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B Casu

Publications and source records attributed to B Casu.

At least 37 records · Page 2Linked to original sources
PubMed1994

Preparation and characterization of deuterium-labeled glycosaminoglycans.

Heparin, NAcHep, DS, and CS were labeled with deuterium by N-reacetylating, with the deuterated acetic anhydride (CD3CO)2O, GAGs previously N-deacetylated (by hydrazinolysis) to the desired extent. Degrees of deuteration of the present preparations, as determined by 2H- and 1H-NMR were 15%, 51%, 49%, and 79% for heparin, NAcHep, DS, and CS, respectively. The NMR analysis (including the 13C spectra) of the labeled products indicated that deuterium labeling did not involve any substantial modification of the GAG structures. Also NMR signals associated with specific sequences of heparin for antithrombin and of DS for heparin cofactor II were essentially the same in the unlabeled and in the deuterated GAGs. The substantial retention of the original structure was confirmed by data on the degree of sulfation (by conductimetry) and on the electrophoretic mobility in acid buffer. On the other hand, HPLC/SEC data indicated some depolymerization of heparin and DS in the N-deacetylation step of the labeling reactions. HPLC/MS spectrometry permitted a clear identification of disaccharide and tetrasaccharide fragments obtained from deuterated GAGs by enzymic (heparinase, chondroitinase ABC) or chemical depolymerization (deaminative cleavage, Smith degradation), opening new prospects for studies of human pharmacokinetics, with differentiation of exogenous from endogenous GAGs.

Acetic Anhydrides↗
PubMed1994

Semisynthesis and analysis of lipophilically modified unfractionated and low molecular mass heparins.

Unfractionated heparin and LMMH were substituted with different lipophilic organic compounds. Specifically endpoint attached (LMMH-tyramine and LMMH-tyramine-FITC) and nonspecifically substituted heparins (acylated heparins, and LMMH-biotin and LMMH-cholesterol hemisuccinate) were obtained. The lipophilically substituted heparins were analysed by HPSEC and showed different retention times, high peak purity, different UV/VIS absorbances, and areas under the absorbance time curve. The determination of the average molecular mass Mn, Mm, and Mz and the polydispersity P was performed by PAGE. The substituted heparins showed an increase in their molecular mass Mm, ranging from 2.9 to 129.7% unfractionated heparin and 3.9 to 224.0% (LMMH) compared with the parent compounds (unfractionated heparin and LMMH). The anticoagulant activity was measured by anti-Factor Xa. Lipophilically modified heparin had an aXa activity ranging from 52 to 168 U/mg (unfractionated) and 60 to 108 U/mg (LMMH) and antithrombin activity ranging from 31 to 270 U/mg (unfractionated) and 5 to 15 U/mg (LMMH). The thrombin generation inhibition assay demonstrated an effective anticoagulant potency of the modified compounds. They were neutralized by different amounts (1.1 to 4.1, w/w) of protamin. 1H NMR spectroscopy revealed the specific endpoint attachment of tyramine to LMMH and FITC to LMMH-tyramine. The lipophilically modified heparins showed intact anticoagulant properties and are now used for pharmacokinetic investigations.

Acylation↗
PubMed1993

Structure and contribution to the heparin cofactor II-mediated inhibition of thrombin of naturally oversulphated sequences of dermatan sulphate.

Dermatan sulphate (DS) obtained from bovine and pig mucosa and pig skin, and charge-enriched fractions of a selected DS preparation, were characterized in terms of charge density, M(r) and disaccharide composition of chondroitin ABC lyase digests, and by 13C-n.m.r. spectroscopy. Besides the major IdoA-GalNAc4SO3 sequences, all DS preparations contain about 10% disulphated disaccharide sequences (mostly IdoA2SO3-GalNAc4SO3, with minor amounts of IdoA-GalNAc4,6SO3). DS fragments (prepared by radical-catalysed depolymerization of DS and retaining the internal structure of the parent polysaccharide) as well as Smith degraded fragments [SD-DS, obtained by controlled degradation of periodate-oxidized and borohydride-reduced DS (RO-DS)] with the general structure GalNAc4SO3(IdoA2SO3-GalNAc4SO3)n-R (where R is the remnant of a glycol-split uronic acid, and n = 2-3 and 3-4) were characterized by one- and two-dimensional 1H-n.m.r., 13C-n.m.r. and disaccharide composition analysis. In accordance with previous findings [Maimone and Tollefsen (1990) J. Biol. Chem. 265, 18263-18271], only fragments with n > or = 3 significantly enhance the heparin cofactor II-mediated inhibition of thrombin. In natural DS preparations and their fractions, this activity (as well as the antithrombotic activity in an animal model) appears to require IdoA2SO3-containing sequences. The heparin cofactor II activity of DS, RO-DS and SD-DS fragments decreases with decreasing M(r). However, RO-DS fragments are more active than DS fragments of similar M(r), probably because of the extra flexibility endowed by glycol-split IdoA residues.

Animals↗
PubMed1993

Minimal sequence in heparin/heparan sulfate required for binding of basic fibroblast growth factor.

Experiments based on interaction in free solution between basic fibroblast growth factor (FGF-2) and saccharides related to heparin/heparan sulfate showed that the growth factor binds to heparin and to selectively glucosaminyl 6-O-desulfated heparin but poorly to iduronosyl 2-O-desulfated heparin. 2-O-sulfate groups thus are essential to the interaction, whereas 6-O-sulfates are not required nor do they interfere with FGF-2 binding. Comparison of various bound/nonbound oligosaccharides implicated a minimal pentasaccharide sequence for FGF-2 binding, with the structure: -hexuronic acid-glucosamine N-sulfate-hexuronic acid-glucosamine N-sulfate-iduronic acid 2-O-sulfate- (reducing terminus to the right). Such (overlapping) sequences are abundant in heparin, albeit heavily obscured by irrelevant O-sulfate groups, and occur also in heparan sulfate, with or without additional O-sulfates.

Animals↗
PubMed1990

Biosynthesis of heparin. Availability of glucosaminyl 3-O-sulfation sites.

Heparin preparations isolated from pig intestinal mucosa and from bovine lung were fractionated with regard to affinity for antithrombin. The resulting fractions, with high (HA) or low (LA) affinity for the proteinase inhibitor, were analyzed by 13C NMR or by identification of di- and tetrasaccharides obtained through deaminative cleavage with nitrous acid. Structural differences between corresponding HA and LA fractions were essentially restricted to minor constituents, in particular 3-O-sulfated glucosamine units that occurred (1 or 2 residues/chain) in all HA preparations but were scarce or absent in LA heparin. The HA fractions also consistently showed higher contents of nonsulfated iduronic acid and, to a lesser extent, N-acetylated glucosamine units than the LA fractions. The two tetrasaccharide sequences, -IdoA-GlcNAc(6-OSO3)-GlcA-GlcNSO3- and -IdoA-GlcNAc(6-OSO3)-GlcA-GlcNSO3(6-OSO3)- , recently implicated as part of the acceptor site for glucosaminyl 3-O-sulfate groups (Kusche, M., Bäckström, G., Riesenfeld, J., Petitou, M., Choay, J., and Lindahl, U. (1988) J. Biol. Chem. 263, 15474-15484), were identified in mucosal LA heparin; it was calculated that the preparation contained approximately one potential acceptor site/polysaccharide chain. Yet this material did not yield any labeled HA components on incubation with adenosine 3'-phosphate 5'-phospho-[35S]sulfate in the presence of glucosaminyl 3-O-sulfotransferase, solubilized from a mouse mastocytoma microsomal fraction. The failure to incorporate any 3-O-sulfate groups could conceivably be explained by the occurrence of a D-glucuronic rather than L-iduronic acid unit linked at the reducing ends of the above tetrasaccharide sequences. Alternatively, 3-O-sulfation may be restricted by other, as yet unidentified, inhibitory structural elements that are preferentially expressed in polysaccharide sequences selected for the generation of LA heparin.

Animals↗
PubMed1990

Conformer populations of L-iduronic acid residues in glycosaminoglycan sequences.

The 1H-n.m.r. 3J values for the L-iduronic acid (IdoA) residues for solutions in D2O of natural and synthetic oligosaccharides that represent the biologically important sequences of dermatan sulfate, heparan sulfate, and heparin have been rationalized by force-field calculations. The relative proportions of the low-energy conformers 1C4, 2S0, and 4C1 vary widely as a function of sequence and of pattern of sulfation. When IdoA or IdoA-2-sulfate units are present inside saccharide sequences, only 1C4 and 2S0 conformations contribute significantly to the equilibrium. This equilibrium is displaced towards the 2S0 form when IdoA-2-sulfate is preceded by a 3-O-sulfated amino sugar residue, and towards the 1C4 form when it is a non-reducing terminal. For terminal non-sulfated IdoA, the 4C1 form also contributes to the equilibrium. N.O.e. data confirm these conclusions. Possible biological implications of the conformational flexibility and the counter-ion induced changes in conformer populations are discussed.

Carbohydrate Conformation↗
PubMed1990

Anticoagulant and antithrombotic effects of chemically modified heparins and pentosanpolysulfate.

Pig mucosal heparin (GAG 98), in which the binding site for antithrombin had been inactivated by periodate oxidation (GAG 262), a supersulfated low-molecular-weight heparin (GAG 869), a low-molecular-weight heparin (Fragmin), and sodium pentosanpolysulfate have been investigated on their anticoagulant effects in vitro and ex vivo and in an animal thrombosis model in which rat mesenteric venules are damaged by defined laser energy. GAG 262 and pentosanpolysulfate had a markedly reduced anticoagulant effect compared to heparin, Fragmin, and the supersulfated low-molecular-weight heparin fragment. Similarily, the doses necessary to inhibit thrombus formation in the laser model were much higher for GAG 262 and for pentosanpolysulfate compared to heparin and the low-molecular-weight heparin Fragmin, but much lower for the supersulfated heparin fragment. The antithrombotic effect of the low-molecular-weight heparin Fragmin and the supersulfated heparin fragment after subcutaneous injection lasted much longer than the ex vivo detectable anticoagulant effect. Although some correlation between the antithrombotic and the anticoagulant effect in the laser model is evident, there seems to be no direct correlation between amount and duration of factor IIa or factor Xa inhibition and extent and duration of the inhibition of thrombus formation.

Animals↗
PubMed1990

Heparin structure.

Heparins used in therapy are largely constituted by sequences of the trisulfated disaccharide L-iduronic acid-2-sulfate----D-glucosamine-N,6-disulfate. These regular sequences are interrupted by undersulfated (occasionally, oversulfated) sequences containing D-glucuronic acid and N-acetylated D-glucosamine. Different heparin sequences are binding domains for heparin cofactors and plasma proteins. The active site for antithrombin is a specific pentasaccharide sequence containing 3-O-sulfated D-glucosamine. Heparin cofactor-II binds, less specifically, mostly to the regular sequences. The conformational flexibility of iduronic acid residues contributes to the binding versatility and to the 'biological reactivity' of heparin.

Carbohydrate Conformation↗
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↗