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Biomedical subjects

M Petitou

Publications and source records attributed to M Petitou.

At least 91 records · Page 5Linked to original sources
PubMed1988

Is there a unique sequence in heparin for interaction with heparin cofactor II? Structural and biological studies of heparin-derived oligosaccharides.

To study the structural requirements in heparin for interaction with heparin cofactor II (HC II) we have analyzed the properties of oligosaccharide fractions obtained after digestion of heparin by heparinase and gel filtration. No activation of HC II was detected in the presence of di-, tetra-, hexa-, octa-, deca-, or do-decasaccharides. The hexasaccharide pool was fractionated by ion-exchange chromatography, and the structure of the major species, obtained in a homogeneous state, was investigated by NMR. All the resonances were unambiguously assigned using correlation by homonuclear and heteronuclear scalar coupling. The six monosaccharide residues of this hexasaccharide were thus easily identified. The sequence was established through two-dimensional nuclear Overhauser effect experiments. The results indicate that this product is a hexasaccharide recently described by Linhardt et al. (Linhardt, R. J., Rice, K. G., Merchant, Z. M., Kim, Y. S., and Lohse, D. L. (1986) J. Biol. Chem. 261, 14448-14454). However, we could not confirm the anticoagulant activity observed by these authors. Moreover, none of the individual components obtained after fractionation of the hexasaccharide pool was able either to activate HC II against thrombin or to inhibit HC II activation by heparin. Thus, our data led us to conclude that no unique sequence is involved in heparin for binding to HC II and inactivation of thrombin. The interaction merely results from the highly anionic character of heparin.

Antithrombins↗
PubMed1988

Inhibition of leucocyte elastase by heparin and its derivatives.

Leucocyte proteinases, e.g. leucocyte elastase and cathepsin G, are inhibited by heparin. The activities of pig pancreatic and Pseudomonas aeruginosa elastases are unaffected by this polysaccharide. Heparin derivatives of known Mr and degree of sulphation were isolated. The inhibition of leucocyte elastase by these oligosaccharides can be classified as tight-binding hyperbolic non-competitive. Ki values ranged from 40 nM to 100 microM and were found to be inversely correlated with the chain length of the oligosaccharides. Desulphated compounds lacked inhibitory potential towards leucocyte elastase. Over-O-sulphated di- and tetra-saccharides are more potent inhibitors than their over-N-sulphated counterparts. It is proposed that the therapeutic use of heparin and its derivatives could be extended to disease states such as emphysema and rheumatoid arthritis, where the role of leucocyte elastase has been clearly established.

Animals↗
PubMed1988

Standard heparin enhances the antithrombotic activity of dermatan sulfate in the rabbit but CY 216 does not.

Standard heparin (SH) and dermatan sulfate (DS) two glycosaminoglycans with different pharmacological targets are effective antithrombotic agents in the rabbit. We have investigated the antithrombotic activity of the association DS plus SH. It was found that doses as low as 25 micrograms/kg for DS and 10 micrograms/kg for SH were ineffective when injected separately but generated a high and significant antithrombotic activity when injected together. These results were confirmed when higher doses of each compound were delivered in association. Further experiments were performed to determine if the enhancement of the antithrombotic activity of DS by HS resulted from its anti-factor IIa or anti-factor Xa activity or from its moiety without affinity to AT III. A low molecular weight heparin (CY 216) with an anti-factor Xa/anti-factor IIa ratio of 5, the synthetic pentasaccharide bearing the minimum binding sequence to antithrombin III, and a low affinity fraction of SH to AT III did not increase the antithrombotic activity of DS; in contrast a high affinity fraction of SH to AT III had the same effect than SH. We conclude that the enhancement of the antithrombotic activity of DS by SH mainly results from its anti-factor IIa activity.

Animals↗
PubMed1988

Structure-function relationships in the inhibitory effect of heparin on complement activation: independency of the anti-coagulant and anti-complementary sites on the heparin molecule.

Fluid phase heparin inhibits formation of the classical and alternative pathway C3 convertase of complement in assays performed either with purified complement proteins or in whole serum. Experiments using oligosaccharides of homogeneous mol. wt obtained by mild nitrous hydrolysis of heparin, demonstrated that the inhibitory activity of heparin increased exponentially with mol. wt for fragments containing between 4 and 14 saccharidic units and that fragments of mol. wt above 4700 (greater than 14 saccharidic units) had a similar anti-complementary activity to that of native heparin. Fragments of homogeneous mol. wt (octasaccharides) separated by ion exchange chromatography on the basis of negative charges, exhibited increasing inhibitory activity with increasing sulfate content. Over-sulfation of fragments of defined mol. wt resulted in a constant enhancement of the relative capacity of each fragment species to inhibit formation of the classical and alternative pathway C3 convertases. A synthetic pentasaccharide representing the minimal critical sequence responsible for the binding of heparin to anti-thrombin III exhibited a similar inhibitory capacity on formation of the C3 convertases as another synthetic pentasaccharide that was devoid of anti-Xa activity. These studies contribute to define a minimal structure of the heparin molecule with C3b- and C4b-binding capacity and definitively establish the independency of the anti-coagulant and anti-complementary sites on the heparin molecule.

Blood Coagulation↗
PubMed1987

Contribution of 3-O- and 6-O-sulfated glucosamine residues in the heparin-induced conformational change in antithrombin III.

The role of 3-O- and 6-O-sulfated glucosamine residues within the heparin octasaccharide critical for biological activity, iduronic acid----N-acetylglucosamine 6-O-sulfate----glucuronic acid----N-sulfated glucosamine 3,6-di-O-sulfate----iduronic acid 2-O-sulfate----N-sulfated glucosamine 6-O-sulfate----iduronic acid 2-O-sulfate----anhydromannitol 6-O-sulfate, was determined by comparing its ability to bind antithrombin, induce a conformational change in this protease inhibitor as monitored by the enhancement of intrinsic fluorescence, and accelerate (at saturation) the interaction of this protein with human factor Xa. The octasaccharide produced a maximum 48% increase in intrinsic fluorescence at 37 degrees C and a rate of factor Xa inhibition of 6 X 10(5) M-1 s-1 as measured by stopped-flow fluorometry at 25 degrees C. The basal rate of the antithrombin-factor Xa interaction observed in the absence of oligosaccharide was 2 X 10(3) M-1 s-1. The synthetic pentasaccharide, consisting of residues 2-6, produced fluorescence enhancement and rate of inhibition equal to those of the octasaccharide. However, a similar pentasaccharide, identical in all respects except that it lacked the 3-O-sulfate on residue 4, produced less than a 5% fluorescence enhancement and a rate of factor Xa inhibition of 8 X 10(3) M-1 s-1. The tetrasaccharide consisting of residues 2-5 produced a 35% fluorescence enhancement and a rate of factor Xa inhibition of 3 X 10(5) M-1 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Heparin degradation in the endothelial cells.

We analyzed the molecular weight distribution of (125I) heparin fractions bound or internalized by human endothelial cells, using gel permeation chromatography. Our results showed that high molecular weight heparin chains are selectively bound and internalized by endothelial cells. Endocytosis is followed by depolymerization of internalized heparin. Chloroquine prevented depolymerization of internalized heparin, indicating that lysosomal enzymes are involved in this process. Degradation of high molecular weight heparin chains by endothelial cells may contribute to the inactivation of the drug, especially as concerns the antifactor IIa activity.

Biological Transport, Active↗
PubMed1987

Antithrombotic activity of a synthetic heparin pentasaccharide in a rabbit stasis thrombosis model using different thrombogenic challenges.

A synthetic pentasaccharide, representing the critical sequence required in heparin for binding to antithrombin III (AT III), produces strong anti-factor Xa activity in vitro in the presence of AT III and is devoid of any activity directed towards thrombin. This pentasaccharide provides a unique tool to study the question of whether an agent capable of inhibiting factor Xa but devoid of anti-factor IIa activity in vitro, has the capacity to produce an antithrombotic effect in vivo. We have previously demonstrated in a rabbit stasis thrombosis model using a human serum challenge, a significant antithrombotic effect of the pentasaccharide. This finding and discrepancies with some earlier reports on the antithrombotic actions of other oligosaccharide fragments, led us to extend these studies. Four modifications of the stasis thrombosis model were developed using the following thrombogenic challenges selected for their specified induction sites of thrombosis, thromboplastin, an activated prothrombin complex concentrate, a non-activated prothrombin complex concentrate administered simultaneously with Russell's viper venom, and factor Xa. Dose-dependent antithrombotic responses were obtained in all four systems with ED50 values between 25-43 ug/kg for pentasaccharide as compared to 16-47 ug/kg for heparin. Complete inhibition of induced thrombosis was obtained in all four systems for pentasaccharide. Ex vivo analysis revealed expected anti-factor Xa levels but no anti-factor IIa activity. It is concluded that an oligosaccharide with high anti-factor Xa activity and devoid of anti-factor IIa activity is capable of inhibiting thrombosis induced in rabbit stasis models, but that higher dosages than heparin are required for this effect.

Animals↗
PubMed1987

Structural characterization of sulfated glycosaminoglycans by fast atom bombardment mass spectrometry: application to heparin fragments prepared by chemical synthesis.

We report herein the results of f.a.b.-m.s. experiments conducted on synthetic fragments of glycosaminoglycans, one of them representing the pentasaccharidic sequence present in heparin and responsible for the binding to antithrombin III, and the others being related to this sequence. The results indicate that f.a.b.-m.s. can be very useful for the structural analysis of sulfated glycosaminoglycans. The relatively small amounts of sample required enable molecular characterization at physiologically significant levels. In contrast to the chondroitin sulfates, the heparin saccharides analyzed and reported here do not provide sequence information. The data indicate that glycosidic rupture is not a process competing with the much more facile loss of N-sulfite residues. Dominating the spectra are a series of molecular-weight-related ions (distributed to indicate the associated countercation composition), and fragments related directly to sulfite elimination. This f.a.b.-induced, facile loss of sulfite may impose limitations in molecular-weight analysis for the larger oligomers.

Carbohydrate Conformation↗
PubMed1986

Heparin fractions with high and low affinities to antithrombin III are cleared at different rates.

The clearance characteristics of standard heparin (SH) and its fractions with high and low affinity to antithrombin III (HAH and LAH respectively) were studied by injecting 125I-labelled SH, HAH and LAH intravenously into the rabbits in increasing doses. Serial blood samples were then collected from an indwelling cannula for measurement of clearance, based both on radioactivity and anticoagulant activity. For equivalent weights injected, the radioactivity of HAH was cleared more slowly from the circulation than that of either SH or LAH. The radioactivity of SH, in turn, was cleared more slowly than that of LAH. The clearance of HAH, measured both by radioactivity and by anticoagulant activity were similar, whereas the clearance of the radioactivity of SH was more rapid than its anticoagulant activity (i.e. anti-factor Xa activity). These observations suggest that the radioactivity clearance curves of SH reflect a "net" estimation of the more complex clearance curves of the different heparin moieties whereas the anticoagulant clearance curves of SH reflects the clearance of the anticoagulant activity of a specific heparin fraction, in this case, the clearance of the anti-factor Xa activity of HAH. These differences suggest that the HAH:LAH ratio is, at any given time, greater in vivo than the same ratio measured in vitro.

Animals↗
PubMed1986

Endothelial binding sites for heparin. Specificity and role in heparin neutralization.

The specificity of endothelial binding sites for heparin was investigated with heparin fractions and fragments differing in their Mr, charge density and affinity for antithrombin III, as well as with heparinoids and other anionic polyelectrolytes (polystyrene sulphonates). The affinity for endothelial cells was estimated by determining I50 values in competition experiments with 125I-heparin. We found that affinity for endothelial cells increases as a function of Mr and charge density (degree of sulphation). Binding sites are not specific receptors for heparin. Other anionic polyelectrolytes, such as pentosan polysulphates and polystyrene sulphonates, competed with heparin for binding to endothelial cells. Fractions of standard heparin with high affinity for antithrombin III also had greater affinity for endothelium. However, these two properties of heparin (affinity for antithrombin III and affinity for endothelial cells) could be dissociated. Oversulphated heparins and oversulphated low-Mr heparin fragments had lower anticoagulant activity and higher affinity for endothelial cells than did their parent compounds. Synthetic pentasaccharides, bearing the minimal sequence for binding to antithrombin III, did not bind to endothelial cells. Binding to endothelial cells involved partial neutralization of heparin. Bound heparin exhibited only 5% and 7% of antifactor IIa and antifactor Xa specific activity, respectively. In the presence of 200 nM-antithrombin III, and in the absence of free heparin, a limited fraction (approx. 30%) of bound heparin was displaced from endothelial cells during a 1 h incubation period. These data suggested that a fraction of surface-bound heparin could represent a pool of anticoagulant.

Antithrombin III↗
PubMed1986

Synthesis of heparin fragments. A chemical synthesis of the pentasaccharide O-(2-deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranosyl)-(1-4 )-O-(beta-D-glucopyranosyluronic acid)-(1-4)-O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-alpha-D-glu copyranosyl)-(1-4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-(1-4)-2-deoxy-2-sulfamido-6-O-sulfo-D-glucopyranose decasodium salt, a heparin fragment having high affinity for antithrombin III.

Known allyl 4,6-O-benzylidene-alpha-D-glucopyranoside was first converted into methyl (prop-1-enyl 2,3-di-O-benzyl-4-O-chloroacetyl-alpha-D-glucopyranosid)-uronate. Acid hydrolysis, followed by treatment with (bromomethylene)dimethyl-ammonium bromide, gave methyl (2,3-di-O-benzyl-4-O-chloroacetyl-alpha-D-glucopyranosyl bromide)uronate. Condensation of this bromide with 3-O-acetyl-1,6-anhydro-2-azido-2-deoxy-4-O-(methyl 2,3-di-O-benzyl-4-O-chloroacetyl-beta-D-glucopyranosyluronate)-bet a-D-glucopyranose. Acetolysis, followed by treatment with titanium tetrabromide, then gave 3,6-di-O-acetyl-2-azido-2-deoxy-4-O-(methyl 2,3-di-O-benzyl-4-O-chloroacetyl-beta-D-glucopyranosyluronate)-alp ha-D-glucopyranosyl bromide. Condensation of this bromide with benzyl 6-O-acetyl-3-O-benzyl-2-benzyloxy- carbonylamino-2-deoxy-4-O-(methyl 2-O-acetyl-3-O-benzyl-alpha-L- idopyranosyluronate)-alpha-D-glucopyranoside provided benzyl O-(methyl 2,3-di-O-benzyl-4-O-chloroacetyl-beta-D-glucopyranosyluronate)-(1- ---4)-O-(3,6-di-O-acetyl- -2-azido-2-deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2-O-acetyl-3-O-benzyl-alpha-L-idopyranosyluronate)-(1----4)-6-O-ac etyl-3-O- acetyl-3-O-benzyl-2-benzyloxycarbonylamino-2-deoxy-alpha-D-gluc opyranoside. O-Dechloroacetylation followed by condensation with 6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl bromide provided benzyl O-(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-beta-D-glucopyranosyl)- (1----4)-O-(methyl 2,3-di-O-benzyl-beta-D-glucopyranosyluronate)-(1----4)- O-(3,6-di-O-acetyl-2-azido-2-deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(m ethyl 2-O-acetyl-3-O-benzyl-alpha-L-idopyranosyluronate)-(1----4)-6-O-ac etyl-3-O- benzyl-2-benzyloxycarbonylamino-2-deoxy-alpha-D-glucopyranoside in 70% yield. O-Deacetylation followed by re-esterification, O-sulfation, saponification, catalytic hydrogenolysis, and N-sulfation gave the decasodium salt of O-(2-deoxy-2-sulfamido-6-O-sulfo-alpha-D- glucopyranosyl)-(1----4)-O-(beta-D-glucopyranosyluronic acid)-(1----4)-O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-alpha-D-gl ucopyranosyl)-(1----4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic+ ++ acid)-(1----4)-2-deoxy-2-sulfamido-6-O-sulfo-D-glucopyranose. This synthetic pentasaccharide binds to antithrombin III with an association constant similar to that of high-affinity heparin and elicits a potent anti-factor Xa activity in plasma.

Antithrombin III↗
PubMed1986

Structural determinants of the capacity of heparin to inhibit the proliferation of vascular smooth muscle cells. II. Evidence for a pentasaccharide sequence that contains a 3-O-sulfate group.

Earlier work from our laboratory demonstrated that heparin inhibited the proliferation of vascular smooth muscle cells in vivo and in vitro. Both anticoagulant and non-anticoagulant heparin species were equally effective as antiproliferative agents. Previous structure-function studies indicated that hexasaccharide and larger fragments retained antiproliferative activity, whereas tetra- and disaccharides were inactive. These experiments also suggested that both N- and O-sulfates of heparin were necessary for growth inhibitory capacity. In this paper, we have further analyzed the structural determinants of the antiproliferative activity of heparin. These experiments were done using synthetically prepared and therefore chemically defined heparin oligosaccharides. We present evidence that a pentasaccharide fragment retains antiproliferative activity, and that the 3-O-sulfate on the internal glucosamine residue is critical for growth inhibitory capacity of the pentasaccharide. We also show that heparins obtained from different manufacturers differ significantly in their ability to suppress smooth muscle cell proliferation.

Animals↗
PubMed1986

Respective role of antithrombin III and heparin cofactor II in the in vitro anticoagulant effect of heparin and of various sulphated polysaccharides.

The in vitro anticoagulant effects of standard heparin (SH) and of seven other sulphated polysaccharides (SPS) were investigated by measuring activated partial thromboplastin time (APTT) prolongation of normal plasma and of plasmas selectively depleted of antithrombin III (AT III), of heparin cofactor II (HC II) and of both heparin cofactors. This allowed the determination of the relative contribution of each of the two heparin cofactors to the SPS anticoagulant effect. The SPS varied in their relative activities as catalysts of thrombin inhibition by purified AT III or HC II. The anticoagulant activities of heparin and dermatan sulphate were primarily attributable to their ability to enhance thrombin inhibition by AT III and HC II respectively. Heparin had an additional minor anticoagulant activity which was independent of both AT III and HC II. Pentosan polysulphate, high molecular weight dextran sulphate, heparin with low affinity for AT III and a sulphated heparin derivative had weaker anticoagulant activities in normal plasma than standard heparin. The anticoagulant activities of these last four SPS in plasma depleted of both AT III and HC II were similar to their respective activities in normal plasma. This suggests that these SPS act by directly preventing thrombin generation rather than by enhancing thrombin inhibition.

Antithrombin III↗
PubMed1985

Contribution of monosaccharide residues in heparin binding to antithrombin III.

The importance of 3-O- and 6-O-sulfated glucosamine residues within the heparin octasaccharide iduronic acid(1)----N-acetylglucosamine 6-O-sulfate(2)----glucuronic acid(3)----N-sulfated glucosamine 3,6-di-O-sulfate(4)----iduronic acid 2-O-sulfate(5)----N-sulfated glucosamine 6-O-sulfate(6)----iduronic acid 2-O-sulfate(7)----anhydromannitol 6-O-sulfate(8) was determined by comparing with synthetic tetra- and penta-saccharides its ability to bind human antithrombin. The octasaccharide had an affinity for antithrombin of 1 X 10(-8) M (10.2 kcal/mol) measured by intrinsic fluorescence enhancement at 6 degrees C. The synthetic pentasaccharide, consisting of residues 2-6, had an affinity of 3 X 10(-8) M (9.6 kcal/mol). The same pentasaccharide, except lacking the 3-O-sulfate on residue 4, had an affinity of 5 X 10(-4) M (4.5 kcal/mol) measured by equilibrium dialysis. The tetrasaccharide, consisting of residues 2-5, bound antithrombin with an affinity of 5 X 10(-6) M (6.8 kcal/mol). The tetrasaccharide, consisting of residues 3-6, had an affinity of 5 X 10(-5) M (5.5 kcal/mol). Since the loss of either the 6-O-sulfated residue 2 or the 3-O-sulfate of residue 4 results in a 4-5 kcal/mol or a 40-50% loss in binding energy of the pentasaccharide, these two residues must be the major contributors to the binding and must be linked to the biologic activity of the octasaccharide.

Animals↗