Tesi sul tema "Heparinoid"

Heparin glycosaminoglycans mediate a range of biological events, including anticoagulation as well as a diversity of cell proliferation and differentiation processes. Heparin saccharides have been shown to act as inhibitors against angiogenesis and metastasis of tumour cells. This thesis describes work developing chemistry towards varying length oligosaccharide sequences with potential to offer variable sulfation patterns. The main synthetic components to this work were contribution to developing scalable syntheses of an orthogonally protected L-Iduronic acid unit and a differentially protected D-glucosamine unit. The synthetic work also evaluated a recently reported diazo transfer reagent, which allowed for earlier placement of azide protection over that of previously developed routes within the group. This provided a cheaper, more atom efficient route towards protected D-glucosamine building blocks. Glycosylation of the developed D-GlcN donor units with the L-Ido acceptor allowed the production of key disaccharides which facilitated an efficient iterative glycosylation strategy towards longer oligosaccharides, ultimately providing a differentially protected pentasaccharide. The project evaluated methods towards generating various dimeric heparin type systems through forming new O4 ether linkages between GlcN residues across various short linker fragments. The most successful of these dimerisations used a methallyl dichloride core which allowed for further derivatisation towards dihydroxylated species, the analysis of which highlighted some interesting proton NMR data. The final aspect of this project began development of chemistry towards non-reducing end-labelled oligosaccharide sequences by implementation of a masked aldehyde unit on the C4 hydroxyl of GlcN synthesised from the allylated GlcN precursor via dihydroxylation chemistry. Incorporation of this moiety (protected as a 1,2-dibenzyl glycol) within both a trisaccharide and a pentasaccharide was achieved. Further development of this chemistry should allow for late step oxidative cleavage to reveal the reactive aldehyde, potentially allowing for attachment of various amine functionalised fluorophores via reductive amination. Radiolabelling of such a species should also be possible through sodium borotritide reduction for example.

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The occurrence of bioactive compounds in marine organisms comes awaking the interest of the pharmaceutical industry. Heparin, a sulfated polysaccharide which presence was already identified in several marine invertebrates, is very attractive due its remarkable functional versatility. Besides to intervene in blood coagulation, this molecule has a great anti-inflammatory potential. However, its strong anticoagulant activity difficult the clinical exploitation of its anti-inflammatory properties. Thus, the aims of this work were to evaluate the effect of a heparin-like compound (heparinoid), isolated from the cephalotorax of the Litopenaeus vannamei shrimp, on the inflammatory response, hemostasia and synthesis of antithrombotic heparan sulfate by endothelial cells, besides studying some aspects concerning its structure. The purified heparinoid was structurally characterized following an analytical boarding, involving electrophoresis and chromatography. The structural analysis have shown that this compound possess a high content of glucuronic acid residues and disulfated disaccharide units. In contrast to mammalian heparin, the heparinoid was incapable to stimulate the synthesis of heparan sulfate by endothelial cells in the tested concentrations, beyond to show reduced anticoagulant activity and hemorrhagic effect. In a model of acute inflammation, the compound isolated from the shrimp reduced more than 50% of the cellular infiltration. Besides reduce the activity of MMP-9 and proMMP-2 of the peritoneal lavage of inflamed animals, the heparinoid also reduced the activity of MMP-9 secreted by activated human leukocytes. These results demonstrate the potential of heparinoid from L. vannamei to intervene in the inflammatory response. For possessing reduced anticoagulant activity and hemorrhagic effect, this compound can serve as a structural model to direct the development of more specific therapeutical agents to the treatment of inflammatory diseases
A ocorr?ncia de compostos bioativos em representantes da biodiversidade marinha vem despertando o interesse da ind?stria farmac?utica. Heparina, um polissacar?deo sulfatado cuja presen?a j? foi identificada em v?rios invertebrados marinhos, destaca-se por sua extraordin?ria versatilidade funcional. Al?m de interferir na coagula??o sangu?nea, essa mol?cula possui grande potencial antiinflamat?rio. No entanto, sua forte atividade anticoagulante dificulta o aproveitamento cl?nico de sua propriedade antiinflamat?ria, o que estimula a pesquisa por an?logos de heparina com efeitos colaterais reduzidos. Diante disso, este trabalho teve por objetivos avaliar o efeito de um composto semelhante ? heparina (heparin?ide), isolado do cefalot?rax do camar?o Litopenaeus vannamei, sobre a resposta inflamat?ria, hemostasia e s?ntese de heparam sulfato antitromb?tico pelas c?lulas endoteliais, al?m de estudar alguns aspectos relevantes a cerca de sua estrutura. O heparin?ide purificado foi estruturalmente caracterizado seguindo uma abordagem anal?tica, envolvendo eletroforeses e cromatografias. As an?lises estruturais mostraram que esse composto possui um elevado conte?do de res?duos de ?cido glucur?nico e de dissacar?deos dissulfatados. Ao contr?rio da heparina de mam?feros, o heparin?ide foi incapaz de estimular a s?ntese de heparam sulfato pelas c?lulas endoteliais nas concentra??es testadas, al?m de apresentar atividade anticoagulante in vitro e efeito hemorr?gico reduzidos. Em um modelo de inflama??o aguda, o composto isolado do camar?o reduziu mais de 50% da infiltra??o celular. Al?m de reduzir a atividade de MMP-9 e proMMP-2 no lavado peritoneal dos animais submetidos ao modelo de inflama??o, o heparin?ide tamb?m reduziu a atividade de MMP-9 secretada por leuc?citos humanos ativados. Esses resultados demonstram o potencial do heparin?ide de L. vannamei em interferir em v?rios eventos da resposta inflamat?ria. Por possuir atividade anticoagulante e efeito hemorr?gico reduzidos, esse composto pode servir como um modelo estrutural para direcionar o desenvolvimento de agentes terap?uticos mais espec?ficos para o tratamento de doen?as inflamat?rias

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In recent years the heparin has been the subject of several studies that aim to expand its use as a therapeutic agent, due to its ability to modulate the activity of various proteins that play important roles in the regulation of pathophysiological processes. In several experiments and preclinical trials, heparin has demonstrated an anti-inflammatory role. However, its clinical use is limited, due to its strong anticoagulant activity and hemorrhagic complications. For this reason, considerable efforts have been employed in discovery of heparin analogous (heparinoid) with reduced side effects, that retain the anti-inflammatory properties of heparin. In this context, a heparinoid obtained from the head of Litopenaeus vannamei shrimp, which presents a structural similarity to heparin, showed, in previous studies, anti-inflammatory activity in a model of acute peritonitis with reduced anticoagulant effect in vitro and low hemorrhagic activity. Thus, the present work had as objective to evaluate the effect the heparinoid of the cephalothorax of gray shrimp on the acute inflammatory response in different times (3 or 6 hours after the induction of inflammatory stimulus), using the model of acute peritonitis induced in mice. It was also analyzed the HL effect over the activity of elastase, an enzyme involved in leukocyte recruitment. Furthermore to check if the different doses of heparin and heparinoid change the hemostatic balance in vivo, was assessed the effect of these compounds on the plasma clotting time in animals submitted to inflammation. The results show that in 3 hours, all doses of heparinoid were able to prevent efficiently in the acute inflammatory process without any anticoagulant effects, unlike the extrapolation dose of heparin, which has induced a large hemorrhage due its high anticoagulant activity. However, 6 hours after induction of inflammation, only the dosages of 0.1 and 1.0 μg/Kg of heparin and 1.0 μg/Kg of heparinoid kept anti-migratory effect, without changing of the hemostatic balance. These results indicate that the anti-migratory effect of theses compounds depends on the dosage and time of inflammatory stimulus. The HL and heparin were also able to inhibit the activity of the enzyme elastase. The discovery of this bioactive compound in the cephalothorax of shrimps can arouse great interest in biotechnology, since this compound could be useful as a structural model interesting for the development of new therapeutic agents for peritonitis
Nos ?ltimos anos a heparina tem sido alvo de diversos estudos que visam ampliar seu uso como agente terap?utico, devido ? sua habilidade de modular a atividade de v?rias prote?nas que desempenham pap?is importantes na regula??o de processos fisiopatol?gicos. Em diversos experimentos e ensaios pr?-cl?nicos, a heparina tem demonstrado papel anti-inflamat?rio. Entretanto, seu uso cl?nico ? limitado, devido ? sua forte atividade anticoagulante e complica??es hemorr?gicas. Por essa raz?o, consider?vel esfor?o tem sido empregado na descoberta de an?logos da heparina (heparin?ide) com reduzidos efeitos colaterais, que retenham as propriedades anti-inflamat?rias da heparina. Nesse contexto, um heparin?ide (HL) obtido da cabe?a do camar?o Litopenaeus vannamei, de semelhan?a estrutural ? heparina, apresentou em estudos pr?vios atividade anti-inflamat?ria em modelo de peritonite aguda, com reduzido efeito anticoagulante in vitro e baixa atividade hemorr?gica. Assim, o presente trabalho teve como objetivo avaliar o efeito deste heparin?ide sobre a resposta inflamat?ria aguda em diferentes tempos (3 ou 6 horas ap?s a indu??o do est?mulo inflamat?rio), utilizando o modelo de peritonite aguda induzida em camundongos. Foi analisado tamb?m o efeito do HL sobre a atividade da elastase, uma enzima envolvida no recrutamento de leuc?citos. Al?m disso, para verificar se as diferentes doses do heparin?ide e da heparina alteram o equil?brio hemost?tico in vivo, foi avaliado o efeito desses compostos sobre o tempo de coagula??o do plasma nos animais submetidos ? inflama??o. Os resultados revelam que em 3 horas, todas as doses do heparin?ide foram capazes de interferir de forma eficiente no processo inflamat?rio agudo sem apresentar efeito anticoagulante e interfer?ncia no equil?brio hemost?tico, ao contr?rio da dose de extrapola??o da heparina que induziu forte hemorragia, al?m de apresentar alta atividade anticoagulante. Entretanto, no tempo de 6 horas ap?s a indu??o da inflama??o, apenas as doses de 0,1 e 1,0 μg/Kg da heparina e 1,0 μg/Kg do heparin?ide mantiveram o efeito antimigrat?rio, sem alterar o equil?brio hemost?tico. Esses resultados indicam que o efeito antimigrat?rio depende do tempo e da dose administrada. O HL e a heparina tamb?m foram capazes de inibir a atividade da enzima elastase. A descoberta desse composto bioativo no cefalot?rax do camar?o poder? despertar grande interesse biotecnol?gico, pois este composto poderia servir como um modelo estrutural interessante para o desenvolvimento de novos agentes terap?uticos espec?ficos para a peritonite

Immune heparin-induced thrombocytopenia (HIT) is a potentially serious complication of heparin therapy and is associated with antibodies directed against a complex of platelet factor 4 (PF4) and heparin. Early diagnosis of HIT is important to reduce morbidity and mortality. I developed an enzyme immunoassay that detects the binding of HIT IgG to PF4-heparin in the fluid phase. This required techniques to purify and biotinylate PF4. The fluid phase assay produces consistently low background and can detect low levels of anti-PF4-heparin. It is suited to testing alternative anticoagulants because, unlike in an ELISA, a clearly defined amount of antigen is available for antibody binding. I was able to detect anti-PF4-heparin IgG in 93% of HIT patients. I also investigated cross-reactivity of anti-PF4-heparin antibodies with PF4 complexed to alternative heparin-like anticoagulants. Low molecular weight heparins cross-reacted with 88% of the sera from HIT patients while half of the HIT sera weakly cross-reacted with PF4-danaparoid (Orgaran). The thrombocytopenia and thrombosis of most of these patients resolved during danaparoid therapy, indicating that detection of low affinity antibodies to PF4-danaparoid by immunoassay may not be an absolute contraindication for danaparoid administration. While HIT patients possess antibodies to PF4-heparin, I observed that HIT antibodies will also bind to PF4 alone adsorbed on polystyrene ELISA wells but not to soluble PF4 in the absence of heparin. Having developed a technique to affinity-purify anti-PF4-heparin HIT IgG, I provide the first estimates of the avidity of HIT IgG. HIT IgG displayed relatively high functional affinity for both PF4-heparin (Kd=7-30nM) and polystyrene adsorbed PF4 alone (Kd=20-70nM). Furthermore, agarose beads coated with PF4 alone were almost as effective as beads coated with PF4 plus heparin in depleting HIT plasmas of anti-PF4-heparin antibodies. I conclude that the HIT antibodies which bind to polystyrene adsorbed PF4 without heparin are largely the same IgG molecules that bind PF4-heparin and thus most HIT antibodies bind epitope(s) on PF4 and not epitope(s) formed by part of a PF4 molecule and part of a heparin molecule. Binding of PF4 to heparin (optimal) or polystyrene/agarose (sub-optimal) promotes recognition of this epitope. Under conditions that are more physiological and sensitive than previous studies, I observed that affinity-purified HIT IgG will cause platelet aggregation upon the addition of heparin. Platelets activated with HIT IgG increased their release and surface expression of PF4. I quantitated the binding of affinity-purified HIT 125I-IgG to platelets as they activate in a plasma milieu. Binding of the HIT IgG was dependent upon heparin and some degree of platelet activation. Blocking the platelet Fc??? receptor-II with the monoclonal antibody IV.3 did not prevent HIT IgG binding to activated platelets. I conclude that anti-PF4-heparin IgG is the only component specific to HIT plasma that is required to induce platelet aggregation. The Fab region of HIT IgG binds to PF4-heparin that is on the surface of activated platelets. I propose that only then does the Fc portion of the bound IgG activate other platelets via the Fc receptor. My data support a dynamic model of platelet activation where released PF4 enhances further antibody binding and more release.

Kidney transplantation is the only therapy for patients with end-stage kidney failure. There is a shortage of suitable organs from living donors and heart-beating donors (HBDs) leading to an increased use of marginal organs, including those from non- heart-beating donors (NHBD) to expand the donor pool. However, such kidneys are exposed to periods of warm and cold ischaemia plus reperfusion (IR) injury which can be associated with acute tubular necrosis leading to primary non function (PNF), delayed graft failure (DGF) with an increased risk of acute and chronic allograft rejection. Therefore, minimising ischaemic injury will be beneficial to improve immediate allograft function and reduce the incidence of PNF and DGF. Perfusate solutions can ameliorate the damage caused by IR injury. This study examined the possible protective effect of heparinoids, low molecular weight (LMW) sodium heparin (HS-3400) and sodium pentosan polysulfate (NaP PS), as supplements in perfusate solutions. An in vivo rodent surgical model of NHB kidney donation was used to investigate the effects of heparinoids as immune modulators during kidney preservation. It was demonstrated that heparinoids significantly reduced oedema but did not have any effects on pressure, intra-renal vascular resistance (I RVR) or osmolarity of the perfusate solution. Microarray and qPCR analysis revealed that many genes involved in IR injury were differentially expressed after warm oxygenated perfusion. Network analysis revealed that the pro-inflammatory cytokine TNF-a was the common denominator and a potential key regulator of IR injury. Data obtained strongly demonstrate that these networks were removed by the addition of heparinoids. In vitro biological assays investigated the effects of heparinoids on class 11 MHC antigen expression on primary endothelial cells (ECs) treated with IFN-y. It was found that NaPPS and the majority of its derivatives were more efficient in antagonising IFN-y activity. In addition, NaPPS and its derivatives significantly inhibited transendothelial migration of leukocytes across an IFN-y-activated endothelial monolayer. The anti-inflammatory properties of heparinoids can be exploited for better preservation of NHBD organs to improve short and long-term allograft survival and function. Significantly, this is the first study to reveal the potential of heparinoids as TNF-a antagonists and additives to perfusate solutions.

It is the first time in Lithuania that practical application of semen filtration by Sephadex G-15 for the needs of artificial insemination industry was assessed. The efficacy of semen filtration was assessed applying a battery of sperm quality assays to study bovine spermatozoa quality before and after filtration, as well as after cryopreservation. The estimation of effect of different concentrations of progesterone, oestradiol, heparin separately or in combination, on different functional parameters of bull spermatozoa after thawing was performed. These are the first published studies applying integrated approach to study the effects of sperm challenge with several biologically active substances.

In recent years, many countries have struggled with the fact that expenditures on health care are growing much faster than the overall level of wealth. Research objectives: 1) to conduct a meta-analysis of heparins by the means of their efficacy, safety parameters and treatment outcomes; 2) to conduct pharmacoepidemiological assessment of long-term heparins utilization in Lithuania; 3) to develop a pharmacoeconomic cost-minimization model for low-molecular-weight heparins based on reference pricing methodology; 4) to investigate heparins prescribing trends and to evaluate heparins prescription adherence to international clinical guidelines at a secondary level clinical hospital. Meta-analysis results showed that low-molecular-weight heparins could be considered interchangeable due to similar therapeutic profiles in some indications. In Lithuania consumption of heparins and corresponding costs were constantly increasing during the period of investigation; therefore it would be relevant to implement modern pharmacoeconomic methodologies to regulate costs. Cost-minimization model suggested that expenditures on this group of medicines could be decreased by nearly 70 percent. Analysis of pharmacoepidemiological study data confirmed that heparins prescription practices at the clinical hospital were insufficiently regulated. In addition this study conducted at the clinical hospital revealed non-compliance of heparins safety monitoring practices with clinical guidelines.
Pastaraisiais metais daugelyje šalių sveikatos priežiūros išlaidos augo daug greičiau nei bendras gerovės lygis, todėl yra nuolat diskutuojama, kaip šį išlaidų augimą reikėtų kontroliuoti. Darbo uždaviniai: 1) atlikti heparinų preparatų meta-analizę, palyginant jų efektyvumo ir saugumo parametrus bei gydymo baigtis; 2) atlikti heparinų preparatų ilgalaikio suvartojimo Lietuvoje farmakoepidemiologinį tyrimą; 3) suformuluoti farmakoekonominį kaštų mažinimo sprendimų modelį mažos molekulinės masės heparinų preparatų grupei, remiantis referentinės kainos metodika; 4) ištirti heparinų preparatų skyrimo tendencijas antrinio lygio klinikinėje ligoninėje ir palyginti heparinų preparatų skyrimo atitikimą tarptautinėms gairėms. Meta-analizės rezultatai parodė, jog mažos molekulinės masės heparinai gali būti tarpusavyje pa¬keičiami dėl analogiškų terapinių savybių tam tikrose indikacijose. Heparinų preparatų suvartojimas ir atitinkamos išlaidos tiriamuoju laikotarpiu Lietuvoje nuolat didėjo, todėl būtų aktualu taikyti šiuolaikines farmakoekonomines išlaidų reguliavimo metodikas. Pritaikius kaštų mažinimo modelį heparinų preparatų grupei, būtų galima sumažinti išlaidas šios grupės preparatams beveik 70 procentų. Farmakoepidemiologinio tyrimo rezultatai atskleidė, jog heparinų preparatų skyrimo praktika klinikinėje ligoninėje buvo nepakankamai reglamentuota. Taip pat heparinų preparatų saugumo parametrų stebėjimo praktika ligoninėje neatitiko tarptautinių rekomendacijų.

Background and Objectives: To perform the analysis of medicines consumption in HKUM from 2001 to 2005; to analyze more comprehensively the usage of Heparins and Antibiotics. Design and Setting: The data about medicines consumption was taken from the hospital pharmacy database in Hospital of Kaunas University of Medicine. Main Outcome Measures: The consumption of medicines was evaluated from the financial stand-point and by the mean of DDD methodology. Also the Meta-analysis of Heparins was performed. In the end, the usage of Antibiotics was evaluated from the financial and DDD stand-points. Results: The total drug expenses in HKUM increased dramatically during the five year period i.e. more than 46%, from 5.261 thousand Lt in 2001 to 9.804 thousand Lt in 2005. The mostly consumed medicines by the means of the number of DDDs per 1000 hospitalization days managed to remain almost the same during the mentioned period. After the performed Meta-analysis of heparins, the price of Dalteparinum natricum was set as the reference one. It was done, because UFH showed significantly lower efficacy in comparison with different LMWHs. The further calculations demonstrated that it could be possible to rationalize the usage of 93-144 thousand Lt yearly, if the reference pricing methodology for heparins were adopted. Besides, total expenses of Antibiotics increased more than 70% during the five-year period, from 1.229 thousand Lt in 2001 to 2.112 thousand Lt in 2005. This is extremely... [to full text]

La suramine est un compose polyanionique et polyaromatique, possedant des proprietes heparinoides. Sa puissante activite antitumorale, en particulier sur les cancers prostatiques, a inscite au developpement d'etudes pour mettre en evidence ses mecanismes d'action antitumoraux, ce qui permettrait le developpement de derives plus cibles et moins toxiques pour l'organisme. Il semble que les proprietes heparinoides de ce compose soient a l'origine de son interaction avec des composants aussi bien extracellulaires que cytoplasmiques et nucleaires. Ainsi, les effets de la suramine sur certains facteurs de croissance, des recepteurs membranaires, des enzymes lysosomales et des enzymes nucleaires (impliquees dans la regulation de l'expression de l'adn) ont ete decrits sans pouvoir avec certitude correler ces interactions aux effets cytostatiques, differenciants, cytotoxiques et antimetastatiques de cet heparinoide. Nous avons recemment montre que la suramine est un puissant inhibiteur de topoisomerase ii in vitro et sur culture cellulaire (dc-3f) et qu'elle agit sans induire la formation de complexes clivables adn-topoisomerase ii. Cet effet est le premier pouvant expliquer l'action cytotoxique de la suramine sur certaines lignees de cellules tumorales ; il indique egalement que ce compose, stable dans les cellules, pourrait servir d'outil pour l'etude de l'effet des heparinoides (impliques dans la regulation de la proliferation cellulaire et du phenotype metastatique) au niveau des topoisomerases, fondamentales pour la regulation de la topologie de l'adn et de l'expression genique. Les cellules de fibrosarcome, dc-3f/su 1000, 10 fois resistantes a l'action cytotoxique de la suramine, developpees et etudiees au cours du travail de these montrent des caracteristiques uniques pour des cellules resistantes a un inhibiteur de topoisomerase ii: (1) ces cellules ont augmente leur potentiel invasif et metastatique par rapport aux cellules dc-3f parentales. (2) l'evolution de la resistance a l'action cytotoxique de la suramine peut etre mise en parallele avec l'evolution du potentiel metastatique. Cette modification phenotypique particuliere confirme que la suramine intervient au niveau du potentiel metastatique des cellules tumorales. (3) les cellules dc-3f/su 1000 sont 2 fois resistantes aux inhibiteurs classiques de topoisomerase ii tels que l'etoposide, l'amsacrine et la doxorubicine (technique de clonage en continu) et elles possedent effectivement un fonctionnement altere de leur topoisomerase ii. En particulier, la topoisomerase ii des cellules resistantes montre une activite catalytique accrue associee a une diminution d'induction de formation des complexes adn-topoisomerase ii, sans que cela puisse s'expliquer par une alteration quantitative de l'enzyme. L'augmentation d'activite enzymatique semble etre due a une augmentation de phosphorylation (environ 4 fois) de l'isoforme 180 kda de la topoisomerase ii. Ces resultats confirment l'implication de cette enzyme nucleaire dans le mecanisme d'action cytotoxique de la suramine. (4) les cellules dc-3f/su 1000 presentent une modification de leur structure dans tous les compartiments associes a la transmission des signaux: la matrice extracellulaire, le cytosquelette et l'organisation chromatinienne dans laquelle la topoisomerase ii est fortement impliquee. Finalement, nous suggerons que ces cellules pourraient etre utilisees comme modele pour l'etude de l'effet des heparinoides sur la topoisomerase ii. Nos resultats laissent en effet esperer que l'exploration du role de la topoisomerase ii au niveau de la matrice nucleaire qui est le cur du systeme de regulation de la replication et de la transcription de l'adn, pourrait ouvrir la voie au developpement d'antitumoraux particuliers

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior
Heparan sulfate (HS) and Heparin (Hep) glycosaminoglycans (GAGs) are heterogeneous and highly charged polysaccharides. HS is structurally related to Hep but is much less substituted with sulfo groups than heparin and has a more varied structure (or sequence). Because of structural similiarities between these two polymers, they have been described together as heparinoids . Both chains bind a variety of proteins and mediate various physiologically important processes including, blood coagulation, cell adhesion and growth factor regulation. Heparinoids with structural characteristics similar to these described from HS and/or Hep from mammalian tissues have been isolated from different species of invertebrates, although only a few heparinoids from unusual sources have been characterized. The present study describes the presence of unusual heparinoids population from Artemia franciscana, isolated after proteolysis and fractionation by ion exchange resin and named, F-3.0M. The study model in vivo were hemostasis (rat tail scarification) and inflamatoty activity. The tests in vitro were used for coagulations assays (PT and APTT). The analyse of the heparinoids eluted with 3,0M NaCl showed electrophoretic migration in different buffer systems a single band with a behaviour intermediate between those of mammalian HEP and HS. The main products obtained from Artemia heparinoids after enzymatic degradation with heparitinases I and II from F. heparinum were N-sulphated disaccharides (?U-GlcNS,6S/ ?U,2S-GlcNS and ?U-GlcNS) and N-acetylated disaccharides (?U, GlcNAc). This heparinoid had a lower hemorrhagic effect (400?g/ml) when compared to unfractiionated heparins(25?g/ml).The results also suggest a negligible APTT activity of this heparinoid (62.2s). No action was observed on PT indicating that F-3.0M haven t action on the extrinsic pathway. The results showed that the fraction F- 3.0M have inhibitory effect on migration of leukocytes, 64.5% in the concentration of 10 ?g/ml (P<0.001). The search for new heparin and/or heparan sulphates analogs devoid of anticoagulant activity is an atractive alternative and may open up a wide variety of new therapeutic applications
Heparam sulfato (HS) e Heparina (Hep) s?o glicosaminoglicanos (GAGs), heterog?neos e altamente poliani?nicos. HS ? estruturalmente relacionado a heparina, mas cont?m uma quantidade menor de grupos sulfatados que a Hep. Devido ?s similaridades estruturais entre estes dois pol?meros, eles t?m sido denominados conjuntamente de heparin?ides. Ambas as cadeias ligam-se a uma variedade de prote?nas mediadas por v?rios processos fisiol?gicos importantes, incluindo a coagula??o sangu?nea, ades?o celular e regula??o dos fatores de crescimento. O presente estudo descreve a presen?a de uma popula??o de heparin?ides (F-3.0M) diferenciados presentes em Artemia franciscana os quais foram isolados por prote?lise e fracionados por cromatografia de troca i?nica com variadas concentra??es de NaCl. Neste trabalho, utilizamos modelos in vivo para avaliar a homeostasia e a atividade anti-inflamat?ria destes heparinoides. A hemostasia foi avaliada por modelo de escarifica??o em caudas de ratos Wistar enquanto que o processo inflamat?rio foi observado atrav?s de peritonite induzida por tioglicolato de s?dio a 3%. Os heparin?ides eluidos com 3,0M de NaCl mostraram por eletroforese em diversos tamp?es uma ?nica banda com comportamento intermedi?rio entre a Hep de mam?fero e HS. O principal produto obtido da fra??o F-3.0M de Artemia franciscana ap?s a degrada??o enzim?tica com heparitinases I e II de Flavo bacterium Heparinum foram dissacar?deos N-sulfatados (?U-GlcNS,6S/ ?U,2S-GlcNS e ?U-GlcNS) e dissacar?deos N-acetilados (?U, GlcNAc). Estes heparin?ides apresentaram uma baixa atividade hemorr?gica somente na concentra??o de 400?g/ml, ? similar a heparina na concentra??o de 25?g/ml. Os resultados sugerem ainda, que esta fra??o tem baixa a??o anticoagulante APTT (62,2s); n?o apresentando a??o sobre PT, indicando que n?o tem a??o sobre a via extr?nseca da cascata. Os testes de atividade anti-inflamat?ria, desta fra??o tem efeito inibit?rio sobre a migra??o dos leuc?citos da ordem de 64,5% na concentra??o de 10 ?g/ml (P<0.001)Heparam sulfato (HS) e Heparina (Hep) s?o glicosaminoglicanos (GAGs), heterog?neos e altamente poliani?nicos. HS ? estruturalmente relacionado a heparina, mas cont?m uma quantidade menor de grupos sulfatados que a Hep. Devido ?s similaridades estruturais entre estes dois pol?meros, eles t?m sido denominados conjuntamente de heparin?ides. Ambas as cadeias ligam-se a uma variedade de prote?nas mediadas por v?rios processos fisiol?gicos importantes, incluindo a coagula??o sangu?nea, ades?o celular e regula??o dos fatores de crescimento. O presente estudo descreve a presen?a de uma popula??o de heparin?ides (F-3.0M) diferenciados presentes em Artemia franciscana os quais foram isolados por prote?lise e fracionados por cromatografia de troca i?nica com variadas concentra??es de NaCl. Neste trabalho, utilizamos modelos in vivo para avaliar a homeostasia e a atividade anti-inflamat?ria destes heparinoides. A hemostasia foi avaliada por modelo de escarifica??o em caudas de ratos Wistar enquanto que o processo inflamat?rio foi observado atrav?s de peritonite induzida por tioglicolato de s?dio a 3%. Os heparin?ides eluidos com 3,0M de NaCl mostraram por eletroforese em diversos tamp?es uma ?nica banda com comportamento intermedi?rio entre a Hep de mam?fero e HS. O principal produto obtido da fra??o F-3.0M de Artemia franciscana ap?s a degrada??o enzim?tica com heparitinases I e II de Flavo bacterium Heparinum foram dissacar?deos N-sulfatados (?U-GlcNS,6S/ ?U,2S-GlcNS e ?U-GlcNS) e dissacar?deos N-acetilados (?U, GlcNAc). Estes heparin?ides apresentaram uma baixa atividade hemorr?gica somente na concentra??o de 400?g/ml, ? similar a heparina na concentra??o de 25?g/ml. Os resultados sugerem ainda, que esta fra??o tem baixa a??o anticoagulante APTT (62,2s); n?o apresentando a??o sobre PT, indicando que n?o tem a??o sobre a via extr?nseca da cascata. Os testes de atividade anti-inflamat?ria, desta fra??o tem efeito inibit?rio sobre a migra??o dos leuc?citos da ordem de 64,5% na concentra??o de 10 ?g/ml (P<0.001)

La heparina es una familia de cadenas de glicosaminoglicanos (GAGs) formados por residuos de glucosamina y ácido urónico, cuya eficacia y seguridad en la profilaxis y tratamiento de las enfermedades trombóticas están bien establecidas.

El descubrimiento de que parecía posible desdoblar el efecto inhibidor del factor Xa de la inhibición de la trombina, con las fracciones de bajo peso molecular de la heparina despertó grandes expectativas y condujo a la introducción en terapéutica de las heparinas y heparinoides de bajo peso molecular. Debido a los distintos procedimientos de obtención y a las diferentes unidades utilizadas por los fabricantes para expresar la actividad de sus productos, las comparaciones entre los distintos preparados eran complicadas.

Esta situación indujo a la realización de la presente tesis en la que se comparó el efecto de distintos GAGs sobre la hemostasia en un modelo de trombosis in vitro.

Los productos estudiados fueron: Fragmin® (Kabi) y Fraxiparinc® (Sanofi), dos heparinas de bajo peso molecular que se obtienen a partir de la heparina por despolimerización y purificación; Lomoparán® (Organon) un hiparinoide de bajo peso molecular que se produce directamente de mucosa intestinal porcina; Org 31550 es un derivado sintético del pentasacárido natural de unión a la ATIII y está exento de toda acción inhibidora de la trombina; la heparina no fraccionada estudiada (HNF, Laboratorios Rovi) es la disponible comercialmente y se obtiene de mucosa intestinal porcina.

Debido a las distintas unidades empleadas por los fabricantes, en primer lugar hubo que seleccionar la concentración óptima que sería utilizada en las perfusiones, para cada uno de los productos. De forma arbitraria se decidió utilizar la mínima necesaria que inhibiera la formación de trombina en sangre mantenida en reposo a temperatura ambiente. Como indicador de la formación de trombina se determinó la concentración de fibrinopéptido A (FPA).

A continuación se estudió el efecto de los GAGs sobre el funcionalismo plaquetario a las dosis seleccionadas. Para ello se realizaron perfusiones en las que se cuantificó el depósito de plaquetas sobre la superficie expuesta al flujo y se evaluó la formación de microagregados plaquetarios.

Las perfusiones se realizaron en una cámara de perfusión desarrollada por Sakariassen. La superficie adhesiva era matriz extracelular (MEC) de células endoteliales (CE) cultivadas in vitro. Expuesta al flujo esta matriz induce la adhesión plaquetaria, pero no la formación de trombos. Para obtener una matriz trombogénica que permitiera estudiar la formación de fibrina y trombos plaquetarios, las células fueron estimuladas con un éster de forbol que induce en las CE la síntesis y depósito de factor tisular en la MEC que activará la cascada de la coagulación que conducirá a la producción de trombina.

En la evaluación en face de la adhesión plaquetaria se cuantificó la superficie cubierta (SC) por plaquetas. Los trombos plaquetarios se evaluaron en sección transversal que permite cuantificar la superficie cubierta por trombos de diferentes alturas. Al finalizar las perfusiones se midió la formación de microagregados (SPD) realizando recuentos en EDTA y en glutaraldehído en un contador automático fijando la ventana de recuento entre 3,2 y 16 fl. La idea es que los microagregados formados durante la perfusión se deshacen al colocarlos en una EDTA, y por el contrario son fijados en glutaraldehído y no son valorados por el contador.

El siguiente paso consistió en encontrar un método fiable que permitiera cuantificar la fibrina depositada sobre la matriz. En primer lugar se comparó la técnica que se pensaba utilizar, que es la del Fg-PO con la del Fg marcado con iodo 125 que se considera el patrón pero que tiene el inconveniente de que obliga a trabajar con material radioactivo. Para el estudio comparativo se realizaron perfusiones sobre matriz trombogénica en las que la fibrina se midió con las dos técnicas a la vez.

Una vez establecida la técnica a utilizar se estudió el efecto de los glicosaminoglicanos y de las condiciones de flujo sobre la formación de fibrina, utilizando las mismas condiciones de perfusiones que antes.

RESULTADOS

Se observa que Fragmin y Fraxiparine son casi igualmente efectivas inhibiendo la formación de trombina en la sangre en reposo en términos de molaridad (33,2 Y 36,8 nmol/ml aunque difieren considerablemente cuando se expresan en las U del fabricante (20 y 40 U/mi). Mucha mayor cantidad se requiere de Lomoparan (146,4 nmol/ml) y del pentasacárido 31550 (95,2 nmol/ml) posiblemente debido a su menor actividad inhibidora de la trombina. Como cabía esperar por su actividad anti-trombina mucha menor cantidad de HNF fue necesaria (1,9 nmol/ml).

Con la sangre anticoagulada con Lomoparan, la SC por plaquetas es significativamente mayor a los otros GAGs. Respecto al SPD la cifra de Lomoparan es menor aunque sólo es estadísticamente significativa comparada con la obtenida con Fragmin. Al estudiar la relación entre SC y SPD en cada una de las perfusiones realizadas, se observa que presentan una correlación lineal inversa estadísticamente significativa. Parece probable que la mayor SC observada con Lomoparan sea debido a su menor capacidad de formación de microagregados.

Cuando se estudió el efecto sobre la formación de agregados, sólo en la superficie cubierta por agregados mayores de 10 milimicras se observaron diferencias, siendo menor para heparina no fraccionada y Lomoparan que para Fragmin y Fraxiparine. Sabemos que la formación de trombos plaquetarios en estas condiciones es un proceso trombina dependiente. Por lo tanto las diferencias observadas en los agregados de mayor tamaño posiblemente sea debida a diferencias en la cantidad de trombina formada. Presentando la HNF la mayor capacidad de inhibición, y tras ella Lomoparan, Fragmin y Fraxiparine.

Tras realizar los estudios comparativos se decidió utilizar el marcaje con yodo pues a pesar de que la manipulación del material radioactivo es más engorrosa, proporciona unos resultados fiables.

Una vez seleccionada la técnica a utilizar, se estudió el efecto de los glicosaminoglicanos y del flujo sobre el depósito de fibrina.
La cantidad de fibrina depositada en las perfusiones realizadas con sangre anticoagulada con la HNF, a ambos coeficientes de cizalladura es casi inexistente. Ni la microscopía óptica ni la electrónica, mostraron fibrina en absoluto; estas cifras probablemente muestran los valores de fondo correspondientes al Fg-Yodo asociado a la MEC y/o a las plaquetas.

Por el contrario, la fibrina formada con los otros GAGs varió considerablemente dependiendo del tipo de anticoagulante y el coeficiente de cizalladura. A 300 s(-1) el pentasacárido Org 31550 proporcionó las cantidades mayores de fibrina formada, seguido por Fraxiparine y Fragmin. Lomoparan inhibió el depósito de fibrina en mayor proporción.

Cuando el coeficiente de cizalladura se aumentó a 1.300 s(-1) la cantidad de fibrina disminuyó. Para el pentasacárido y la HNF, la reducción no alcanzó significación estadística. Sí lo fue para Fraxiparine, Fragmin y Lomoparán, en los que rondó alrededor del 80%.

La generación del FPA siguió el mismo patrón que la fibrina, es decir el menor aument o correspondió a la HNF, ya continuación Lornoparan, Fragmin y Fraxiparine y el mayor al pentasacárido. El aumento en los niveles de fibrinopéptido A se detuvo al fmalizar la perfusión en todos los GAGs excepto para el pentasacárido. En este caso, a los 15' de acabar la perfusión los niveles de FPA habían crecido aproximadamente un 30%. Esto probablemente indica que una vez se ha formado trombina escapa a los mecanismos de inhibición y continua ejerciendo su acción en la fase líquida.

Entre los GAGs de bajo peso molecular Lomoparán posee un efecto antitrombina mayor que Fragmin y Fraxiparine. Este mayor potencial antitrombólico podría deberse a la presencia de dermatán sulfato, pues recientemente se ha demostrado que los complejos de dermatán sulfato-cofactor JI de la heparina son capaces de inhibir a la trombina unida a la fibrina mientras que los complejos de antitrombina III-heparina no lo son. El perfil de acción de Fragmin y Fraxiparine es más o menos igual con una tendencia de Fraxiparine hacia un menor efecto antitrombótico.

Las diferencias observadas en la generación de FPA comparando los dos coeficientes de cizalladura no fueron estadísticamente significativas. Esto sugiere que la menor cantidad de fibrina depositada a alto coeficiente de cizalladura posiblemente se debe a una menor polimerización de los monómeros de fibrina inducida por el flujo que a un defecto en la formación de trombina.
Low molecular weight heparin(oid)s (LMWH) differ markedly in terms of molecular composition and activity. In order lo gel more insight into the effect on platelets and fibrin deposition we have compared Fragmin® (KabiVitrum, FA), Fraxiparine® (Choay Laboratories, FP), Lornoparan®, a LMW heparinoid (Organon, LO), a synthetic derivative of rhe natural pentasaccharide, Organon 31550 (PE) and unfractionated heparin (Rovi Laboratories, UH) in a perfusion systern. The concentration selected for perfusion studies was the lowest which inhibited thrombin generation (measured as increase in fibrinopeptide A, FPA) in blood stored for 3 hours at room temperature. Blood from healthy donors was anticoagulated with FA 20 U/ml, FP 40 U/ml, LO 15 U/ml, PE 200 U/ml and UH 5 U/ml. Perfusions were carried out in a rectangular chamber at a shear rate of 300 and 1300 s(-1) for 5 minutes. The extracellular matrices (ECM) used as adhesive surface were obtained from cultured human umbilical vein endothelial cells. A part of these cells were stimulated with phorbol myristate acetate for 16 hours to induce tissue factor synthesis and thrombin formation in the ECM.

The extent of platelet aggregation during perfusion was measured as single platelet disappearance (SPD) by comparing platelet counts in EDTA and glutaraldehyde. Fibrin deposition was measured with IUI-fibrinogen. FPA generation during perfusion was assessed with a RIA kit. In the perfusions performed al 1300 s(-1)' for 5 min, over non stimulated ECM, LO showed a significantly higher platelet adhesion than the other heparins. This correlated with a significantly lower SPD during perfusion. In perfusions carried out at 300 s(-1) over stimulated ECM, all LMWH's and PE gave significantly more fibrin deposition than UH. LO gave less fibrin than the others LMWH's. When the shear rate was increased to 1,300 s(-1) the amount of fibrin deposited on !he ECM decreased, with no changes in the amount of FPA generated, indicating that probably it was due to a defect on fibrin polymerisation more than to a impairment of thrombin formation. In conclusion: in this experimental model, LO exerts less effect on platelets than the other heparins studied. All LMWH's tested and PE, but not UH, a1low local thrombin generation and fibrin deposition on subendothelium. LO inhibited fibrin deposition to a larger extent than the other LMWH's.

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior
Sulfated Polysaccharides with unique chemical structures and important biological activities has been found in a diversity of sea invertebrates. For that, to exist a huger interest on the biotechnology field in the research theses sulfated compounds isolated from sea organisms. Despite the privileged brazilian position for these compounds attainment, there are still a few scientific informations about the isolated substances and their biological activities. A head the displayed, the present work has for objectives, to evaluate the pharmacological properties of the glycosaminoglycans isolated from the sea shrimp Litopenaeus schimitti on homeostasis, blood coagulation, leukocytes migration and platelet/leukocyte adhesion. For this, yhe glycosaminoglycans were extracted from crustacean tissues by proteolysis, fractionation with acetone and later submitted to pharmacological assays. The crustacean tissues showed compounds heparin-like, with anticoagulant activity of 45 IU/mg and 90 IU/mg, respectively. These molecules showed low residual hemorrhagic effects in the tested concentration (100 ?g/mL), when compared to unfractionated commercial heparin (UFH). Another dermatan sulfate-like compound, predominately constituted for disulfated disaccharides, was isolated from crustacean abdomen. This compound showed an efficient effect on leukocytes migration inhibition, in the concentration of 15 ?g/mL, reducing the cellular infiltration in 65% when compared to the controlled animals. In this same concentration, the DS reduced in 60% the protein concentration of the peritoneal exudates. In the concentration, this compound of 0.5 mg/mL, it was capable to reduce in 40% platelet/leukocytes adhesion. Our data demonstrate that these sulfated polysaccharides isolated from the shrimp L. schimitti will can be used as bioactive compounds, appearing as active principles for pharmacological development, anticoagulants and inflammatory response regulators
Polissacar?deos sulfatados com caracter?sticas estruturais distintas e importantes atividades biol?gicas t?m sido encontrados em uma diversidade de invertebrados marinhos. Por isso existe um grande interesse no campo da biotecnologia na pesquisa destes compostos sulfatados isolados de organismos aqu?ticos. No entanto, apesar da posi??o privilegiada do Brasil para a obten??o destes compostos, ainda s?o poucas as informa??es cient?ficas sobre as subst?ncias isoladas e suas atividades biol?gicas. Diante do exposto, este presente trabalho teve por objetivos avaliar os potenciais farmacol?gicos dos glicosaminoglicanos (GAGs) isolados do camar?o marinho Litopenaeus schimitti, sobre a hemostasia, coagula??o sang??nea, migra??o leucocit?ria e ades?o celular. Para isso os GAGs foram extra?dos dos tecidos do crust?ceo mediante prote?lise, fracionamento com acetona e posteriormente submetidos aos ensaios farmacol?gicos. Os tecidos do crust?ceo, abd?men e cefalot?rax, apresentaram compostos semelhantes ? heparina (heparin?ides) com atividade anticoagulante de 45 UI/mg e 90 UI/mg, respectivamente. Estas mol?culas apresentaram baixo efeito hemorr?gico residual na concentra??o de 100 ?g/mL, quando comparada com a heparina comercial n?o fracionada (HNF). Um outro composto semelhante ao dermatam sulfato (DS), constitu?do predominantemente por dissacar?deos dissulfatados foi isolado do abd?men do crust?ceo. Este composto apresentou, na concentra??o de 15 ?g/?L, uma inibi??o significativa (P<0.01) da migra??o leucocit?ria, reduzindo a infiltra??o celular em 65% quando comparado com os animais controle. Nessa mesma concentra??o o DS reduziu em 60% a concentra??o de prote?nas do lavado peritonial. As an?lises qualitativas da composi??o celular do exudato peritonial foram similares ao encontrado para os animais controles em todas as concentra??es testadas. Na concentra??o de 0,5 mg/mL foi capaz de reduzir em 40% a ades?o das plaquetas aos leuc?citos. Os dados obtidos demonstram que estes polissacar?deos sulfatados isolados do camar?o L.schimitti podem vir a ser utilizados como compostos bioativos, podendo surgir como princ?pios ativos para o desenvolvimento de f?rmacos, anticoagulantes e moduladores da resposta inflamat?ria

Patients in the intensive care unit are in critical condition which is often accompanied by a coagulation disorder. Sepsis as a leading cause of death in critically ill patients may be associated with both hypercoagulable state with microtrombi formation in microcirculation and with increased production of endogenous heparinoids with inhibitory effects on blood clotting. Central venous catheter and arterial catheter are established in patients for hemodynamic monitoring and these are flushed with heparin to prevent their closure. Both inputs are used for blood sampling for laboratory tests such as blood count and coagulation parameters, including thromboelastography (TEG). In the first step of the work, arterio-venous differences in coagulation parameters were investigated in patients with sepsis. Higher concentration of D-dimers and lower antithrombin activity were found in venous blood. This finding can be explained by increased antithrombin consumption in hypercoagulable state and reactive hyperfibrinolysis. Inconsistency in the site of blood sampling may then lead to misinterpretation of the pathophysiological processes in the body. No significant differences were found in TEG parameters. In the second step of the work we examined how heparin commonly used for catheter flushing affects TEG-assessed...

Heparin resistance occurs in about 20% of population. In that case, after application of a usual dose of heparin (2-3 mg/kg) ACT will not be sufficiently extended. Aim: To determine the real incidence of heparin resistance in patients scheduled for a cardiac surgical procedure. To determine whether there is an association between preoperative treatment with heparin and the incidence of heparin resistance. Also, to establish whether there is an association between platelet count, patients' age, and heparin resistance. Patients and methods: A total of 624 patients scheduled for on-pump surgery were included into a prospective study over a period of three years. Preoperative and intraoperative activated clotting time (ACT) values were recorded. Additionally, four factors referred to in the relevant literature as potential causes of developing heparin resistance were monitored in all patients: age 65 years, preoperative platelet count ≥ 300 × 109/l, preoperative administration of various types of heparin, antithrombin concentration ≤ 60%, and a combination of all. Patients were considered heparin-resistant if a heparin dose ≥ 5 mg/kg had not produced an anticoagulation response with ACT ≥ 480 s. Our data were evaluated using the test of agreement of relative frequency and the χ2 test. Results: In our...