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Biomedical subjects

B Casu

Publications and source records attributed to B Casu.

At least 55 records · Page 3Linked to original sources
PubMed1987

"Supersulfated" heparin fragments, a new type of low-molecular weight heparin. Physico-chemical and pharmacological properties.

A new type of low-molecular-weight heparin (ss-LMW-H) was prepared (by controlled depolymerization and concurrent sulfation of heparin with a mixture of sulfuric and chlorosulfonic acid), to test the influence of extra-sulfate groups on biological properties of heparin fragments. The fragments had an average molecular weight ranging from 5000 to 10,000, a sulfate-to-carboxyl molar ratio of 2.8-3.1, and electrophoretic mobilities and NMR spectra distinctly different from those of the parent heparins. Depolymerization with oversulfation reduced the anticoagulant activity of heparin (ex vivo, in rats) much more than depolymerization alone, to about 10% of the original APTT and 25-30% of the original a.Xa units. By contrast, the antithrombotic activity (venous stasis model, in rats) was still comparable to that of heparin, and bleeding times were not significantly increased. The lipasemic (lipoprotein-lipase-releasing) activity of ss-LMW-H fragments was more than twice that of heparin. Results are discussed in terms of contribution of charge-density effects to different activities and to different mechanisms for the same activity of heparin.

Animals↗
PubMed1986

Retention of antilipemic activity by periodate-oxidized non-anticoagulant heparins.

Heparin preparations with different anticoagulant and antilipemic (fat-clearing) activities were oxidized with periodate under conditions of cleavage of all the C(2)-C(3) bonds of non-sulfated uronic acid residues, while preserving the original molecular weight of the polysaccharide. Periodate-oxidised heparins (oxyheparins, O-HEP) and the corresponding borohydride-reduced products (reduced oxyheparins, RO-HEP) were compared with the original heparins for their content in trisulfated disaccharide sequences (as determined by 13C-nuclear magnetic resonance) and in active sites for antithrombin-III (as determined indirectly by affinity chromatography), and for their anticoagulant and antilipemic (lipoprotein lipase-releasing) activities. The drop of anticoagulant activity induced by periodate oxidation was paralleled by a substantial decrease of affinity for antithrombin, and is thought to arise from glycol splitting at the level of the D-glucuronic acid residue that is part of the active site for antithrombin. The trisulfated disaccharide sequences and the associated antilipemic activities were substantially unaffected by periodate oxidation. The residual anticoagulant activity of periodate-oxidized heparins obtained from preparations - such as those from beef lung - rich in trisulfated disaccharide sequences is discussed in terms of the influence of charge density on heparin-protease interactions not mediated by antithrombin.

Animals↗
PubMed1985

Antithrombotic and bleeding effects of a low molecular weight heparin fraction.

Low molecular weight (LMW) heparin prevents venous thrombosis by potentiating the inhibition of coagulation factor Xa. Heparin, however, has other biological properties whose role in the prevention of thrombosis is still unknown. The aim of our study was to compare the antithrombotic activity of a LMW heparin and its parent molecule in an attempt to understand better the mechanism and structural requirements for heparin's antithrombotic effect. We studied a preparation of an unfractionated pig mucosal heparin pure by any accepted criteria (electrophoresis in various systems, conductimetric titration and NMR spectra) and a LMW heparin fraction obtained from the former by fractional precipitation with ethanol. Both heparins completely prevented thrombus formation in an experimental model of stasis-induced venous thrombosis in rats. When administered intravenously to rats, the unfractionated heparin had an ex vivo anti-Xa/APTT ratio of 1.67, versus 6.60 of the LMW heparin fraction. Unexpectedly, both heparins induced a significant prolongation of tail bleeding time, performed by two different techniques, the "transection" (mostly exploring blood clotting) and the "template" (exploring the platelet/vessel wall interactions). This study suggests that, beside anticoagulation, other effects may play a role in both the antithrombotic and haemorrhagic effects of some heparins and LMW heparin fractions.

Animals↗
PubMed1985

Mono- and bidimensional 500 MHz 1H-NMR spectra of a synthetic pentasaccharide corresponding to the binding sequence of heparin to antithrombin-III: evidence for conformational peculiarity of the sulfated iduronate residue.

1H-NMR spectra of the synthetic pentasaccharide (N-sulfate-6-0-sulfate-alpha-D-glucosamine) 1----4 (beta-D-glucuronic acid) 1----4 (N-sulfate-3,6-di-0-sulfate-alpha-D-glucosamine) 1----4 (2-0-sulfate-alpha-L-iduronic acid) 1----4 (N-sulfate-6-0-sulfate-alpha-D-glucosamine), corresponding to the active site of heparin for antithrombin (AT-III), have been resolved at 500 MHz and assigned by mono- and bidimensional techniques. Vicinal proton coupling constants of the D-glucosamine residues are similar to those in the regular sequences of heparin, indicating that the 4C1 conformation of the ring, and preference for the g,g conformation of the sulfated hydroxymethyl groups of these residues are neither affected by the unique 3-0-sulfo group nor by sequence effects. By contrast, an unusually large coupling between H-2 and H-3 of the sulfated L-iduronic acid residue suggests a greater departure from the 1C4 conformation of this residue. when present in the binding sequence to AT-III than in the regular sequences. Such a departure, leading to different orientation and spacing of essential sulfate groups, may have implications for high-affinity binding to AT-III.

Antithrombin III↗
PubMed1985

Fractionation and structural features of two heparin families with high antithrombotic, antilipemic and anticoagulant activities.

15 heparin preparations from bovine intestine, pancreas and lung and hog intestine were fractionated in two main components by selective barium precipitation. The ones that precipitated at room temperature with barium (slow moving (SM)-heparins) had a high anticoagulant activity measured by the USP and APTT (activated partial thromboplastin time) assay and low antithrombotic activity by the Yin and Wessler method. The fractions precipitated at 5 degrees C with barium (fast moving (FM)-heparins) had a low anticoagulant action and high antithrombotic activity. The maximum anti-Xa activity (chromogenic method) was present in heparins with molecular weights around 12-15 X 10(3) daltons whereas high APTT and LPL releasing activities were present in SM-heparins with molecular weights of 30-40 X 10(3) and 15-25 X 10(3) daltons, respectively. FM-heparins had a higher anti-Xa activity and lower lipoprotein lipase (LPL)-releasing activity when compared with the SM-heparins with the same molecular weights. Significant structural differences were observed between SM- and FM-heparins by 13C-NMR spectra and enzymatic degradation with heparinase and heparitinase from Flavobacterium heparinum. Also, significant differences were observed for anti-Xa and anticoagulant activities for the two types of heparins depending on the pharmacological assay used.

Animals↗
PubMed1984

Structure of heparins and their fragments.

Different methods of depolymerization of heparin affording biologically-active fragments are described and discussed in terms of the influence of the specific fragmentation approach and structural heterogeneity of the parent heparins on the structure and biological activities of the fragments.

Chemical Fractionation↗
PubMed1983

Structure-activity relationship in heparin: a synthetic pentasaccharide with high affinity for antithrombin III and eliciting high anti-factor Xa activity.

The structures of the tetrasaccharide (beta-D-glucuronic acid)1 leads to 4 (N-sulfate-3,6-di-0-sulfate-alpha-D-glucosamine)1 leads to 4(2-0-sulfate-alpha-L-iduronic acid)1 leads to 4(N-sulfate-6-0-sulfate-D-glucosamine) and of the pentasaccharide (N-sulfate-6-0-sulfate-alpha-D-glucosamine)1 leads to 4(beta-D-glucuronic acid)1 leads to 4(N-sulfate-3,6-di-0-sulfate-alpha-D-glucosamine)1 leads to 4(2-0-sulfate-alpha-L-iduronic acid)1 leads to 4(N-sulfate-6-0-sulfate-D-glucosamine), both prepared for the first time, by chemical synthesis from D-glucose and D-glucosamine, have been confirmed by nuclear magnetic resonance. The synthetic tetrasaccharide neither binds to AT-III nor induces anti-factor Xa activity enhancement of this inhibitor. In contrast, the synthetic pentasaccharide strongly binds to AT-III (Ka: 7.10(6)M-1) forming an equimolar complex and also enhances the AT-III inhibitory activity towards factor Xa. These results confirm that the synthetic pentasaccharide with the above structure corresponds to the actual minimal sequence required in heparin for binding to AT-III.

Antithrombin III↗
PubMed1983

Correlation between structure, fat-clearing and anticoagulant properties of heparins and heparan sulphates.

Heparins and heparan sulphates from different organs, of high purity by accepted criteria, were characterized by chemical and physical (including electrophoretic and 13C-NMR spectroscopic) methods. The fat-clearing activity of these preparations was shown to be correlated with their content of the trisulphated disaccharide units I2S-ANS,6S (L-iduronic acid 2-O-sulphate-N-sulphated D-glucosamino 6-O sulphate). Species with low anti-lipemic activity contained significant proportions (greater than or equal to 30%) of nonsulphated uronic acids (especially D-glucuronic acid), and of D-glucosamino residues undersulphated at C-6, these latter residues being partially N-acetylated. Heparins from beef lung and sheep mucosa, predominantly consisting of trisulphated disaccharide units, displayed consistently higher antilipemic activity than the more heterogeneous pig mucosal heparins. The anticoagulant activity (as determined by the U.S.P., APTT and anti-Xa tests) was not a simple function of the measured physicochemical parameters. Trends were confirmed for pig mucosal heparins being more anticoagulant than beef lung preparations, and low molecular weight (usually undersulphated) species being more active in the anti-Xa test than in the U.S.P. and APTT tests.

Amino Acids↗
PubMed1981

The structure of heparin oligosaccharide fragments with high anti-(factor Xa) activity containing the minimal antithrombin III-binding sequence. Chemical and 13C nuclear-magnetic-resonance studies.

The chemical composition and the 13C n.m.r. spectra of heparin oligosaccharides (essentially octasaccharides), having high affinity for antithrombin III and high anti-(Factor Xa) activity, prepared by three independent approaches (extraction, partial deaminative cleavage with HNO2 and partial depolymerization with bacterial heparinase), leading to different terminal residues, have been studied and compared with those of the corresponding inactive species. Combined wit chemical data, the spectra of the active oligosaccharides and of their fragmentation products afforded information on composition and sequence. The three types of active oligosaccharides were shown to have the common hexasaccharide core I-Aa-G-As*-Is-As, where I and alpha-L-idopyranosyl-uronic acid, Aa = 2-acetamido-2-deoxy-alpha-D-glucopyranose, G = beta-D-glucopyranosyl-uronic acid, Is = alpha-L-idopyranosyluronic acid 2-O-sulphate, As = 2-deoxy-2-sulphamino-alpha-D-glucopyranose 6-O-sulphate. The fourth residue (As*) is an unusually substituted amino sugar resistant to mild deamination. The 13C spectra of the active species are characterized by signals from the above atypical amino sugar, the most evident of which is at 57.7 p.p.m. These signals, compared with those of appropriate synthetic model compounds, are compatible with the recently proposed 3-O-sulphation of the residue As* [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555].

Animals↗
PubMed1980

Glycosaminoglycans from pig duodenum.

A polysaccharide extract from pig duodenum, used in therapy as antilipemic, was shown by chromatographic and electrophoretic methods to be a mixture of the glycosaminoglycans (GAG) heparin (HEP), heparan sulfate (HS), dermatan sulfate (DeS), chondroitin sulfates (ChS) and hyaluronic acid (HA), in the ratio 24:31:23:9:13. The GAG mixture was fractionated with alkyl-ammonium salts, and, for DeS, with copper salts. Further purification of the fractions either by repeated complexation or by removal of residual impurities using specific enzymes or chemical reactions, permitted obtaining individual GAG more than 97% pure by electrophoretic and 1H-NMR analysis. These preparations will be used to assess the contribution of individual GAG to the biological activity of duodenal GAG extracts.

Animals↗