Progress in Application of Heparin Coating in Blood Contact Medical Devices / 中国医疗器械杂志

Blood compatibility is the main restriction of blood-contacting medical devices in clinical application, especially long-term blood-contacting medical devices will stimulate the immune defense mechanism of the host, resulting in thrombosis. Heparin anticoagulant coating links heparin molecules to the surface of medical device product materials, improves the compatibility between the material surface interface and the body, and reduces the host immune defense reactions. This study reviews the structure and biological properties of heparin, the market application status of heparin-coated medical products, the insufficiency and improvement of heparin coating, which can provide a reference for the application research of blood contact medical devices.

Heparan sulphate, its derivatives and analogues share structural characteristics that can be exploited, particularly in inhibiting microbial attachment

Heparan sulphate (HS) and the related polysaccharide, heparin, exhibit conformational and charge arrangement properties, which provide a degree of redundancy allowing several seemingly distinct sequences to exhibit the same activity. This can also be mimicked by other sulphated polysaccharides, both in overall effect and in the details of interactions and structural consequences of interactions with proteins. Together, these provide a source of active compounds suitable for further development as potential drugs. These polysaccharides also possess considerable size, which bestows upon them an additional useful property: the capability of disrupting processes comprising many individual interactions, such as those characterising the attachment of microbial pathogens to host cells. The range of involvement of HS in microbial attachment is reviewed and examples, which include viral, bacterial and parasitic infections and which, in many cases, are now being investigated as potential targets for intervention, are identified.

Comparison between heparin-conjugated fibrin and collagen sponge as bone morphogenetic protein-2 carriers for bone regeneration

Bone morphogenetic protein-2 (BMP-2) is used to promote bone regeneration. However, the bone regeneration ability of BMP-2 relies heavily on the delivery vehicle. Previously, we have developed heparin-conjugated fibrin (HCF), a vehicle for long-term delivery of BMP-2 and demonstrated that long-term delivery of BMP-2 enhanced its osteogenic efficacy as compared to short-term delivery at an equivalent dose. The aim of this study was to compare the bone-forming ability of the BMP-2 delivered by HCF to that delivered by clinically utilized BMP-2 delivery vehicle collagen sponge. An in vitro release profile of BMP-2 showed that HCF released 80% of the loaded BMP-2 within 20 days, whereas collagen sponge released the same amount within the first 6 days. Moreover, the BMP-2 released from the HCF showed significantly higher alkaline phosphatase activity than the BMP-2 released from collagen sponge at 2 weeks in vitro. Various doses of BMP-2 were delivered with HCF or collagen sponge to mouse calvarial defects. Eight weeks after the treatment, bone regeneration was evaluated by computed tomography, histology, and histomorphometric analysis. The dose of BMP-2 delivered by HCF to achieve 100% bone formation in the defects was less than half of the BMP-2 dose delivered by collagen sponge to achieve a similar level of bone formation. Additionally, bone regenerated by the HCF-BMP-2 had higher bone density than bone regenerated by the collagen sponge-BMP-2. These data demonstrate that HCF as a BMP-2 delivery vehicle exerts better osteogenic ability of BMP-2 than collagen sponge, a clinically utilized delivery vehicle.

Farmacovigilância da heparina no Brasil / Heparin pharmacovigilance in Brazil

OBJETIVO: Investigar a origem das preparações de heparina, na forma farmacêutica injetável, disponíveis no mercado brasileiro, discutindo o impacto do perfil dos produtos comercializados e das alterações na monografia da heparina na segurança do fármaco. MÉTODOS: Pesquisou-se o banco de dados de Produtos Registrados das Empresas de Medicamentos da Anvisa e o Dicionário de Especialidades Farmacêuticas (DEF 2008/2009). Foi realizado inquérito com as indústrias com autorização ativa para o comércio do fármaco no Brasil. RESULTADOS: Cinco indústrias possuem autorização para o comércio de heparina não fracionada no Brasil. Três são de origem suína e duas de origem bovina, sendo que apenas uma possui essa informação explicitada na bula. A efetividade e a segurança da heparina, estudadas em populações estrangeiras, podem não representar a nossa realidade, já que a maioria dos países não produz a heparina bovina. A heparina atualmente comercializada tem, ainda, aproximadamente 10 por cento menos atividade anticoagulante que a anteriormente produzida, e essa alteração pode ter implicações clínicas. CONCLUSÃO: Evidências acerca da ausência de intercambialidade de doses entre as heparinas de origem bovina e suína e o diferenciado perfil de segurança entre esses fármacos indicam necessidade de acompanhamento do tratamento e da resposta dos pacientes. Eventos que ameacem a segurança do paciente devem ser comunicados ao sistema da farmacovigilância do país.

OBJECTIVE: To investigate the biological origin of injectable unfractioned heparin available in Brazilian market by discussing the impact of the profile of commercial products and the changes in heparin monograph on the drug safety. METHODS: The Anvisa data base for the Registered Products of Pharmaceutical Companies and the Dictionary of Pharmaceutical Specialties (DEF 2008/2009) were searched. A survey with industries having an active permission for marketing the drug in Brazil was conducted. RESULTS: Five companies were granted a permission to market unfractioned heparin in Brazil. Three of them are porcine in origin and two of them are bovine in origin, with only one explicitly showing this information in the package insert. The effectiveness and safety of heparin studied in non-Brazilian populations may not represent the Brazilian reality, since most countries no longer produce bovine heparin. The currently marketed heparin has approximately 10 percent less anticoagulant activity than that previously produced and this change may have clinical implications. CONCLUSIONS: Evidence about the lack of dose interchangeability between bovine and porcine heparins and the unique safety profile of these drugs indicates the need to follow the treatment and the patients' response. Events threatening the patient's safety must be reported to the pharmacovigilance system in each particular country.

Controle da qualidade das preparações de heparina disponíveis no Brasil: implicações na cirurgia cardiovascular / Heparin quality control in the Brazilian market: implications in the cardiovascular surgery

INTRODUÇÃO: A mudança na marca da heparina rotineiramente utilizada nas cirurgias cardíacas no Brasil tem sido acompanhada por aumento do número de casos de discrasia sanguínea, aumento de reoperações e efeitos adversos em nossa Instituição e em outras. MÉTODOS: Foram avaliadas no Laboratório de Tecido Conjuntivo do HUCFF/UFRJ, quatro preparações disponíveis e comparadas à heparina retirada do mercado (Liquemine) e ao padrão de controle internacional. As preparações de heparina foram submetidas à ressonância nuclear magnética para avaliação da integridade estrutural, bem como avaliação de sua eficácia anticoagulante. RESULTADOS: Houve diferença significativa quanto à atividade anticoagulante entre as amostras. Também se observou a presença de contaminação com dermatam sulfato, amostras degradadas quimicamente e com significativa alteração do peso molecular. CONCLUSÃO: Das amostras estudadas, nenhuma atendeu aos requisitos de segurança para utilização em cirurgias cardíacas com circulação extracorpórea. Nenhuma delas apresentou a qualidade semelhante ao Liquemine, não mais disponível no mercado brasileiro.

INTRODUCTION: The change in the heparin solution trade mark in Brazil that had been commonly used in cardiac surgery has shown increased number in the coagulopathy, re-exploration and other side effects in our Institution and others. METHODS: All four different heparin solutions available in the Brazilian market were studied in the Connective Tissue Lab, HUCFF, UFRJ and compared to the Liquemine (out of the market) and the international control solution. All samples were evaluated by magnetic nuclear resonance as well as their anticoagulant effectiveness. RESULTS: There were significant differences among them regarding the anticoagulant activity. It was also observed contamination with other dermatan sulfate, samples chemically degraded and with significant change in the molecular weight. CONCLUSION: Among the studied samples, none of them can offer security in cardiac surgeries on pump. None of them has demonstrated similar quality to Liquemine, which is not available in the Brazilian market.

The specificity of interactions between proteins and sulfated polysaccharides

Os polissacarídeos sulfatados são capazes de se ligar às proteínas com diferentes níveis de especificidade. São macromoléculas altamente ácidas que podem se ligar de forma inespecífica a qualquer domínio básico da superfície de uma proteína em soluções com baixa força iônica, contudo tais interações não parecem ser fisiologicamente significativas. Por outro lado, foram identificados vários sistemas nos quais componentes estruturais muito específicos dos polissacarídeos sulfatados conferem alta afinidade para algumas proteínas. O exemplo mais conhecido é o pentassacarídeo da heparina com alta afinidade pela antitrombina. Outros exemplos podem ser observados no estudo de invertebrados marinhos, tais como a importância da estrutura fina do dermatam sulfato para sua interação com o cofator II da heparina e o envolvimento defucanas sulfatadas encontradas no gel que envolve osóvulos dos ouriços-do-mar na espécie especificidade da fertilização. Um terceiro exemplo de interação específica é aquele descrito para o glicosaminoglicano heparam sulfato encontrado na superfície celular. Neste caso, o padrão de sulfatação pode determinar diferentes afinidades do carboidrato por citoquinas, fatores de crescimento e outras proteínas encontradas na superfície celular e na matriz extracelular. Estas interações complexas entre proteínas e carboidratos são capazes de influenciar a difusão das proteínas através dos tecidos, assim como modelar a resposta celular a estas moléculas.

Extraction of heparin and heparin-like substance from marine mesogastropod mollusc Turritella attenuata (Lamarck, 1779).

Heparin was extracted from marine gastropod T. attenuata through the sequential precipitation with methanol and ethanol. The metachromatic dye method using toluidine blue was used to estimate colorimetrically the amount of heparin present in the sample. The anticoagulant activity of the sample was calculated as per United States of Pharmacopoeia standard procedure using sheep blood. After the purification, samples were analyzed, for the presence of heparin, with agarose-gel electrophoresis and HPLC and the mobility of the sample and the peak respectively were compared with standard heparin. The results of the present study shall help in finding out alternate source.

Heparina de alto peso molecular. Uma alternativa nas operaçöes com circulaçäo extracorpórea: estudo experimental / High molecular weight heparin. An alternative in extracorporeal circulation surgery: an experimental study

Introduçäo: As síndromes hemorrágicas no intra e pós-operatório de operaçöes com circulaçäo extracorpórea (CEC) constituem motivo de preocupaçäo e, parte delas, pode ser atribuída à heparina näo fracionada (HNF), droga indispensável e, até hoje, insubstituível nesse tipo de procedimento. Alguns autores consideram a açäo anticoagulante da HNF como o principal responsável pelo sangramento e investem em drogas antifibrinolíticas ou que alteram a atividade plaquetária para tentar substituí-la. Toda HNF contém fraçöes de heparina de baixo peso molecular (HBPM), näo neutralizáveis pela protamina, que, em doses elevadas, e/ou em pacientes heparino-sensíveis, podem causar vasoplegia e aumento no sangramento pós-operatório em operaçöes com CEC. Material e Métodos: Isolamos uma heparina de alto peso molecular (HAPM - peso modal de 25.000 Daltons), com 11 por cento de fraçöes de HBPM (< 7.000 Daltons), para experiências "in vitro" e "in vivo", e comparamos com HNF (peso modal de 15.000 Daltons), com 21 por cento de fraçöes de HBPM. Resultados: A atividade específica anticoagulante, por massa, foi superior quando comparada com a HNF tanto "in vitro", 273 ui/mg contra 181 ui/mg e TTPA mais elevado nas várias diluiçöes, como "in vivo", em cäes, durante CEC, comprovado pelo TCA, TTPA e heparinemia. A vida média da HNF foi de 60 minutos e acima de 90 minutos para a HAPM, na situaçäo de experimentaçäo. Conclusäo: Acreditamos que esta experiência, inédita na literatura indexada, nos habilite ao uso da HAPM, em seres humanos, para averiguaçäo da sua melhor neutralizaçäo pela protamina e menor incidência de hemorragia

Structure and function of fibroblast growth factor

We describe some structural requirements od the fibroblast growth factor (FGF) signaling system for the stimulation of the mitogenic response in terms of the design, synthesis and mitogenic activity of linear peptides related to the human FGF-1 sequence and the structural requirements of heparin for the potentiation of the mitogenic activity of FGF-1. The best mitogenic peptide we have synthesized so far is Ac-WFVGLKKNGSSKRGPRT-NH2, that has been shown: 1)to bind to heparin-Sepharose columns with moderate affinity, requiring about 0.5 M NaCl to be eluted from the resin; 2) to be mitogenic upon BALB/c 3T3 fibroblasts in culture (ED50=10-20 muM) and 3)to compete with human FGF-1 for cellular binding (ID50=30-50 muM). The potentiating activity of heparin upon FGF-1 has shown to be dependent on the oligosaccharide size, degree of sulfation and carboxylation. Apparently, these same requirements hold for the heparan sulfate molecules. Based on the reported studies, ee propose some important requirements of an oligosaccharide to potentiate FGF-1 mitogenic activity: 1) to have a minimum of twelve units, organized as disaccharides where one of the units is a uronic acid and the second is glycosamine; 2) to have at least one iduronic acid sulfated at position 2 and 3) to have N-sulfated glycosamines, preferentially 6-O-sulfated. To have groups of negative charges is not enough: they need to be localized in a correct conformation.

Distribution of phospholipase C isozymes in normal human lung tissue and their immunohistochemical localization

Phospholipase C(PLC) plays a central role in signal transduction and it is important in cellular growth, differentiation and transformation. There are currently ten known mammalian isozymes of PLC identified and cloned. However, there are no report of PLC distribution in human lung tissue or their significances in pulmonary diseases. Presence of various PLC isozymes in normal human lung tissue was studied from surgical specimens. PLC isozymes in tissue extracts of the lung were partially purified by successive chromatographic steps on heparin-sepharose CL-6B conventional and TSKgel heparin-5PW HPLC columns and their activities were assayed. PLC activity peaks identified in the chromatography were immunoblotted with specific antibodies against ten known mammalian PLC isozymes(PLC-beta 1-4, -gamma 1-2, and -delta 1-4). In addition, immunohistochemical staining of the lung tissue was performed to determine subcellular and histological localization of PLC isozymes. The results indicate that normal human lungs contain beta 1, beta 3, gamma 1, and delta 1, isozymes of PLC. The order of amount present in the lung tissue was PLC-delta 1 > gamma 1 >beta 1 >> beta 3, in descending order. On immunohistochemistry, PLC-gamma 1 was most widely distributed and was present in bronchiolar epithelium, in type I and type II pneumocytes as well as in fibroblasts of the interstitial tissue. PLC-delta 1 was present in the cytoplasm of the bronchiolar epithelium whereas PLC-beta 1 was localized to the apical membranous portion of the same epithelium. PLC-beta 3 was seen in the nucleus of the respiratory and alveolar lining epithelium as well as in the nucleus of lung fibroblasts.

The rat thymus contains a heparin-binding factor that modulates steroidogenesis in the testis

Thymus development and function are under the influence of hormones secreted by the gonads and pituitary. On the other hand, thymurus is crucial for the development of reproductive capacitities in female and male rats and we have shown that a factor derived from the prepubertal rat thy mus has antigonadotropic effect in ovarian and testis cells in vitro. In the present paper we show that the rat thymic factor which modulates gonadotropin action in the gonads is an heparin-binding factor. This capacity was also used as a useful tool to obtain this activity from semipure extracts. An acetone extract was prepared from 15 day old male rats and subjected to molecular filtration chromatography. The activity, of those fractions was investigated in a testis cells biossay, by measuring testosterone secretion under basal and hCG-stimulation. Active fraction were processed in a heparin-Sepharose affinity column. We found that fractions that eluted with 0.6 and 2M NaCl/10mM Tris had biological specific activity. The electrophoretic procedure showed that the apparent molecular weight of the Heparin Sephadex binding factor is 60 kDa. Since this factor was obtained from a protein peak that eluted in the volume of carbonic anhidrase a dimerization process could be involved. Present results show that the rat thymus has an heparin-binding factor that interacts with hCG in testis cells. This factor could play an interesting role in the mutual influence between thymus and gonads.

Anticoagulant, antithrombotic and antihemostatic activities of heparin: structural requeriments, mechanism of action and clinical applications

The structural features for the anticoagulant, antithrombotic and antihemostatic activities of the heparin molecule as well as the resulting clinical applications are reviewed. For anticlotting activity, an intact heparin molecule with a minimum molecular weight of 8 kDa is necessary. On the other hand, for the antithrombotic activity, a heparin hexasaccharide fragment already exhibits 60 percent of the activity of heparin. Also compounds like heparan sulfate, without anticlotting activity, show the same antithrombotic effect of heparin. Heparin, besides its favorable anticoagulant and antithrombotic actions, has also a strong hemorrhagic activity. This effect is related to special structures of the damaged vessel wall and is not related to the anticoagulant and antithromboic actions. The minimum structure for the production of hemorrhage is a disaccharide composed of glucosamine C-6 sulfate and uronic acid with and 1 (4 glycosidic linkage. The hemorrhagic effect of heparin and fragments, including disaccharides, is abolished by ATP and/or myosin. The hemorrhagic disaccharides resemble the molecular conformation of ATP. Topical use of ATP in patients subjected to cardiovascular surgery with extracorporeal circulation significantly reduced the blood loss caused by heparin.

Determinacao da atividade anticoagulante da heparina / Determination of anticoagulant activity of heparin

Foi realizada a dosagem biologica de produtos comerciais de heparina e de amostras do Primeiro Padrao Nacional Argentino de Heparina da mucosa intestinal suina. Os resultados foram comparados com o Quarto Padrao Internacional. Foi empregado o metodo de Reinert e Winterstein, modificado preconizado pelas Farmacopeias Americana e Brasileira. Foram discutidas as metodologias descritas na literatura e indicada a importancia destas para a avaliacao da atividade anticoagulante das heparinas nao-fracionadas e das de baixo peso molecular.