 9 por ciento de los casos en los que se utilizaron las HBPM como tratamiento, fueron abordados según los lineamientos. Conclusión: En el Hospital Clínica Bíblica, el uso las HBPM se apegó a las recomendaciones establecidas por la normativa de la ACCP, a pesar de la no existencia en ese momento de un protocolo en el hospital. El análisis farmacoterapéutico por parte del farmacéutico clínico, puede suministrar información importante al médico para que se tomen las medidas correctivas y/o preventivas asociadas a la correcta utilización de estos medicamentos. Realizar una correcta estratificación de riesgo e individualización del tratamiento facilita la implementación adecuada de la farmacoterapia con HBPM en este hospital, donde existe una alta proporción de pacientes que pueden desarrollar eventos tromboembólicos.

Binding activity, structure, and immunogenicity of synthetic peptides derived from Plasmodium falciparum CelTOS and TRSP proteins.

Several sporozoite proteins have been associated with Plasmodium falciparum cell traversal and hepatocyte invasion, including the cell-traversal protein for ookinetes and sporozoites (CelTOS), and thrombospondin-related sporozoite protein (TRSP). CelTOS and TRSP amino acid sequences have been finely mapped to identify regions specifically binding to HeLa and HepG2 cells, respectively. Three high-activity binding peptides (HABPs) were found in CelTOS and one HABP was found in TRSP, all of them having high α-helical structure content. These HABPs' specific binding was sensitive to HeLa and HepG2 cells' pre-treatment with heparinase I and chondroitinase ABC. Despite their similarity at three-dimensional (3D) structural level, TRSP and TRAP HABPs located in the TSR domain did not compete for the same binding sites. CelTOS and TRSP HABPs were used as a template for designing modified sequences to then be assessed in the Aotus monkey experimental model. Antibodies directed against these modified HABPs were able to recognize both the native parasite protein by immunofluorescence assay and the recombinant protein (expressed in Escherichia coli) by Western blot and ELISA assays. The results suggested that these modified HABPs could be promising targets in designing a fully effective, antimalarial vaccine.

Unique extracellular matrix heparan sulfate from the bivalve Nodipecten nodosus (Linnaeus, 1758) safely inhibits arterial thrombosis after photochemically induced endothelial lesion.

Heparin-like glycans with diverse disaccharide composition and high anticoagulant activity have been described in several families of marine mollusks. The present work focused on the structural characterization of a new heparan sulfate (HS)-like polymer isolated from the mollusk Nodipecten nodosus (Linnaeus, 1758) and on its anticoagulant and antithrombotic properties. Total glycans were extracted from the mollusk and fractionated by ethanol precipitation. The main component (>90%) was identified as HS-like glycosaminoglycan, representing approximately 4.6 mg g(-1) of dry tissue. The mollusk HS resists degradation with heparinase I but is cleaved by nitrous acid. Analysis of the mollusk glycan by one-dimensional (1)H, two-dimensional correlated spectroscopy, and heteronuclear single quantum coherence nuclear magnetic resonance revealed characteristic signals of glucuronic acid and glucosamine residues. Signals corresponding to anomeric protons of nonsulfated, 3- or 2-sulfated glucuronic acid as well as N-sulfated and/or 6-sulfated glucosamine were also observed. The mollusk HS has an anticoagulant activity of 36 IU mg(-1), 5-fold lower than porcine heparin (180 IU mg(-1)), as measured by the activated partial thromboplastin time assay. It also inhibits factor Xa (IC(50) = 0.835 microg ml(-1)) and thrombin (IC(50) = 9.3 microg ml(-1)) in the presence of antithrombin. In vivo assays demonstrated that at the dose of 1 mg kg(-1), the mollusk HS inhibited thrombus growth in photochemically injured arteries. No bleeding effect, factor XIIa-mediated kallikrein activity, or toxic effect on fibroblast cells was induced by the invertebrate HS at the antithrombotic dose.

The presence of heparan sulfate in the mammalian oocyte provides a clue to human sperm nuclear decondensation in vivo.

BACKGROUND: Previous results from our laboratory have led us to propose heparan sulfate (HS) as a putative protamine acceptor during human sperm decondensation in vivo. The aim of this paper was to investigate the presence of glycosaminoglycans in the mammalian oocyte in an effort to better support this contention. METHODS: Two experimental approaches are used: oocyte labeling to identify the presence of HS and analysis of sperm decondensing ability of fresh oocytes in the presence or absence of specific glycosidases. RESULTS: Staining of mouse zona-intact oocytes with the fluorescent cationic dye, Rubipy, at pH 1.5 allowed for the detection of sulfate residues in the ooplasm by confocal microscopy. HS was detected in the ooplasm by immunocytochemistry. A sperm decondensation microassay using heparin and glutathione was successfully developed. The same level of sperm decondensation could be attained when heparin was replaced by mouse zona-free oocytes. Addition of heparinase to the oocyte/glutathione mixture significantly reduced sperm decondensation (P = 0.0159), while there was no effect following addition of either chondroitinase ABC or hyaluronidase. CONCLUSIONS: The results presented in this paper demonstrate for the first time that HS is present in the mammalian oocyte and show that HS is necessary for fresh oocytes to express their sperm decondensing ability in vitro.

Properties of carbodiimide treated heparin.

Heparin, a well-known anticoagulant, has been frequently used to coat surfaces for attaining blood compatibility of polymeric materials. Since carbodiimides are often used for immobilization of heparin on these biomaterials, the present study intended to evaluate some properties of carbodiimide-treated heparin. It was observed that the properties of heparin were altered after treatment with carbodiimide, mainly in the molar excess of this reagent. Thus, dye fixation and electrophoretic behaviour of heparin were modified, as well as its degradability by specific enzymes. Also, these modifications resulted in loss of anticoagulant activity. Infrared spectra of this carbodiimide-treated heparin presented evidence that can confirm its modification.

Human neutrophil migration in vitro induced by secretory phospholipases A2: a role for cell surface glycosaminoglycans.

The purpose of this study was to examine the ability of type I- (porcine pancreas and Naja mocambique mocambique venom), type II- (bothropstoxin-I, bothropstoxin-II, and piratoxin-I), and type III- (Apis mellifera venom) secretory phospholipases A2 (sPLA2s) to induce human neutrophil chemotaxis, and the role of the cell surface proteoglycans, leukotriene B4 (LTB4), and platelet-activating factor (PAF), in mediating this migration. The neutrophil chemotaxis assays were performed by using a 48-well microchemotaxis chamber. Piratoxin-I, bothropstoxin-I, N. m. mocambique venom PLA2 (10-1000 microg/mL each), bothropstoxin-II (30-1000 microg/mL), porcine pancreas PLA2 (0.3-30 microg/mL), and A. mellifera venom PLA2 (30-300 microg/mL) caused concentration-dependent neutrophil chemotaxis. Heparin (10-300 U/mL) concentration-dependently inhibited the neutrophil migration induced by piratoxin-I, bothropstoxin-II, and N. m. mocambique and A. mellifera venom PLA2s (100 microg/mL each), but failed to affect the migration induced by porcine pancreas PLA2. Heparan sulfate (300 and 1000 microg/mL) inhibited neutrophil migration induced by piratoxin-I, whereas dermatan sulfate and chondroitin sulfate (30-1000 microg/mL each) had no effect. Heparitinase I and heparinase (300 mU/mL each) inhibited by 41.5 and 47%, respectively, piratoxin-I-induced chemotaxis, whereas heparitinase II and chondroitinase AC failed to affect the chemotaxis. The PAF receptor antagonist WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6H-thienol-[3,2-f] -triazolo-[4,3-a] -diazepine-2-yl]-1-(4-morpholynil)-1-propionate) (0.1-10 microM) and the LTB4 synthesis inhibitor AA-861 [2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone] (0.1-10 microM) significantly inhibited the piratoxin-I-induced chemotaxis. Piratoxin-I (30-300 microg/mL) caused a concentration-dependent release of LTB4. Our results suggest that neutrophil migration in response to sPLA2s is independent of PLA activity, and involves an interaction of sPLA2s with cell surface heparin/heparan binding sites triggering the release of LTB4 and PAF.

Anticoagulant and antiprotease effects of a novel heparinlike compound from shrimp (Penaeus brasiliensis) and its neutralization by heparinase I.

Heparin is usually obtained from mammalian organs, such as beef lung, beef mucosa, porcine mucosa, and sheep intestinal mucosa. Because of the increased use of heparin in the production of low-molecular-weight heparin (LMWH), there is a growing shortage of the raw material needed to produce LMWHs. A previous report described the structural features of a novel LMWH from the shrimp (Penaeus brasiliensis). In order to compare anticoagulant and antiprotease effects of this heparin, global anticoagulant tests, such as the prothrombin time, activated partial thromboplastin time, thrombin time, and Heptest, were used. Amidolytic anti-Xa and anti-IIa activities were also measured. The relative susceptibility of this heparin to flavobacterial heparinase was also evaluated. The United States Pharmacopeia (USP) potency of shrimp heparin (SH) was found to be 28 U/mg. SH produced a concentration-dependent prolongation of all of the clotting tests and exhibited marked inhibition of FXa and FIIa. Heparinase treatment resulted in a marked decrease of the anticoagulant effects and neutralized the in vitro anti-IIa actions. However, the anti-Xa activities were only partially neutralized. Protamine sulfate was only partially effective in neutralizing the anticoagulant and antithrombin effects of SH. SH also produced marked prolongation of activated clotting time, which was neutralized by heparinase but not by protamine sulfate. These results suggest that SH is a strong anticoagulant with comparable properties to mammalian heparins and can be used in the development of clinically useful antithrombotic-anticoagulant drugs.

Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2'-bipyridine) ruthenium (II).

The paper shows the ability of the fluorochrome tris(2,2'-bipyridine) ruthenium (II) (Rubipy) to detect heparan sulfate, heparin, and heparinase activity of M3 murine mammary adenocarcinoma cells as well as bacterial heparinases I, II, and III in native polyacrylamide gel electrophoresis (PAGE). The technique is based on the electrophoretic mobility of high molecular weight heparins and subsequent staining with Rubipy (50 micrograms/mL). The minimum content of heparin detected by fluorescence in a UV transilluminator was 25-50 ng. The number of Rubipy molecules bound to heparin, determined in relationship to the number of disaccharide units (DU), showed that two to six heparin disaccharide units are bound by each fluorochrome molecule. Scatchard plot analysis showed one Rubipy-binding site (Kd = (8.56 +/- 2.97) x 10(-5) M). Heparinase activity was determined by densitometric analysis of the fluorescence intensity of the heparin-containing band of the gel. While heparinase I (EC 4.2.2.7.) degraded heparin and, to a lower degree, partially N-desulfated N-acetylated heparin (N-des N-Ac), heparinase II (no EC number) could efficiently degrade heparan sulfate (HS) and partially N-des N-Ac heparin. Finally, heparinase III (EC 4.2.2.8.) degraded HS almost exclusively. Only heparin and N-des N-Ac heparin were substrates for M3 tumor cell heparinases. We describe a qualitative, sensitive and simple method to detect heparinase activity and determine its substrate specificity using Rubipy fluorescence with heparin and heparan sulfate in multiple biological samples tested in parallel.