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.

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.

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.

Glycosaminoglycans and proteoglycans of normal and tumoral cartilages of humans and rats.

Differences in the glycosaminoglycans and proteoglycans synthesized by "young," "adult," and tumoral chondrocytes are reported. Young cartilage and human chondrosarcoma contain chondroitin 4- and 6-sulfates, whereas adult human cartilage contains almost exclusively chondroitin 6-sulfate. High keratan sulfate content is reported in adult cartilage, whereas it is almost absent in young and tumoral cartilages. The electrophoretic pattern and keratan sulfate content in these proteoglycans from adult cartilage are clearly distinct from those of the young and tumoral cartilages. The high molecular weight is the distinguishing property of the glycosaminoglycan synthesized by tumoral chondrocytes.

Effects of detergents on the sulfation of chondroitin sulfate by sulfotransferase from chicken embryo epiphyseal cartilage.

Homogenates of chicken embryo epiphyseal cartilage were prepared in buffered saline. The bulk of the sulfotransferase was found in the supernatant. However, small amounts of sulfotransferase were consistently found in the particulate fraction. Detergents (Triton X-100 and C12E8) added to the incubation mixture activated the sulfation of exogenous sulfate acceptor by the particulate fraction, whereas detergent treatment during homogenization increased sulfotransferase activity in the supernatant at the expense of that in the particulate fraction. Since sulfotransferase activities of the supernatant and particulate fractions had similar properties concerning specificity, affinity for chondroitin with different degrees of sulfation, thermal stability and activation by protamine, we conclude that the same enzyme is present in both fractions and that detergent activates indirectly, by releasing it to the medium.

Urinary excretion of sulfated polysaccharides administered to Wistar rats suggests a renal permselectivity to these polymers based on molecular size.

Sulfated polysaccharides were administered to Wistar rats and their elimination from the blood as well as their urinary excretion were evaluated. Sulfated polysaccharides with differences in molecular mass, charge density and molecular structure were obtained from algae, marine invertebrates and vertebrates. A simple methodology based on the metachromatic property of these polysaccharides with 1,9-dimethylmethylene blue was used to estimate their concentration in urine and blood. Renal permselectivity to these macromolecules was based on molecular size, but the upper limit of molecular mass for excretion of a sulfated polysaccharide in urine varies among polymers with different structures. For dextran sulfates the upper limit is approximately 8 kDa. Chondroitin 4- and 6-sulfates were excreted as fragments of approximately 30 kDa, which is smaller than the injected polysaccharide. This suggests that they were degraded enzymatically in vivo. Large synthetic polymers (dextran sulfate > 8 kDa) were not excreted in urine, but slowly disappeared from the blood. Evaluation of their tissue distribution after intravenous administration indicated that these molecules are preferentially accumulated in the kidney.

Increase of chondroitin 4-sulfate concentration in the endochondral ossification cartilage of normal dogs.

The absolute concentrations of chondroitin 4- and 6-sulfate are compared in articular and endochondral ossification cartilage from normal dogs. In newborn dogs, the absolute concentration of chondroitin 4-sulfate decreases nearly 3-fold from the deeper endochondral ossification cartilage to the articular surface, whereas that of chondroitin 6-sulfate does not change. In cartilage from the articular surface of the epiphysis in adults, where the ossification process is complete, the concentration of chondroitin 4-sulfate is also low. These observations suggest that chondroitin 4-sulfate may be important in the ossification process. The pathogenesis of heritable disorders involving the sulfation of chondroitin sulfate is discussed in view of these findings.

The binding of chondroitin 6-sulfate to plasma low density lipoprotein.

The interaction in vitro of several sulfated glycosaminoglycans with low density lipoproteins (LDL) has been studied. Chondroitin 6-sulfate and heparin were the only ones to produce turbidity when added to LDL in presence of Ca2+. However, when these two glycosaminoglycans were applied to LDL-affinity columns in presence of Ca2+, only chondroitin 6-sulfate was retained. Partially desulfated chondroitin 6-sulfate was not retained on LDL-affinity column, indicating the relevance of sulfate groups in the binding of LDL. Since chondroitin 4-sulfate and heparin, with a sulfate content respectively equal to and greater than that of chondroitin 6-sulfate, are not retained on LDL-affinity columns, the factors relevant to the binding of LDL are probably the conformation of the glycan in solution and the orientation of its sulfate groups.

Sulfated glycosaminoglycans synthesized by human smooth muscle cells isolated from different organs.

The sulfated glycosaminoglycans synthesized by human smooth muscle cells isolated from different organs were identified on the basis of electrophoretic mobility, enzymatic degradation with specific mucopolysaccharidases and by the type of degradation products formed. The results obtained indicated that chondroitin sulfate and heparan sulfate were the main glycosaminoglycans found, that most of the labeled glycosaminoglycans were found in the pericellular pool, and that no marked differences were observed in the sulfated glycosaminoglycan composition of the smooth muscle cells obtained from different organs. 'Liver connective tissue cells', isolated from pathological livers (which had been shown to possess biochemical and physiological features typical of smooth muscle cells) showed a pattern of glycosaminoglycan synthesis similar to that of the smooth muscle cells.

Membrane-associated and secreted proteoglycans from a continuous cell line derived from fibrotic schistosomal granulomas.

Proteoglycans were isolated from a continuous murine cell line (GRX) established from fibrotic granulomas induced in mouse liver by schistosomal infection, representative of liver connective tissue cells. The proteoglycans were labelled with 35SO4, extracted by guanidine-HCl + Triton X-100 in the presence of proteinase inhibitors, and purified by anion-exchange, gel-filtration and affinity-column chromatography. The major fractions of cell-associated and secreted proteoglycans are heparan sulfate proteoglycans. Gel-filtration chromatography on Sephacryl S-400 revealed Kav values of 0.20 and 0.30 for the cell-associated and secreted heparan sulfate proteoglycans, respectively. About 50% of the cell-associated heparan sulfate proteoglycans contained hydrophobic regions, as evidenced by their ability to bind to octyl-Sepharose, while only about 20% of secreted proteoglycans bound to this resin. In addition, no proteoglycan was competitively displaced from the cell surface by heparin. Taken together with other reports on proteoglycan synthesis by a variety of cell types in culture, these observations suggest that cell-surface heparan sulfate proteoglycans possibly contain a hydrophobic domain that functions as a membrane anchor in their attachment to cells. Addition of beta-D-xyloside to the cultures greatly enhanced the release of 35S-dermatan sulfate to the medium. Interestingly, dermatan sulfate is the major glycosaminoglycan found in the schistosoma-induced granuloma, from which the GRX cell line is derived. These studies provide the first biochemical description of the proteoglycans produced by a liver connective tissue cell line derived from schistosomal granulomas.

Proteoglycans synthesized by the hepatic granulomas isolated from schistosome-infected mice and by the granuloma-derived connective tissue cell lines.

Proteoglycans synthesized in vitro by periovular granulomas isolated from livers of schistosome-infected mice were compared with those produced by granuloma-derived cell lines: the primary cell line GR and the permanent cell line GRX. Proteoglycans were metabolically labelled with 35S-sulfate and extracted with 4 M guanidine-HCl containing 2.0% Triton X-100, in the presence of proteinase inhibitors. The radiolabelled proteoglycans were purified and characterized by anion-exchange, gel-filtration and affinity-column chromatography. Heparan sulfate proteoglycans (HS-PGs) and chondroitin sulfate/dermatan sulfate-containing proteoglycans (CS/DS-PGs) were detected in both the culture medium and the cell-associated fractions obtained from GR cells. More than 90% of the cell-associated HS-PG from these cells contained a hydrophobic portion, as evidenced by their ability to bind to octyl-Sepharose. In contrast, among the secreted proteoglycans, it was the CS/DS-PG and not the HS-PG that bound to this resin. The major fractions of cell-associated and secreted proteoglycans from GRX cells were HS-PGs. Similar to HS-PGs from GR cells, 50% of the cell-associated HS-PG bound to octyl-Sepharose, while only 20% of secreted proteoglycans (HS-PGs) bound to this resin. The proteoglycans purified from the whole granuloma were composed mainly of DS-PG, of a size and hydrophobicity similar to the CS/DS-PG from GR cells. Possible correlations among the structure, secretion, distribution and function of proteoglycans in granulomatous reactions are discussed.

Acidic polysaccharides of the ascidian Styela plicata. Biosynthetic studies on the sulfated L-galactans of the tunic, and preliminary characterization of a dermatan sulfate-like polymer in body tissues.

Histochemical and chemical analyses reveal important differences between tunic and body of the ascidian Styela plicata. The body contains hydroxyproline, nucleic acid and hexuronic acid. Analyses of the hexuronic acid-containing molecules indicates the presence of dermatan sulfate and heparan sulfate. The tunic, on the other hand, contains no hydroxyproline and small amounts of nucleic acid and hexuronic acid. Large amounts of sulfated polysaccharides, identified by agarose gel electrophoresis, are also present in the tunic. In vitro, incorporation of 35S-sulfate and 14C-glucose and 35S-sulfate pulse-chase experiments show that sulfate and glucose are incorporated into the sulfated polysaccharides of the tunic. The radioactivity is associated mainly with the region of the tunic containing the epidermal cells; however, a small amount of radioactivity is also detected in other regions of the tunic, suggesting that the sulfated polysaccharides are synthesized mainly by the epidermal cells and, to a small extent, by other cell types present in the tunic. Conversion of D-glucose to L-galactose, previously observed in the tunic of Styela plicata (Biochemistry 30, 3458-3464, 1990) is more intense in the region of the tunic containing the epidermal cells.

Protamine increases the affinity of 3'-phosphoadenosine 5'-phosphosulfate toward a sulfotransferase from chicken embryo epiphyseal cartilage.

A 3' -phosphoadenosine 5' -phosphosulfate (PAPS):chondroitin sulfate sulfotransferase from chicken embryo epiphyseal cartilage, which was partially purified, exhibited a molecular mass of 150 kDa. The enzymatic sulfation of totally desulfated chondroitin was activated up to 12-fold by protamine while the sulfation of partially sulfated chondroitin was activated only 3-fold. Protamine increased the affinity of the enzyme for PAPS about 4-fold when partially desulfated chondroitin was used as sulfate acceptor. The S 0.5 for the totally desulfated chondroitin was not affected by protamine, while high PAPS concentration slightly increased the affinity of the enzyme for the same sulfate acceptor. The possible role of these substances in the regulation of the sulfation of chondroitin sulfate is discussed.

Isolation and characterization of a highly sulfated heparan sulfate from ascidian test cells.

Several sulfated polysaccharides have been isolated from the test cells of the ascidian Styela plicata. The preponderant polysaccharide is a highly sulfated heparan sulfate with the following disaccharide composition: (1) UA(2SO4)-1-->4 GlcN(SO4)(6SO4), 53%; (2) UA(2SO4)-1-->4-GlcN(SO4), 22%; (3) UA-1-->4-GlcNAc(6SO4), 14% and (4) UA-1-->4-GlcN(SO4), 11%. Two others unidentified sulfated polysaccharides and a glycogen polymer are also present in the ascidian eggs. Histochemistry with the cationic dye 1,9-dimethyl-methylene blue and biochemical analysis of the 35S-sulfate incorporation into the eggs reveal that the sulfated glycans are present exclusively in the test cells. Possibly these sulfated polysaccharides are involved in important functions of these cells, such as to confer an external and hydrophilic layer which protect the eggs and the larvae of ascidians.

The distribution of chondroitin sulfates in articular and growth cartilages of human bones.

The relative contents of chondroitin 4- and 6-sulfates in cartilages of different human bones are reported. Articular and vertebral body cartilages contain almost exclusively chondroitin 6-sulfate, whereas growth and subarticular cartilages contain nearly equal amounts of chondroitin 4-sulfate and chondroitin 6-sulfate. Adult cartilages, where the calcification process is complete, contain only chondroitin 6-sulfate. These results that chondroitin 4-sulfate may be an important component for the calcification process, whereas chondroitin 6-sulfate seems to be related to the integrity of the articular surfaces. A chemical defect of chondroitin 6-sulfate in a new mucopolysaccharidosis, characterized by platyspondyly and irregularities of articular surfaces, is in agreement with these results.

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.

Searching for alternatives to heparin: sulfated fucans from marine invertebrates.

We describe a variety of sulfated polysaccharides with regular and well-defined structures which are useful tools for elucidating structure/biological function relationship. Several of these compounds have anticoagulant and antithrombotic activities. The most studied and promising polysaccharide is a fucosylated chondroitin sulfate, composed of a chondroitin sulfate-like backbone, substituted at position 3 of the beta-D-glucuronic acid with heavily sulfated fucose side chains. The anticoagulant activity of this polysaccharide is mediated by both antithrombin and heparin cofactor II; it has antithrombotic activity when targeted at the intrinsic coagulation pathway.

High affinity of a fucosylated chondroitin sulfate for plasma low density lipoprotein.

Factors that influence the binding of sulfated polysaccharides to plasma low density lipoprotein (LDL) were investigated. Among the naturally occurring polysaccharides tested, a fucosylated chondroitin sulfate from an echinoderm exhibited the strongest interaction with LDL. Defucosylation and desulfation totally abolished the interaction with LDL while reduction of carboxyl groups had little effect. These data indicate that the sulfated fucose branches are essential for binding of fucosylated chondroitin sulfate to LDL. In addition, there was a positive correlation between the binding to LDL and increasing length of the sulfated polysaccharide chains. The possibility of a practical use of this fucosylated chondroitin sulfate for the binding of LDL is discussed.

Interaction of high molecular weight chondroitin sulfate from human aorta with plasma low density lipoproteins.

Aortic glycosaminoglycans were separated into fractions of increasing molecular weights containing heparan sulfate or chondroitin 4/6-sulfate + dermatan sulfate. When these fractions were added to plasma low density lipoproteins (LDL) in the presence of Ca2+, only chondroitin 4/6 sulfate + dermatan sulfate of high relative molecular weight produced turbidity. Treatment with testicular hyaluronidase abolished totally the formation of insoluble complex. When these glycosaminoglycans were applied to LDL-affinity columns in the presence of Ca2+, higher NaCl concentrations were necessary for the elution of the high relative molecular weight chondroitin sulfate. Heparan sulfate fractions did not produce turbidity when added to LDL solutions and were eluted from LDL-affinity columns at low NaCl concentrations. All these results suggest that besides the structure (or charge density), the molecular weight of the chondroitin sulfate chains is a relevant factor for the binding of this compound to LDL.