Recommendations on biosimilar low-molecular-weight heparins.

Based on the results of large clinical trials, several low-molecular-weight heparins (LMWHs) have been approved for prophylaxis and the treatment of venous and arterial thromboembolism. As a result of expiration or pending expiration of patent protection of the originator LMWHs, many generic or biosimilar LMWHs have been approved in some countries and more are likely to be approved elsewhere. Their greater availability may reduce the treatment costs. The Working Party on Requirements for Development of Biosimilar LMWHs of the Subcommittee on Control of Anticoagulation, Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis has reached a consensus on recommendations to ensure the quality of biosimilar LMWHs as compared with the originator LMWHs.

Endometriosis of the round ligament: description of a clinical case and review of the literature.

We report a case of endometriosis of the round ligament in a 29-year-old woman, who complained of a lump with a diameter of about 2.5 cm in the right inguinal region, which increased in bulk and was accompanied by intense pain during the menstrual period. The clinical suspicion of inguinal endometriosis, supported by ultrasonography and Magnetic Resonance (MR), was confirmed by histological examination of the surgical specimen, which included the mass and the extraperitoneal segment of the round ligament. The authors conclude that the appearance of a lump in the inguinal region associated with subjective and objective changes of the lesion in relation to the menstrual cycle must raise the suspicion of endometriosis among the possible diagnoses.

[Left pyo-pneumothorax: a rare complication of colon carcinoma]. / Pio-pneumotorace sinistro: rara complicanza di un carcinoma del colon.

A case of pio-pneumothorax complicating a splenic flexure colonic carcinoma is herein presented. The patient was a 58 years old male and was submitted 3 months earlier to a colo-colic bypass for a locally advanced tumor infiltrating stomach, spleen, tail of the pancreas and left emidiaphragm. Few days before the admittance in our ward, he experienced fever, anorexia, and severe dispnoea. Treatment was a water seal drainage of the chest evacuating nearly 8 Liters of purulent material where Escherichia coli was found. Death occurred 2 weeks after drainage. From the analysis of the literature thoracic empyema is an extremely rare complication of colonic carcinoma: 5 other cases have been reported so far. Pathogenesis in half of the cases was due to septicemia and in the others to infectious local spreading.

Generation of anti-factor Xa active, 3-O-sulfated glucosamine-rich sequences by controlled desulfation of oversulfated heparins.

In the framework of a project aimed at generating heparin-like sulfation patterns and biological activities in biotechnological glycosaminoglycans, different approaches have been considered for simulating the alpha(1-->4)-linked 2-O-sulfated L-iduronic acid (IdoA2SO(3))-->N,6-O-sulfated D-glucosamine (GlcNSO(3)6SO(3)) disaccharide sequences prevalent in mammalian heparins. Since the direct approach of sulfating totally O-desulfated heparins, taken as model compounds for C-5-epimerized sulfaminoheparosan (N-deacetylated, N-sulfated Escherichia coli K5 polysaccharide), preferentially afforded heparins O-sulfated at C-3 instead than at C-2 of the iduronate residues, leading to products with low anticoagulant activities, the problem of re-generating a substantial proportion of the original IdoA2SO(3) residues was circumvented by performing controlled solvolytic desulfation (with 9:1 v/v DMSO-MeOH) of extensively sulfated heparins. The order of desulfation of major residues of heparin GlcN and IdoA and of the minor one D-glucuronic acid was: GlcNSO(3)>GlcN6SO(3)>IdoA3SO(3) congruent with GlcA2SO(3) congruent with GlcN3SO(3)>IdoA2SO(3) congruent with GlcA3SO(3). Starting from a 'supersulfated' low-molecular weight heparin, we obtained products with up to 40% of iduronate residues O-sulfated exclusively at C-2 and up to 40% of their glucosamine residues O-sulfated at both C-6 and C-3. Upon re-N-sulfation, these products displayed an in vitro antithrombotic activity (expressed as anti-factor Xa units) comparable with those of current low-molecular weight heparins.

Toward a biotechnological heparin through combined chemical and enzymatic modification of the Escherichia coli K5 polysaccharide.

A process to generate glycosaminoglycans with heparin- and heparan sulfate-like sequences from the Escherichia coli K5 capsular polysaccharide is described. This polymer has the same structure as N-acetylheparosan, the precursor in heparin/ heparan sulfate biosynthesis. The process involves chemical N-deacetylation and N-sulfation, enzymatic conversion of up to 60% of the D-glucuronic acid to L-iduronic acid residues, and chemical O-sulfation. Because direct sulfation afforded unwanted 3-O-sulfated (instead of 2-O-sulfated) iduronic acid residues, a strategy involving graded solvolytic desulfation of chemically oversulfated C5-epimerized sulfaminoheparosans was assessed using persulfated heparin and heparan sulfate as model compounds. The O-desulfation process was shown to increase the anti-factor Xa activity of oversulfated heparin.

Conformation of heparin pentasaccharide bound to antithrombin III.

The interaction, in aqueous solution, of the synthetic pentasaccharide AGA*IA(M) (GlcN,6-SO(3)alpha 1-4GlcA beta 1-4GlcN,3,6-SO(3)alpha 1-4IdoA,2-SO(3)alpha 1-4GlcN,6-SO(3)alpha OMe; where GlcN,6-SO(3) is 2-deoxy-2-sulphamino-alpha-D-glucopyranosyl 6-sulphate, IdoA is l-iduronic acid and IdoA2-SO(3) is L-iduronic acid 2-sulphate), which exactly reproduces the structure of the specific binding sequence of heparin and heparan sulphate for antithrombin III, has been studied by NMR. In the presence of antithrombin there were marked changes in the chemical shifts and nuclear Overhauser effects (NOEs), compared with the free state. On the basis of the optimized geometry of the pentasaccharide the transferred NOEs were interpreted with full relaxation and conformational exchange matrix analysis. An analysis of the three-dimensional structures of the pentasaccharide in the free state, and in the complex, revealed the binding to be accompanied by dihedral angle variation at the A-G and I-A(M) (where G, I, A and A(M) are beta-d-glucuronic acid, 2-O-sulphated alpha-L-iduronic acid, N,6-O-sulphated alpha-D-glucosamine and the alpha-methyl-glycoside of A respectively) glycosidic linkages. Evidence is also provided that the protein drives the conformation of the 2-O-sulphated iduronic acid residue towards the skewed (2)S(0) form.

Influence of glucose on production and N-sulfation of heparan sulfate in cultured adipocyte cells.

Altered lipoprotein lipase regulation associated with diabetes leading to the development of hypertriglyceridemia might be attributed to possible changes in content and the fine structure of heparan sulfate and its associated lipoprotein lipase. Adipocyte cell surface is the primary site of synthesis of lipoprotein lipase and the enzyme is bound to cell surface heparan sulfate proteoglycans via heparan sulfate side chains. In this study, the effect of diabetes on the production of adipocyte heparan sulfate and its sulfation (especially N-sulfation) were examined. Mouse 3T3-L1 adipocytes were exposed to high glucose (25 mM) and low glucose (5.55 mM) in the medium and cell-associated heparan sulfate was isolated and characterized. A significant decrease in total content of heparan sulfate was observed in adipocytes cultured under high glucose as compared to low glucose conditions. The degree of N-sulfation was-assessed through oligosaccharide mapping of heparan sulfate after chemical cleavages involving low pH (1.5) nitrous acid and hydrazinolysis/high pH (4.0) nitrous acid treatments; N-sulfation was found to be comparable between the adipocyte heparan sulfates produced under these glucose conditions. The activity and message levels for N-deacetylase/N-sulfotransferase, the enzyme responsible for N-sulfation in the biosynthesis of heparan sulfate, did not vary in adipocytes whether they were exposed to low or high glucose. While most cells or tissues in diabetic situations produce heparan sulfate with low-charge density concomitant with a decrease in N-sulfation, adipocyte cell system is an exception in this regard. Heparan sulfate from adipocytes cultured in low glucose conditions binds to lipoprotein lipase by the same order of magnitude as that derived from high glucose conditions. It is apparent that adipocytes cultured under high glucose conditions produce diminished levels of heparan sulfate (without significant changes in N-sulfation). In conclusion, it is possible that the reduction in heparan sulfate in diabetes could contribute to the decreased levels of heparan sulfate associated lipoprotein lipase, leading to diabetic hypertriglyceridemia.

A novel heparan sulphate with high degree of N-sulphation and high heparin cofactor-II activity from the brine shrimp Artemia franciscana.

With the aid of heparinase and heparitinases from Flavobacterium heparinum and 13C and IH NMR spectroscopy it was shown that the heparan sulphate isolated from the brine shrimp Artemia franciscana exhibits structural features intermediate between those of mammalian heparins and heparan sulphates. These include an unusually high degree of N-sulphation (with corresponding very low degree of N-acetylation), a relatively high content of iduronic acid residues (both unsulphated and 2-O-sulphated) and a relatively low degree of 6-O-sulphation of the glucosamine residues. The major sequences (glucuronic acid-->N-sulphated glucosamine and glucuronic acid-->N, 6-disulphated glucosamine) are most probably arranged in blocks. Although exhibiting negligible anticlotting activity in the APTT and anti-factor Xa assays the A. franciscana heparan sulphate has a high heparin cofactor-II activity (about 1/3 that of heparin).

New insights on the specificity of heparin and heparan sulfate lyases from Flavobacterium heparinum revealed by the use of synthetic derivatives of K5 polysaccharide from E. coli and 2-O-desulfated heparin.

The capsular polysaccharide from E. Coli, strain K5 composed of ...-->4)beta-D-GlcA(1-->4)alpha-D-GlcNAc(1-->4)beta-D-GlcA (1-->..., chemically modified K5 polysaccharides, bearing sulfates at C-2 and C-6 of the hexosamine moiety and at the C-2 of the glucuronic acid residues as well as 2-O desulfated heparin were used as substrates to study the specificity of heparitinases I and II and heparinase from Flavobacterium heparinum. The natural K5 polysaccharide was susceptible only to heparitinase I forming deltaU-GlcNAc. N-deacetylated, N-sulfated K5 became susceptible to both heparitinases I and II producing deltaU-GlcNS. The K5 polysaccharides containing sulfate at the C-2 and C-6 positions of the hexosamine moiety and C-2 position of the glucuronic acid residues were susceptible only to heparitinase II producing deltaU-GlcNS,6S and deltaU,2S-GlcNS,6S respectively. These combined results led to the conclusion that the sulfate at C-6 position of the glucosamine is impeditive for the action of heparitinase I and that heparitinase II requires at least a C-2 or a C-6 sulfate in the glucosamine residues of the substrate for its activity. Iduronic acid-2-O-desulfated heparin was susceptible only to heparitinase II producing deltaU-GlcNS,6S. All the modified K5 polysaccharides as well as the desulfated heparin were not substrates for heparinase. This led to the conclusion that heparitinase II acts upon linkages containing non-sulfated iduronic acid residues and that heparinase requires C-2 sulfated iduronic acid residues for its activity.

Structural characterization of low molecular weight heparins.

Low molecular weight heparins (LMWHs) obtained by different depolymerization processes can be distinguished from each other by characteristic end-residues, which are easily identified and quantified by nuclear-magnetic-resonance (NMR) spectroscopy. NMR spectroscopy characterizes major sulfation patterns as well as minor sequences such as the antithrombin-binding sequence and the linkage region of LMWHs. Artifacts associated with base-induced modifications such as the formation of iduronic acid epoxide and aziridine derivatives of N-sulfoglucosamine residues can also be detected. The influence of these modifications on the binding of heparins and LMWHs to proteins other than antithrombin are discussed.

"Linkage region" sequences of heparins and heparan sulfates: detection and quantification by nuclear magnetic resonance spectroscopy.

The (13)C NMR spectra of most heparin and heparan sulfate preparations display minor signals not attributable to the glycosaminoglycan chains of these polysaccharides. These signals have been "concentrated" in oligosaccharides isolated from an acid hydrolyzate of heparin and shown to arise from the sequence GlcA-Gal-Gal-Xyl of the "linkage region" (LR) connecting the carbohydrate chains to the peptide chains in the original proteoglycans. Mono- and two-dimensional (1)H and (13)C NMR analysis of the major oligosaccharide (LR-OLIGO) indicated the prevalent structure GlcA-GlcNAc-GlcA-Gal-Gal-Xyl, where GlcNAc is partially 6-O-sulfated. (13)C NMR signals at 84.6 and 85.0 ppm, arising from C-3 of the two Gal residues, lend themselves to easy detection and quantification of the linkage region in heparins and heparan sulfates and can be used to assess the importance of the LR in the modulation of various biological activities of these glycosaminoglycans.

Motional properties of E. coli polysaccharide K5 in aqueous solution analyzed by NMR relaxation measurements.

13C NMR relaxation measurements at three different magnetic field strengths have been used to analyse the motional properties of a low molecular weight K5 polysaccharide (delta UA-[-->4)-beta-D-GlcNAc(1-->4)-beta-D-GlcA(1-->]n-GlcNAcred) from E. coli. Two-dimensional double INEPT spectra with suppression of cross-correlation effects between dipolar and chemical shift anisotropy relaxation mechanisms were collected in order to determine carbon longitudinal and transverse relaxation times. The values of the overall correlation time and the rate of internal motions were obtained using the model free spectral densities. The data indicate that the overall motion of the molecule is non-isotropic and can be approximated with the symmetric top model with an axial ratio of approximately 22. The magnitude of the generalized order parameters (S2 approximately 0.8) and the internal motion correlation time (tau e approximately 30 ps) differ from those found for iduronic acid-containing glycosaminoglycans and suggest that the internal motions in K5 polysaccharide are more limited.

Synthesis and biological effects of N-alkylamine-labeled low-molecular-mass dermatan sulfate.

Dermatan sulfate (DS) is a component of connective tissue and catalyzes the heparin cofactor II-mediated inhibition of thrombin. Low-molecular-mass dermatan sulfates (LMMDS) are produced to prolong the antithrombotic activity of this substance. Cleavage of DS by nitrous acid leads to an LMMDS with a terminal 2,5-anhydrotalose (At) group at the reducing end which can react with primary amines. Tyramine (Tyr) was bound to the terminal At of LMMDS using reductive amination. LMMDS-tyr is produced using DS. LMMDS desacetglated were produced using totally deaminated DS. These compounds were employed as a model for the characterization of DS using NMR spectroscopy. The purity of the compounds was checked using capillary electrophoresis. The structure of the products was proven by 1H- and 13C-NMR spectroscopy. LMMDS-Tyr was radiolabeled with 125I for use in a radioimmunoassay. The anti-Xa activity and antithrombin activity of the tyramine-labeled DS are very low. The clotting assays Heptest, aPTT, thrombin time, and ecarin time indicate a highly anticoagulant-active substance. The heparin cofactor II-mediated inhibition of thrombin is similar to the parent compound. LMMDS were labeled "endpoint-attached." They are a new tool to understand the actions of DS in biologic systems.

Electrostatic interactions between human leukocyte elastase and sulfated glycosaminoglycans: physiological implications.

The influence of ionic strength and composition on the binding and inhibition of human leukocyte elastase by glycosaminoglycans with variable degree and position of sulfation was investigated. The kinetic mechanism of inhibition had a hyperbolic, mixed-type character with a competitive component that was promoted by low ionic strength, reduced by phosphate ions, and which also depended on the substrate and glycosaminoglycan structure. Enzyme binding was a cooperative phenomenon that varied with ionic strength and composition. The inhibition patterns correlated with the cationic character of elastase and with the distribution of arginines on its molecular surface, most notably with residues located in the vicinity of the substrate binding region. The order of affinity for elastase binding was chondroitin 4-sulfate < chondroitin 6-sulfate < dermatan sulfate, iduronate-containing derivatives being superior with respect to the glucuronate-containing counterparts. Additional sulfation at both the 4- and 6- positions or at the N- and 4-positions of the N-acetylgalactosamine moiety decidedly improved the inhibitory efficiency. The results highlight a fundamental physiological role of enzyme-glycosaminoglycan interactions. In the azurophil granule of the human polymorphonuclear neutrophil, elastase and other enzymes are bound to a matrix of chondroitin 4-sulfate because this is the only glycosaminoglycan that simultaneously offers good binding for enzyme compartmentalization together with prompt release from the bound state at the onset of phagocytosis.

1H and 13C NMR spectral assignments of the major sequences of twelve systematically modified heparin derivatives.

The complete 1H and 13C NMR spectral assignments are described for the most prevalent patterns of sulfation and acetylation which can be found in polymeric heparin or can be obtained by standard chemical modifications. These include a number of novel structures containing unsubstituted or acetylated amino groups and the first complete NMR assignments of many of the other derivatives. Beef lung heparin was chosen as a model system and studies were carried out using conditions to control the influences on the chemical shift positions in heparin samples of divalent cations and variations in pH and temperature.

Characterization of sulfation patterns of beef and pig mucosal heparins by nuclear magnetic resonance spectroscopy.

Though differing only slightly in their degrees of sulfation, heparin preparations from pig mucosa and those from beef mucosa have consistently different 13C- and 1H-NMR spectra, which provide useful fingerprints for distinguishing the two types of heparin. Integrated areas of NMR signals associated with minor, undersulfated sequences (assigned by comparison with mono-dimensional spectra of selectively desulfated heparins and by analysis of two-dimensional spectra of heparins prepared from pig and beef mucosa) permit quantitation of differences in sulfation patterns. Undersulfation of pig mucosal heparins at position 6 of the hexosamine units, determined by 13C-NMR and expressed as percent glucosamines nonsulfated at C6 referred to total glucosamines, is substantially lower for pig mucosal heparins than for beef mucosal heparins (16.9-21.7% vs 36.7-40.7%; average values: 18.6% vs 40.3%). By contrast, undersulfation at position 2 of the iduronic acid units, determined by 1H-NMR and expressed as percent nonsulfated iduronic acid referred to total (sulfated + nonsulfated) iduronic acid is significantly higher for pig mucosal preparations (9.6-13.5% vs 2.1-2.7%; average values: 12.7% vs 2.3%). Pig mucosal heparins also have a significantly higher content of 3-O-sulfated glucosamine units, which are markers for the active site of heparin for antithrombin-III.