Chemical and pharmacological aspects of neutralization of heparins from different animal sources by protamine.

Essentials Bovine (HBI) and porcine (HPI) heparins differ in structure and anticoagulant activity. Protamine-neutralization was evaluated on a variety of physical-chemical methods. HBI requires more protamine than HPI to fully neutralize its anticoagulant activity. Protamine preferentially removes higher-sulfated chains of HBI while HPI is evenly precipitated. SUMMARY: Background Protamine neutralization is an essential step for the safe use and inactivation of the unfractionated heparin (UFH) that is widely employed in surgical and non-surgical procedures involving extracorporeal circulation. Objective To compare protamine neutralization of different pharmaceutical-grade UFHs prepared from porcine or bovine intestine (HPI and HBI, respectively). HBI has approximately half the anticoagulant potency of HPI, mostly as consequence of its fraction enriched with N-sulfated α-glucosamine disaccharides. Methods Protamine neutralization of HPI and HBI was evaluated with in vitro, ex vivo and in vivo assays. We also performed in-depth assessments of the complexation of protamine with these distinct UFHs by using nuclear magnetic resonance and mass spectroscopy. Results HPI and HBI interact similarly with protamine on a mass/mass basis; however, HBI requires more protamine than HPI to have its anticoagulant activity fully neutralized, because of its lower potency, which entails the use of higher doses. Nuclear magnetic resonance spectra revealed that HPI precipitates homogeneously with protamine. On the other hand, the low-sulfated fraction of HBI, enriched with N-sulfated α-glucosamine, precipitates at higher concentrations of protamine than the fraction more like HPI, with a preponderance of N,6-disulfated α-glucosamine disaccharides. Finally, mass spectroscopy spectra showed that some of the different peptide components of protamine interact preferentially with the heparins, irrespective of their animal origin. Conclusion Our results have important medical implications, indicating that protamine neutralization of HBI, determined exclusively by point-of-care coagulation assessments, must fail because of its lower-sulfated fraction with reduced anticoagulant activity that could remain in the circulation after the neutralization procedure.

Is the antithrombotic effect of sulfated galactans independent of serpin?

BACKGROUND: Sulfated galactans are polysaccharides with heterogeneous structures that frequently show anticoagulant activity. Their anticoagulant mechanisms are complex and distinct from those observed for heparin. Sulfated galactans act through a combination of effects involving serpin-dependent and serpin-independent mechanisms. Interestingly, these polymers can also induce blood coagulation due to activation of factor XII (FXII). OBJECTIVES: The structure of a complex sulfated galactan from the red alga Acanthophora muscoides was characterized by solution nuclear magnetic resonance. This polysaccharide and another previously characterized algal sulfated galactan from Botryocladia occidentalis were each used in in vitro and in vivo anticoagulant and antithrombotic assays to understand the possible structural determinants of their functional effects. RESULTS AND CONCLUSIONS: The serpin-dependent anticoagulant effects and FXII-related procoagulant effects of the sulfated galactans decreased in parallel with the molecular size. The serpin-independent anticoagulation also correlated with the chemical structure of the sulfated galactans. The sulfated galactan from A. muscoides, which showed mostly serpin-independent anticoagulant activity and reduced activation of FXII, drastically reduced arterial thrombus formation. However, the sulfated galactans produced opposite effects on venous thrombosis; this difference appears to result from the tenuous balance between the various effects on coagulation, including serpin-dependent and serpin-independent anticoagulation and FXIIa-dependent procoagulation. This study of novel sulfated polysaccharides with distinct effects on coagulation and thrombosis helps to establish the minimal structural-function relationship required for the development of antithrombotic drugs.

An MD-PhD program in Brazil: students’ concepts of science and of common sense

In 1995, a pioneering MD-PhD program was initiated in Brazil for the training of medical scientists in experimental sciences at the Federal University of Rio de Janeiro. The program’s aim was achieved with respect to publication of theses in the form of papers with international visibility and also in terms of fostering the scientific careers of the graduates. The expansion of this type of program is one of the strategies for improving the preparation of biomedical researchers in Brazil. A noteworthy absence of interest in carrying out clinical research limits the ability of young Brazilian physicians to solve biomedical problems. To understand the students’ views of science, we used qualitative and quantitative triangulation methods, as well as participant observation to evaluate the students’ concepts of science and common sense. Subjective aspects were clearly less evident in their concepts of science. There was a strong concern about "methodology", "truth" and "usefulness". "Intuition", "creativity" and "curiosity" were the least mentioned thematic categories. Students recognized the value of intuition when it appeared as an explicit option but they did not refer to it spontaneously. Common sense was associated with "consensus", "opinion" and ideas that "require scientific validation". Such observations indicate that MD-PhD students share with their senior academic colleagues the same reluctance to consider common sense as a valid adjunct for the solution of scientific problems. Overcoming this difficulty may be an important step toward stimulating the interest of physicians in pursuing experimental research.

An MD-PhD program in Brazil: students' concepts of science and of common sense.

In 1995, a pioneering MD-PhD program was initiated in Brazil for the training of medical scientists in experimental sciences at the Federal University of Rio de Janeiro. The program's aim was achieved with respect to publication of theses in the form of papers with international visibility and also in terms of fostering the scientific careers of the graduates. The expansion of this type of program is one of the strategies for improving the preparation of biomedical researchers in Brazil. A noteworthy absence of interest in carrying out clinical research limits the ability of young Brazilian physicians to solve biomedical problems. To understand the students' views of science, we used qualitative and quantitative triangulation methods, as well as participant observation to evaluate the students' concepts of science and common sense. Subjective aspects were clearly less evident in their concepts of science. There was a strong concern about "methodology", "truth" and "usefulness". "Intuition", "creativity" and "curiosity" were the least mentioned thematic categories. Students recognized the value of intuition when it appeared as an explicit option but they did not refer to it spontaneously. Common sense was associated with "consensus", "opinion" and ideas that "require scientific validation". Such observations indicate that MD-PhD students share with their senior academic colleagues the same reluctance to consider common sense as a valid adjunct for the solution of scientific problems. Overcoming this difficulty may be an important step toward stimulating the interest of physicians in pursuing experimental research.

A carbohydrate-based mechanism of species recognition in sea urchin fertilization.

In the present review, we describe a systematic study of the sulfated polysaccharides from marine invertebrates, which led to the discovery of a carbohydrate-based mechanism of sperm-egg recognition during sea urchin fertilization. We have described unique polymers present in these organisms, especially sulfated fucose-rich compounds found in the egg jelly coat of sea urchins. The polysaccharides have simple, linear structures consisting of repeating units of oligosaccharides. They differ among the various species of sea urchins in specific patterns of sulfation and/or position of the glycosidic linkage within their repeating units. These polysaccharides show species specificity in inducing the acrosome reaction in sea urchin sperm, providing a clear-cut example of a signal transduction event regulated by sulfated polysaccharides. This distinct carbohydrate-mediated mechanism of sperm-egg recognition coexists with the bindin-protein system. Possibly, the genes involved in the biosynthesis of these sulfated fucans did not evolve in concordance with evolutionary distance but underwent a dramatic change near the tip of the Strongylocentrotid tree. Overall, we established a direct causal link between the molecular structure of a sulfated polysaccharide and a cellular physiological event - the induction of the sperm acrosome reaction in sea urchins. Small structural changes modulate an entire system of sperm-egg recognition and species-specific fertilization in sea urchins. We demonstrated that sulfated polysaccharides - in addition to their known function in cell proliferation, development, coagulation, and viral infection - mediate fertilization, and respond to evolutionary mechanisms that lead to species diversity.

A carbohydrate-based mechanism of species recognition in sea urchin fertilization

In the present review, we describe a systematic study of the sulfated polysaccharides from marine invertebrates, which led to the discovery of a carbohydrate-based mechanism of sperm-egg recognition during sea urchin fertilization. We have described unique polymers present in these organisms, especially sulfated fucose-rich compounds found in the egg jelly coat of sea urchins. The polysaccharides have simple, linear structures consisting of repeating units of oligosaccharides. They differ among the various species of sea urchins in specific patterns of sulfation and/or position of the glycosidic linkage within their repeating units. These polysaccharides show species specificity in inducing the acrosome reaction in sea urchin sperm, providing a clear-cut example of a signal transduction event regulated by sulfated polysaccharides. This distinct carbohydrate-mediated mechanism of sperm-egg recognition coexists with the bindin-protein system. Possibly, the genes involved in the biosynthesis of these sulfated fucans did not evolve in concordance with evolutionary distance but underwent a dramatic change near the tip of the Strongylocentrotid tree. Overall, we established a direct causal link between the molecular structure of a sulfated polysaccharide and a cellular physiological event - the induction of the sperm acrosome reaction in sea urchins. Small structural changes modulate an entire system of sperm-egg recognition and species-specific fertilization in sea urchins. We demonstrated that sulfated polysaccharides - in addition to their known function in cell proliferation, development, coagulation, and viral infection - mediate fertilization, and respond to evolutionary mechanisms that lead to species diversity.

Dermatan sulfate is the major metachromatic glycosaminoglycan in the integument of the anuran Bufo ictericus.

Glycosaminoglycans from the ventral and dorsal integuments of the anuran Bufo ictericus were characterized based on biochemical and histochemical methods. Dermatan sulfate is the major metachromatic glycosaminoglycan found in these tissues, but small amount of heparan sulfate was also detected. The average molecular mass of the dermatan sulfate is approximately 20 kDa, similar to the glycosaminoglycan isolated from mammalian skin. In addition, the amphibian integument contains high amounts of hyaluronic acid, especially in the ventral area. We also observed that the glycosaminoglycans occur in the anuran integument as irregular deposits through the spongious dermis and in the mast cells, as revealed by histochemical analysis using Alcian blue, dimethylmethylene blue and toluidine blue stains. The concentration and composition of glycosaminoglycans found in the amphibian integument resemble those from mammalian skin except for the higher concentration of hyaluronic acid in the amphibian tissue. Possibly, this observation indicates that the function of the sulfated glycosaminoglycan in these tissues has been preserved during evolution, although the amphibian integument and the human skin have their own particular physiology.

Separating surface and solvent effects and the notion of critical adsorption energy in the adsorption of phenolic compounds by activated carbons.

A modified form of the Freundlich equation in which the solute equilibrium concentration is normalized with respect to the solute solubility is analyzed and applied to adsorption isotherms of phenol, 4-nitrophenol, 4-chlorophenol, and 2-chlorophenol at different values of pH on commercial activated carbon before and after oxidation. The analysis confirms the importance of normalizing the solute equilibrium concentration when analyzing the adsorption isotherms, and it is suggested that a parameter, K(F10), obtained by taking 10% solubility as the reference point when applying the Freundlich equation, is probably the best comparative estimate of the relative adsorption capacity of the carbon for different phenolic compounds. In combination with the Freundlich exponent, n(F), estimates of the adsorption capacity at any other reference point can then be obtained. Analysis of the experimental results also indicates a need to distinguish between two regimes of adsorption, characterized by an adsorption energy, E(ads), greater than or less than a critical value, E(ca). When E(ads) > E(ca), the shape of the adsorption isotherm is determined by solute-solid interactions. On the other hand, when E(ads) < E(ca), solute-solution interactions become more important.

Balance between education- and research-oriented publications from a Brazilian University Hospital.

We analyzed the trends of scientific output of the University Hospital, Federal University of Rio de Janeiro. A total of 1420 publications were classified according to pattern and visibility. Most were non-research publications with domestic visibility. With time, there was a tendency to shift from non-research (or education-oriented) publications with domestic visibility to research publications with international visibility. This change may reflect new academic attitudes within the institution concerning the objectives of the hospital and the establishment of scientific research activities. The emphasis of this University Hospital had been on the training of new physicians. However, more recently, the production of new knowledge has been incorporated as a new objective. The analysis of the scientific production of the most productive sectors of the hospital also showed that most are developing non-research studies devoted to the local public while a few of the sectors are carrying out research studies published in journals with international status. The dilemma of quality versus quantity and of education versus research-oriented publication seems, however, to continue to exist within the specialized sectors. The methodology described here to analyze the scientific production of a university hospital can be used as a tool to better understand the evolution of medical research in Brazil and also to help formulate public policies and new strategies to include research among the major objectives of University Hospitals.

Balance between education- and research-oriented publications from a Brazilian University Hospital

We analyzed the trends of scientific output of the University Hospital, Federal University of Rio de Janeiro. A total of 1420 publications were classified according to pattern and visibility. Most were non-research publications with domestic visibility. With time, there was a tendency to shift from non-research (or education-oriented) publications with domestic visibility to research publications with international visibility. This change may reflect new academic attitudes within the institution concerning the objectives of the hospital and the establishment of scientific research activities. The emphasis of this University Hospital had been on the training of new physicians. However, more recently, the production of new knowledge has been incorporated as a new objective. The analysis of the scientific production of the most productive sectors of the hospital also showed that most are developing non-research studies devoted to the local public while a few of the sectors are carrying out research studies published in journals with international status. The dilemma of quality versus quantity and of education versus research-oriented publication seems, however, to continue to exist within the specialized sectors. The methodology described here to analyze the scientific production of a university hospital can be used as a tool to better understand the evolution of medical research in Brazil and also to help formulate public policies and new strategies to include research among the major objectives of University Hospitals.

Distribution of small proteoglycans and glycosaminoglycans in humerus-related articular cartilage of chickens.

The expression of components present in the cartilaginous extracellular matrix is related to development, gender, and genotype, as well as to the biomechanical properties of each type of cartilage. In the present study, we analyzed small proteoglycans and glycosaminoglycans present in different cartilages of the chicken wing after extraction with guanidine hydrochloride or papain. Quantitative analysis of glycosaminoglycans showed a larger amount in humeral cartilage (around 200 mg/g tissue) than in articular cartilage of the radius and ulna, with 138 and 80 mg/g tissue, respectively. Non-collagenous proteins isolated were predominantly from cartilage in the proximal regions of the humerus and radius. D4 fractions obtained by ultracentrifugation were separated by DEAE-Sephacel and Octyl-Sepharose chromatography and analyzed by SDS-PAGE. Two bands of 57 and 70-90 kDa were observed for all samples treated with beta-mercaptoethanol. Immunoblotting of these proteins was positive for the small proteoglycans fibromodulin and decorin, respectively. Apparently, the 57-kDa protein is present in macromolecular complexes of 160 and 200 kDa. Chondroitin sulfate was detected in all regions. HPLC analysis of the products formed by chondroitinase AC and ABC digestion mainly revealed beta-D-glucuronic acid and N-acetyl beta-D-galactosamine residues. The 4-sulfation/6-sulfation ratio was close to 3, except for the proximal cartilage of the radius (2.5). These results suggest functional differences between the scapula-humerus, humerus-ulna, and humerus-radius joints of the chicken wing. This study contributes to the understanding of the physiology of cartilage and joints of birds under different types of mechanical stress.

Distribution of small proteoglycans and glycosaminoglycans in humerus-related articular cartilage of chickens

The expression of components present in the cartilaginous extracellular matrix is related to development, gender, and genotype, as well as to the biomechanical properties of each type of cartilage. In the present study, we analyzed small proteoglycans and glycosaminoglycans present in different cartilages of the chicken wing after extraction with guanidine hydrochloride or papain. Quantitative analysis of glycosaminoglycans showed a larger amount in humeral cartilage (around 200 mg/g tissue) than in articular cartilage of the radius and ulna, with 138 and 80 mg/g tissue, respectively. Non-collagenous proteins isolated were predominantly from cartilage in the proximal regions of the humerus and radius. D4 fractions obtained by ultracentrifugation were separated by DEAE-Sephacel and Octyl-Sepharose chromatography and analyzed by SDS-PAGE. Two bands of 57 and 70-90 kDa were observed for all samples treated with ß-mercaptoethanol. Immunoblotting of these proteins was positive for the small proteoglycans fibromodulin and decorin, respectively. Apparently, the 57-kDa protein is present in macromolecular complexes of 160 and 200 kDa. Chondroitin sulfate was detected in all regions. HPLC analysis of the products formed by chondroitinase AC and ABC digestion mainly revealed ß-D-glucuronic acid and N-acetyl ß-D-galactosamine residues. The 4-sulfation/6-sulfation ratio was close to 3, except for the proximal cartilage of the radius (2.5). These results suggest functional differences between the scapula-humerus, humerus-ulna, and humerus-radius joints of the chicken wing. This study contributes to the understanding of the physiology of cartilage and joints of birds under different types of mechanical stress.

A sulphated fucan from the Laminaria abyssalis inhibits the human T cell lymphotropic virus type 1-induced syncytium formation in HeLa cells.

This work evaluated the effect of a sulphated fucan extracted from the Laminaria abyssalis marine algae on the human T cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation. The experiments were carried out in HeLa cells cocultured with a HTLV-1-infected T cell line (C91/PL cells) in the presence of the sulphated polysaccharide at concentration below that corresponding to the ED50. The sulphated fucan inhibited almost 100% of the syncytium formation at concentration of 100 microg/mI and was still active (>95%) at a concentration of 25 microg/ml. It was also observed that the best inhibition occurred when the compound was added in the first 2 h of the cell-to-cell contact. This is the first report showing that a purified sulphated polysaccharide, extracted from marine algae, is able to inhibit the cell-to-cell contact essential for the spreading of the HTLV-1.

Embryos of the sea urchin Strongylocentrotus purpuratus synthesize a dermatan sulfate enriched in 4-O- and 6-O-disulfated galactosamine units.

Unfertilized eggs of the sea urchin Strongylocentrotus purpuratus are surrounded by a gelatinous layer rich in sulfated fucan. Shortly after fertilization this polysaccharide disappears, but 24 h later the embryos synthesize high amounts of dermatan sulfate concomitantly with the mesenchyme blastula-early gastrula stage when the larval gut is forming. This glycosaminoglycan has the same backbone structure [4-alpha-L-IdoA-1-->3-beta-D-GalNAc-1](n) as the mammalian counterpart but possesses a different sulfation pattern. It has a high content of 4-O- and 6-O-disulfated galactosamine units. In addition, chains of this dermatan sulfate are considerable longer than those of vertebrate tissues. Adult sea urchin tissues contain high concentrations of sulfated polysaccharides, but dermatan sulfate is restricted to the adult body wall where it accounts for approximately 20% of the total sulfated polysaccharides. In addition, sulfation at the 4-O-position decreases markedly in the dermatan sulfate from adult sea urchin when compared with the glycan from larvae. Overall, these results demonstrate the occurrence of dermatan sulfates with unique sulfation patterns in this marine invertebrate. The physiological implication of these oversulfated dermatan sulfates is unclear. One hypothesis is that interactions between components of the extracellular matrix in marine invertebrates occur at higher salt concentrations than in vertebrates and therefore require glycosaminoglycans with increased charge density.

Inactivation of thrombin by a fucosylated chondroitin sulfate from echinoderm.

A polysaccharide extracted from the sea cucumber body wall has the same backbone structure as the mammalian chondroitin sulfate, but some of the glucuronic acid residues display sulfated fucose branches. These branches confer high anticoagulant activity to the polysaccharide. Since the sea cucumber chondroitin sulfate has analogy in structure with mammalian glycosaminoglycans and sulfated fucans from brown algae, we compared its anticoagulant action with that of heparin and of a homopolymeric sulfated fucan with approximately the same level of sulfation as the sulfated fucose branches found in the sea cucumber polysaccharide. These various compounds differ not only in their anticoagulant potencies but also in the mechanisms of thrombin inhibition. Fucosylated chondroitin sulfate, like heparin, requires antithrombin or heparin cofactor II for thrombin inhibition. Sulfated fucans from brown algae have an antithrombin effect mediated by antithrombin and heparin cofactor II, plus a direct antithrombin effect more pronounced for some fractions. But even in the case of these two polysaccharides, we observed some differences. In contrast with heparin, total inhibition of thrombin in the presence of antithrombin is not achieved with fucosylated chondroitin sulfate, possibly reflecting a less specific interaction. Fucosylated chondroitin sulfate is able to inhibit thrombin generation after stimulation by both contact-activated and thromboplastin-activated systems. It delayed only the contact-induced thrombin generation, as expected for an anticoagulant without direct thrombin inhibition. Overall, the specific spatial array of the sulfated fucose branches in the fucosylated chondroitin sulfate not only confer high anticoagulant activity to the polysaccharide but also determine differences in the way it inhibits thrombin.

Unbalanced effects of dermatan sulfates with different sulfation patterns on coagulation, thrombosis and bleeding.

We compared the anticoagulant, antithrombotic and bleeding effects of highly sulfated dermatan sulfates from invertebrates and their mammalian counterpart. An invertebrate dermatan sulfate containing 2-O-sulfated alpha-L-iduronic acid and 4-O-sulfated N-acetyl-beta-D-galactosamine residues is a potent anticoagulant due to a high heparin cofactor II activity. It inhibits thrombin due to the formation of a covalent complex with heparin cofactor II, as in the case of mammalian dermatan sulfate, but the effect occurs at lower concentrations for the invertebrate polysaccharide. Surprisingly, the invertebrate dermatan sulfate has a lower potency to prevent thrombus formation on an experimental model and a lower bleeding effect in rats than the mammalian dermatan sulfate. In contrast, another invertebrate dermatan sulfate, also enriched in 2-O-sulfated alpha-L-iduronic acid, but in this case sulfated at O-6 position of the N-acetyl-beta-D-galactosamine units, has no in vitro or in vivo anticoagulant activity, does not prevent thrombus formation but shows a bleeding effect similar to the mammalian glycosaminoglycan. Overall, these results demonstrate unbalanced effects of dermatan sulfates with different sulfation patterns on coagulation, thrombosis and bleeding, and raise interesting questions concerning the relationship among these three biological actions of sulfated polysaccharides.

Dual effects of sulfated D-galactans from the red algae Botryocladia occidentalis preventing thrombosis and inducing platelet aggregation.

Sulfated D-galactans occur on the red algae Botryocladia occidentalis as three fractions that differ in their sulfate content. Fractions F2 and F3 are potent anticoagulants. Like heparin, they enhance thrombin and factor Xa inhibition by antithrombin and/or heparin cofactor II. The inhibition potency increases simultaneously with the sulfate content of the fractions. The antithrombotic activity of these sulfated D-galactans was investigated on an experimental thrombosis model in which thrombus formation was induced by a combination of stasis and hypercoagulability. In contrast with heparin. the sulfated D-galactans showed a dual dose-response curve preventing thrombosis at doses up to approximately 0.5 mg/ kg body weight but losing the effect at higher doses. This unexpected behavior is probably due to a combined action of the sulfated D-galactan as anticoagulant and also as a strong inducer of platelet aggregation. In platelet-depleted animals the antithrombotic activity at higher dose of sulfated D-galactan is restored and almost total inhibition of thrombus formation is achieved. The sulfated D-galactan has no hemorrhagic effect even at high doses, possibly as a consequence of its effect on platelet aggregation. At comparable dose heparin has an intense bleeding effect. These results indicate that new polysaccharides, with well-defined structures, can help to distinguish events, such as antithrombotic and anticoagulant activities, bleeding and platelet-aggregating effects, which are obscure when induced simultaneously by a single compound.

A wide diversity of sulfated polysaccharides are synthesized by different species of marine sponges.

Sulfated polysaccharides were extracted from four species of marine sponges by exhaustive papain digestion. These compounds were purified by anion-exchange and gel-filtration chromatography. Analysis of the purified polysaccharides revealed a species-specific variation in their chemical composition and also in their molecular masses. In the species Aplysina fulva we found a sulfated glucan with a glycogen-like structure. The other three species contained sulfated polysaccharides with variable proportions of galactose, fucose, arabinose and hexuronic acid and also with different degrees of sulfation. Although the complex nature of these polysaccharides did not allow complete structure determination, we detected the occurrence of 4-sulfated residues of fucose and arabinose in the species Dysidea fragilis. The biological role of these sulfated polysaccharides requires further investigation. They may be involved in the species-specific aggregation of sponge cells and/or in the structural integrity of sponge, resembling the proteoglycans of mammalian connective tissues.