On the catalytic mechanism of polysaccharide lyases: evidence of His and Tyr involvement in heparin lysis by heparinase I and the role of Ca2+.

The structurally diverse polysaccharide lyase enzymes are distributed from plants to animals but share common catalytic mechanisms. One, heparinase I (F. heparinum), is employed in the production of the major anticoagulant drug, low molecular weight heparin, and is a mainstay of cell surface proteoglycan analysis. We demonstrate that heparinase I specificity and efficiency depend on the cationic form of the substrate. Ca(2+)-heparin, in which α-L-iduronate-2-O-sulfate residues adopt (1)C4 conformation preferentially, is a substrate, while Na(+)-heparin is an inhibitor. His and Tyr residues are identified in the catalytic step and a model based on molecular dynamics and docking is proposed, in which deprotonated His203 initiates ß-elimination by abstracting the C5 proton of the α-L-iduonate-2-O-sulfate residue in the substrate, and protonated Tyr357 provides the donor to the hexosamine leaving group.

Testing for urinary hyaluronate improves detection and grading of transitional cell carcinoma.

OBJECTIVE: The purpose of this study is to establish a method for the diagnosis and grading of transitional cell carcinoma (TCC), which is responsible for 90% of bladder tumors, using a recently developed ultrasensitive assay for the measurement of hyaluronan (HA). MATERIALS AND METHODS: Urine samples were collected prior to surgery (cystoscopy, transurethral resection for bladder cancer (TURBT), and cystectomy) in 350 patients. After the procedure, pathologic examination revealed that 160 patients had TCC. HA was measured directly in the urine by a noncompetitive enzyme-linked immunosorbent assay (ELISA)-like fluorometric assay. Using the receiver operator characteristic curve (ROC), t-test, Dunn test, Kruskal-Wallis test, and Mann-Whitney test, we evaluated the differences between groups (those with TCC vs. those without TCC). RESULTS: By analyzing the ROC curve, we chose a urinary HA cutoff value of 13.0 µg/l for indicating risk of TCC. Using the value this of 13.0 µg/l, we found that this test had an overall sensitivity of 82.3% and an overall specificity of 81.2%. The positive predictive value of this assay was 78.9%, the negative predictive negative value was 84.2%, and the predictive accuracy was 81.7%. Logistic regression analysis revealed that every 1 µg/l increase in HA increased a patient's likelihood of having TCC by 3.9%. The sensitivity of this test to detect superficial tumors was 76.6%, whereas its sensitivity for detecting invasive tumors was 94.6%. The urinary HA excretion of patients with TCC, classified according to the TNM staging system and the World Health Organization (WHO) grading system, were compared, and a significant difference was observed between the HA levels of patients with superficial tumors compared with invasive tumors (P = 0.005) as well as between patients with low- vs. high-grade carcinomas (P < 0.001). Patients with urinary HA levels >35 µg/l had a 4.63 times increased risk of having an aggressive, invasive, high grade tumor (P = 0.005). CONCLUSIONS: Our results support the postulate that urinary HA may be used as a tumor marker to aid in the diagnosis and grading of TCC. Additionally, more invasive tumors produce and release more HA in urine than superficial tumors, thus higher HA levels indicate more aggressive disease.

Glycosaminoglycan chains from alpha5beta1 integrin are involved in fibronectin-dependent cell migration.

Alpha5beta1 integrin from both wild-type CHO cells (CHO-K1) and deficient in proteoglycan biosynthesis (CHO-745) is post-translationally modified by glycosaminoglycan chains. We demonstrated this using [35S]sulfate metabolic labeling of the cells, enzymatic degradation, immunoprecipitation reaction with monoclonal antibody, fluorescence microscopy, and flow cytometry. The alpha5beta1 integrin heterodimer is a hybrid proteoglycan containing both chondroitin and heparan sulfate chains. Xyloside inhibition of sulfate incorporation into alpha5beta1 integrin also supports that integrin is a proteoglycan. Also, cells grown with xyloside adhered on fibronectin with no alteration in alpha5beta1 integrin expression. However, haptotactic motility on fibronectin declined in cells grown with xyloside or chlorate as compared with controls. Thus, alpha5beta1 integrin is a proteoglycan and the glycosaminoglycan chains of the integrin influence cell motility on fibronectin. Similar glycosylation of alpha5beta1 integrin was observed in other normal and malignant cells, suggesting that this modification is conserved and important in the function of this integrin. Therefore, these glycosaminoglycan chains of alpha5beta1 integrin are involved in cellular migration on fibronectin.

Internalization and degradation of heparin is not required for stimulus of heparan sulfate proteoglycan synthesis.

In vitro, heparin and antithrombotic drugs specifically stimulate the synthesis of an antithrombotic heparan sulfate proteoglycan (HSPG) produced by endothelial cells. The putative heparin binding site(s) that may be related to this phenomenon were investigated. In the preceding article, using various heparin probes, it was shown that the heparin does not bind to the endothelial cell surface, but only to the extracellular matrix. The present study demonstrated that, when the cells were exposed to heparin at 37 degrees C, the heparin was internalized and with time was localized in lysosomes. However, endocytosis of heparin was not required for the stimulation of HSPG synthesis. The requirement for heparin degradation in the stimulus of HSPG synthesis was also investigated. When the cells were incubated with chloroquine, a lysosomotropic amine that raises the lysosomal pH thus inhibiting enzymatic degradation of internalized compounds, stimulation of HSPG synthesis was still observed. These combined results indicate that neither internalization nor degradation of heparin is required for stimulation of HSPG synthesis, and suggests that its binding to the extracellular matrix could be responsible for this effect.

The binding of heparin to the extracellular matrix of endothelial cells up-regulates the synthesis of an antithrombotic heparan sulfate proteoglycan.

Exposure of endothelial cells to heparin and other antithrombotic drugs specifically stimulates the synthesis of an antithrombotic heparan sulfate (HS). In the present work, biotinylated heparin (BiotHep) was used to characterize the binding site(s) of heparin responsible for the stimulus in HS synthesis on endothelial cells. No differences were observed between biotinylated and non-biotinylated heparin in their ability to increase the synthesis of HS. In kinetic studies the BiotHep showed fast, saturable and specific binding with an apparent K(D) of 83 nM to adherent cells and 44 nM to the extracellular matrix (ECM) in the absence of cells. By confocal and electron microscopy, BiotHep bound only to the ECM, co-localizing with fibronectin. The same pattern of binding to the ECM was observed using heparin conjugated with FITC or Alexa Fluor 488 in the presence or absence of fetal calf serum. However, after degradation of HS, heparin binds to the cell surface, indicating that endogenous HS possibly occupied the heparin binding sites. Analyses by flow cytometry and confocal microscopy of cells with non-associated ECM, showed labeling of the cell surface using syndecan-4 monoclonal antibody as well as wheat germ agglutinin, but no binding of heparin. Furthermore, the stimulation in HS synthesis is not elicited by heparin in the absence of ECM. These results indicate that the stimulus for the synthesis of HS does not require binding of the heparin to the cell surface, and the signaling may be mediated through the ECM.

Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells.

Sulfated glycosaminoglycans were labeled with biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate were labeled using biotin-hydrazide, under different conditions. The structural characteristics of the biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and biotin) and enzymatic methods (susceptibility to degradation by chondroitinases and heparitinases). The binding of biotinylated glycosaminoglycans was investigated both in endothelial and smooth muscle cells in culture, using a novel time resolved fluorometric method based on interaction of europium-labeled streptavidin with the biotin covalently linked to the compounds. The interactions of glycosaminoglycans were saturable and number of binding sites could be obtained for each individual compound. The apparent dissociation constant varied among the different glycosaminoglycans and between the two cell lines. The interactions of the biotinylated glycosaminoglycans with the cells were also evaluated using confocal microscopy. We propose a convenient and reliable method for the preparation of biotinylated glycosaminoglycans, as well as a sensitive non-competitive fluorescence-based assay for studies of the interactions and binding of these compounds to cells in culture.

Sulfated glycosaminoglycans of periurethral tissue in pre- and postmenopausal women.

OBJECTIVE: The objective was to determine the sulfated glycosaminoglycans (GAGs) of the extracellular matrix (ECM) in pre- and postmenopausal women. STUDY DESIGN: Periurethral tissue was obtained from 44 consecutive women who underwent surgery for urinary incontinence, for pelvic organ prolapse, or for other gynecologic benign conditions. Biopsy specimens were assessed by biochemical methods to characterize and quantify sulfated GAG. Measurements were made of total GAG, chondroitin sulfate, dermatan sulfate and of heparan sulfate. Data were compared using the t-test. RESULTS: Patients were divided into two groups (pre- and postmenopausal groups) and dermatan sulfate was the most predominant glycosaminoglycan. Postmenopausal women had significantly less total sulfated glycosaminoglycans (p<0.01), dermatan sulfate (p<0.01) and chondroitin sulfate (p<0.05) than premenopausal women. We did not observe any differences in heparan sulfate. CONCLUSIONS: Postmenopausal women showed quantitative differences in the biochemical characteristics of the ECM in periurethral tissue by analysis of sulfated GAG.

Identification, cloning, expression and functional characterization of an astacin-like metalloprotease toxin from Loxosceles intermedia (brown spider) venom.

Injuries caused by brown spiders (Loxosceles genus) are associated with dermonecrotic lesions with gravitational spreading and systemic manifestations. The venom has a complex composition containing many different toxins, of which metalloproteases have been described in many different species of this genus. These toxins may degrade extracellular matrix constituents acting as a spreading factor. By using a cDNA library from an Loxosceles intermedia venom gland, we cloned and expressed a 900 bp cDNA, which encoded a signal peptide and a propeptide, which corresponded to a 30 kDa metalloprotease, now named LALP (Loxosceles astacin-like protease). Recombinant LALP was refolded and used to produce a polyclonal antiserum, which showed cross-reactivity with a 29 kDa native venom protein. CD analysis provided evidence that the recombinant LALP toxin was folded correctly, was still in a native conformation and had not aggregated. LALP addition to endothelial cell cultures resulted in de-adhesion of the cells, and also in the degradation of fibronectin and fibrinogen (this could be inhibited by the presence of the bivalent chelator 1,10-phenanthroline) and of gelatin in vitro. Sequence comparison (nucleotide and deduced amino acid), phylogenetic analysis and analysis of the functional recombinant toxin revealed that LALP is related in both structure and function to the astacin family of metalloproteases. This suggests that an astacin-like toxin is present in a animal venom secretion and indicates that recombinant LALP will be a useful tool for future structural and functional studies on venom and the astacin family.

Isolation and characterization of heparin from tuna skins.

This study characterized heparin isolated from tuna skins. Glycosaminoglycans were isolated from tuna skin after digestion using anion exchange resin. Heparin was eluted from the resin by sodium chloride gradient and was further fractionated by acetone fractionation. Anticoagulant activity was determined using the activated partial thromboplastin time and Heptest assays. Potency was determined using amidolytic antifactor IIa and antifactor Xa assays. The presence of heparin in the extracted tuna skin glycosaminoglycans was confirmed using (13)C-nuclear magnetic resonance. The activated partial thromboplastin time and Heptest clotting times were doubled at concentrations of about 4 and 1 microg/mL, respectively. The clotting time prolongation and antiprotease activity induced by tuna heparin was readily neutralized by 25 microg/mL protamine sulfate. These results demonstrate that biologically active heparin with properties similar to clinical grade heparin can be derived from tuna skin, a raw material with otherwise relatively little economic value.

Urinary hyaluronan as a marker for the presence of residual transitional cell carcinoma of the urinary bladder.

OBJECTIVE: The purpose of this report is to evaluate the value of urinary hyaluronan (HA) as a maker of residual transitional cell carcinoma (TCC). PATIENTS AND METHODS: Urine samples were collected from 83 patients hospitalized for transurethral resection (TUR). Patient ages ranged from 36 to 86 years. Samples were taken both before and after surgery. HA analysis was performed using an "ELISA-like" fluorometric assay. RESULTS: Patients were divided into two groups: a control group whose previous diagnosis was negative for tumors (n=22) and another with positive diagnosis for tumors (n=61) which was further sub-divided into with and without residual tumor. After the second procedure 47 individuals did not display residual tumor, whereas 14 (23%) did. The average HA in the control group was 8.3 microg/L pre- and 7.1 post-operatively, hence, no change occurred (p=0.471). In the group with TCC patients, the HA dropped from 885.5 microg/L to 215.3 microg/L with residual tumors and from 234.3 microg/L to 11.2 microg/L for those without residual tumor. Using a cut-off value of 20 microg/L, the sensitivity to detect residual tumor is 92.9% and specificity is 83%. CONCLUSION: HA in addition to being one of the best markers for the initial evaluation of bladder carcinoma can be used to determine the presence of a residual tumor. This is associated with poor prognosis.

Sulfated glycosaminoglycans of the periurethral tissue in women with and without stress urinary incontinence, according to genital prolapse stage.

OBJECTIVE: The objective was to determine sulfated glycosaminoglycans (GAG) of the extracellular matrix (ECM) in women with and without stress urinary incontinence according to genital prolapse stage. STUDY DESIGN: Periurethral tissue was obtained from 30 women who underwent surgery for urinary incontinence, for pelvic organ prolapse, or for other benign gynecologic conditions. Biopsy specimens were assessed by biochemical methods to characterize and quantify sulfated glycosaminoglycans. Measurements were made of total glycosaminoglycans, chondroitin sulfate, dermatan sulfate, and of heparan sulfate. Data were compared using the t-test. RESULTS: In two groups, dermatan sulfate was the most predominant glycosaminoglycan. Women with stress urinary incontinence had significantly more total sulfated glycosaminoglycans (p<0.05) and dermatan sulfate (p<0.05) than women without stress urinary incontinence. We did not observe any differences in chondroitin sulfate and heparan sulfate. CONCLUSIONS: Women with stress urinary incontinence showed quantitative and qualitative differences in the biochemical characteristics of the extracellular matrix in periurethral tissue by analysis of sulfated glycosaminoglycans, according to genital prolapse stage.

Structural and hemostatic activities of a sulfated galactofucan from the brown alga Spatoglossum schroederi. An ideal antithrombotic agent?

The brown alga Spatoglossum schroederi contains three fractions of sulfated polysaccharides. One of them was purified by acetone fractionation, ion exchange, and molecular sieving chromatography. It has a molecular size of 21.5 kDa and contains fucose, xylose, galactose, and sulfate in a molar ratio of 1.0:0.5:2.0:2.0 and contains trace amounts of glucuronic acid. Chemical analyses, methylation studies, and NMR spectroscopy showed that the polysaccharide has a unique structure, composed of a central core formed mainly by 4-linked beta-galactose units, partially sulfated at the 3-O position. Approximately 25% of these units contain branches of oligosaccharides (mostly tetrasaccharides) composed of 3-sulfated, 4-linked alpha-fucose and one or two nonsulfated, 4-linked beta-xylose units at the reducing and nonreducing end, respectively. This sulfated galactofucan showed no anticoagulant activity on several "in vitro" assays. Nevertheless, it had a potent antithrombotic activity on an animal model of experimental venous thrombosis. This effect is time-dependent, reaching the maximum 8 h after its administration compared with the more transient action of heparin. The effect was not observed with the desulfated molecule. Furthermore, the sulfated galactofucan was 2-fold more potent than heparin in stimulating the synthesis of an antithrombotic heparan sulfate by endothelial cells. Again, this action was also abolished by desulfation of the polysaccharide. Because this sulfated galactofucan has no anticoagulant activity but strongly stimulates the synthesis of heparan sulfate by endothelial cells, we suggested that this last effect may be related to the "in vivo" antithrombotic activity of this polysaccharide. In this case the highly sulfated heparan sulfate produced by the endothelial cells is in fact the antithrombotic agent. Our results suggested that this sulfated galactofucan may have a potential application as an antithrombotic drug.

Fucan inhibits Chinese hamster ovary cell (CHO) adhesion to fibronectin by binding to the extracellular matrix.

In recent years, sulfated fucans have emerged as an important class of natural biopolymers. In this study, the anti-adhesive activity of a fucan from the brown seaweed Spatoglossum schröederi was analyzed using tumorigenic cells: wild-type Chinese hamster ovary cells (CHO-K1) and the mutant type deficient in xylosyltransferase (CHO-745). Fibronectin (FN) was used as substrate for cell attachment. For both cell types, this fucan has shown a dose-dependent anti-adhesive effect, reaching saturation at around 400 mug/mL. This effect was abolished by desulfation of the fucan. In addition, this polymer exhibited the highest inhibitory effect in comparison to other sulfated polysaccharides. The fucan was biotinylated and used as a probe to identify its action sites. Biotinylated fucan was detected in the extracellular matrix environment by confocal microscopy and flow cytometric analysis, but not at the cell surface. The results suggest that the fucan shows anti-adhesive activity by binding directly to FN, and blocking FN sites that are recognized by cell surface ligands, possibly the integrin family.

Genetic polymorphism in the sulfotransferase SULT1A1 gene in cancer.

Cytosolic sulfotransferases are enzymes that catalyze the conjugation of sulfate groups to a variety of xenobiotic and endogenous substrates. A mutation in the SULT1A1 gene has been associated with decreased sulfotransferase activity. We studied 125 cancer patients and 100 healthy controls from Brazil matched by age and gender. The objective of this study was to assess the impact of the SULT1A1 polymorphism on sulfotransferase activity in a population of cancer patients. Both heterozygous and homozygous individuals for the mutant allele had significantly decreased sulfotransferase enzymatic activity. This decrease was more significant in cancer patients. The frequency of the SULT1A1( *)2 allele was increased in the myeloma group (odds ratio=0.53). These data suggest a functional role for the SULT1A1 gene polymorphism in cancer.

Identification and partial characterisation of hyaluronidases in Lonomia obliqua venom.

By studying Lonomia obliqua (caterpillar) venom we were able to detect a lytic activity on purified hyaluronic acid. The venom hydrolyses purified chondroitin sulphate, but was unable to degrade either heparan sulphate or dermatan sulphate. Moreover, through purified hyaluronic acid-degrading kinetic assays, we observed that this lytic activity was caused by a hydrolase rather than lyase enzyme. In addition, by using the Reissig colorimetric reaction, we detected this hyaluronic acid hydrolase action as a beta-endohexosaminidase enzyme originating terminal N-acetylglucosamine residues rather than beta-endoglucuronidase, which may originate glucuronic acid residues. Zymogram analysis of the venom detected 49 and 53 kDa molecules with hyaluronic acid lytic activity. An examination of these hyaluronic acid degrading activities as a function of pH showed that these hydrolases had no apparent activities at a pH below 5.0 and higher than 8.0 and displayed their optimal activities at pH ranging from 6.0 to 7.0. Finally, through a fluorescence reaction to hyaluronic acid and confocal microscopy, we confirmed this cleaving action upon hyaluronic acid organised on the extracellular matrix of the dermis of rabbit. The data provide experimental evidence of the presence of hyaluronidases in the L. obliqua venom, probably involved in the harmful effects of the venom.

Sulfated glycosaminoglycans of the vagina and perineal skin in pre- and postmenopausal women, according to genital prolapse stage.

The aim of this study was to analyze the amount and types of sulfated glycosaminoglycans (GAGs) of the extracellular matrix (ECM) in the posterior vaginal wall and perineal skin in menacme and postmenopausal women, according to genital prolapse stage. Samples of vaginal tissue and perineal skin were obtained from 40 women who underwent vaginal surgery. Sulfated glycosaminoglycans were extracted by extensive tissue maxatase digestion, submitted to electrophoresis on agarose gel, and their concentrations were determined by densitometry. Dermatan sulphate (DS) was the predominant GAG, followed by chondroitin sulfate (CS) and heparan sulfate (HS). In the vagina there was a significant decrease in total GAGs, CS, DS and HS in postmenopausal women with prolapse stage 2 and 3 compared to the premenopausal group, independent of the stage. In stage 2 and 3 postmenopausal patients there was a significant decrease of DS and HS compared to the stage 1 postmenopausal group. In perineal skin there was no significant difference between total GAG amount, DS and HS. However, the amount of CS in premenopausal stage 1 patients was significantly than that in postmenopausal patients stage 1 and stages 2 and 3. In conclusions, there are quantitative and qualitative differences in GAGs of the ECM in vaginal wall and perineal skin between women in menacme and the postmenopause, according to genital prolapse stage.

Web-based learning in undergraduate medical education: development and assessment of an online course on experimental surgery.

In order to increase the number of practical and discussion classes offered to students in the traditional-curriculum scenario, while decreasing the lecture-based ones and to create an online community to share knowledge on surgery, we developed and assessed the first online course for undergraduate medical students on experimental surgery at the Federal University of Sao Paulo-UNIFESP, Brazil. The purposes of the present study are: describe and discuss the process and the lessons learned involved in developing an undergraduate web-based course and analyze the students' attitude towards this educational environment. A group of medical students was taught online during 5 weeks on the theory of experimental surgery through video quizzes, required readings, collaborative activities using discussion board and asynchronous communication. The students' knowledge gain, their web session variables and the results of the course evaluation were used to support our study. The students have significantly improved their knowledge on experimental surgery after the course. Among factors in the online course that could possibly have contributed to this gain, the interactive activities (video quizzes), key element in our online material, seemed to be promising for candidates. The evaluation results demonstrated high levels of course functionality, effectiveness of its online content and acceptance among medical students. This study indicated that a web-based course for undergraduate students may be successfully developed and implemented in medical settings and the students seem to be quite supportive. We encourage undergraduate medical learning strategies involving the Web.

Heparan sulfate and control of endothelial cell proliferation: increased synthesis during the S phase of the cell cycle and inhibition of thymidine incorporation induced by ortho-nitrophenyl-beta-D-xylose.

The effect of xylosides on the synthesis of [35S]-sulfated glycosaminoglycans by endothelial cells in culture was investigated. Ortho-nitrophenyl-beta-D-xylose (10(-3)M) produces a dramatic enhancement on the synthesis of heparan sulfate and chondroitin sulfate secreted to the medium (20- and 100-fold, respectively). Para-nitrophenylxyloside, at the same concentration, produces an enhancement of only 37- and 3-fold of chondroitin sulfate and heparan sulfate, respectively. These differences of action seem to be related with the higher lipophilic character of ortho-nitrophenyl-xyloside. A lower enhancement of the synthesis of the two glycosaminoglycans is also observed with 2-naphtol beta-D-xylose and cis/trans-decahydro-2-naphtol beta-D-xylose. Besides stimulating the synthesis, O-nitrophenyl-beta-D-xylose as PMA [J. Cell. Biochem. 70 (1998) 563] also inhibits [3H]-thymidine incorporation by quiescent endothelial cells stimulated for growth by fetal calf serum (FCS). The combination of xylosides with PMA produced some cumulative effect. PMA stimulates the synthesis of heparan sulfate mainly at G1 phase whereas the highest enhancement of synthesis produced by the xylosides is in the S phase of the endothelial cell cycle.

Comparison of practical methods for urinary glycosaminoglycans and serum hyaluronan with clinical activity scores in patients with Graves' ophthalmopathy.

OBJECTIVE: Immunosuppressive treatment of Graves' opthalmopathy (GO) should be restricted to patients with active eye disease, but assessing disease activity is difficult. Several methods to evaluate GO activity have been introduced, but none of them is satisfactory. Glycosaminoglycans (GAGs) are complex polysaccharides that participate on the pathogenesis of GO and attempts to correlate its local increase to urinary GAGs (uGAGs) or serum hyaluronan (sHA) have been made, but the available techniques are labourious, time-consuming and difficult for routine use. The aim of the present study is to develop practical and simple methods for uGAGs and sHA and compare them to the activity and severity of thyroid-associated ophthalmopathy. DESIGN, PATIENTS AND MEASUREMENTS: We developed a microelectrophoresis technique for uGAGs and a fluoroassay for sHA and assessed each in 152 patients with Graves' disease, 25 without GO and 127 with GO, classified according to the Clinical Activity Score (CAS). All patients had been euthyroid for > 2 months. RESULTS: Patients with inactive disease (CAS = 2, n = 100) had uGAGs (4.2 +/- 1.3 micro g/mg/creatinine) and sHA (11.1 +/- 7.2 micro g/l) that did not differ from normal subjects (3.1 +/- 1.1 micro g/mg/creatinine, n = 138 and 13.9 +/- 9.6 micro g/l, n = 395). In contrast, patients with active eye disease (CAS = 3, n = 27) had uGAGs (8.4 +/- 2.7 micro g/mg/creatinine) and sHA (32.3 +/- 17.8 micro g/l) 2-3 times higher than those patients with inactive eye disease. Using a cut-off of 6.1 micro g/mg creatinine for uGAGs and 20.7 micro g/l for sHA we found, respectively, 85% and 81% sensitivity and 93% and 91% specificity for each test. The positive and negative predictive values were 77% and 96% for uGAGs and 71% and 95% for sHA. CONCLUSION: Employing these two new methods we have established a significant relationship between the levels of uGAGs and/or sHA and the clinical activity of GO. Therefore, together with CAS, uGAGs determination, and, to a lesser degree, sHA, would be very useful in the discrimination from active and inactive ocular disease and aid in deciding on the best therapy for GO patients.

Heparins and heparinoids: occurrence, structure and mechanism of antithrombotic and hemorrhagic activities.

The correlation between structure, anticlotting, antithrombotic and hemorrhagic activities of heparin, heparan sulfate, low molecular weight heparins and heparin-like compounds from various sources that are in used in clinical practice or under development is briefly reviewed. Heparin-like molecules composed exclusively of iduronic acid 2-O-sulfate residues have weak anticlotting activities, whereas molecules that contain both iduronic acid 2-O sulfate, iduronic acid and small amounts of glucuronic acid, such as heparin, or mixed amounts of glucuronic and iduronic acids (mollusk heparins) possess high anticlotting and anti-Xa activities. These results also suggest that a proper combination of these elements might produce a strong antithrombotic agent. Heparin isolated from shrimp mimics the pharmacological activities of low molecular weight heparins. A heparan sulfate derived from bovine pancreas and a sulfated fucan from brown algae have a potent antithrombotic activity in arterial and venous thrombosis model "in vivo" with a negligible activity upon the serine-proteases of the coagulation cascade "in vitro". These and other results led to the hypothesis that antithrombotic activity of heparin and other antithrombotic agents is due at least in part by their action on endothelial cells stimulating the synthesis of an antithrombotic heparan sulfate. All the antithrombotic agents derived from heparin and other heparinoids have hemorrhagic activity. Exceptions to this are a heparan sulfate from bovine pancreas and a sulfated fucan derived from brown algae, which have no hemorrhagic activity but have high antithrombotic activities "in vivo". Once the structure of these compounds are totally defined it will be possible to design an ideal antithrombotic.