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Biomedical subjects

J C Lormeau

Publications and source records attributed to J C Lormeau.

At least 55 records · Page 3Linked to original sources
PubMed1992

Pharmacologic properties of an unfractionated heparin butyryl derivative with long-lasting effects.

This article reports on the pharmacologic properties of an O-acylated butyryl derivative (C4-UH) of unfractionated heparin (UH). In a purified system, the ability of C4-UH to catalyze the inhibition of thrombin and of factor Xa in the presence of antithrombin III was similar to that of UH. Addition of albumin (10 mg/ml) to the reagents reduced the antithrombin and antifactor Xa catalytic potency of C4-UH 68-fold and 20-fold, respectively, and did not alter those of UH. As judged from the prolongation of the activated partial thromboplastin time and the thrombin clotting time, the anticoagulant activities of C4-UH were two times weaker than those of UH. After calibration against UH, the antifactor Xa-specific and antithrombin-specific activities were two and 6.6 times lower, respectively. After bolus intravenous injection into rabbits, the apparent clearances of C4-UH were reduced 2.4 (antifactor Xa activity) and 3.2 times (antithrombin activity) in comparison with those of UH. This property accounted for the higher plasma concentrations generated during a constant infusion of the same dose. In the Wessler thromboplastin model, the minimum doses providing the maximum antithrombotic effect after bolus injection were equivalent for both compounds when expressed as antifactor Xa units; the duration of the antithrombotic effect of this derivative was prolonged, whereas the hemorrhagic potential was unaffected. This study opens a new concept for heparin derivatives having lower clearances and long-lasting effects. These properties could be linked to nonspecific binding of C4-UH to plasma proteins, thereby reducing the amount of free compound available to interact with antithrombin III.

Animals↗
PubMed1991

Antiproliferative effects of novel, nonanticoagulant heparin derivatives on vascular smooth muscle cells in vitro and in vivo.

The proliferation of vascular smooth muscle cells (VSMC) is strongly inhibited by whole heparin both in vitro and in vivo. To identify and characterize antiproliferative, but nonanticoagulant heparin derivatives, heparin fragments made by periodate treatment were produced and acylated with 2-, 4-, or 6-carbon chain lengths. In culture, the 4- and 6-carbon acylated compounds were more effective than whole heparin in inhibiting serum stimulated VSMC growth at equal mass or approximately equal mean molar concentrations. Further testing was performed in the rat carotid balloon injury model. Myointimal VSMC proliferation produced by balloon catheterization of rat carotid arteries was inhibited by the 4-carbon acylated compound as effectively as heparin at the same mass dose. Importantly, unlike heparin, the 4-carbon acylated compound had no anticoagulant effect in vivo. These experiments suggest nonanticoagulant, acylated heparin derivatives may have a pharmacologic role in preventing myointimal proliferative lesions that are responsible for failures of vascular surgeries and angioplasties.

Animals↗
PubMed1990

Antithrombin III: structural and functional aspects.

Antithrombin III is a plasma glycoprotein responsible for thrombin inhibition in the blood coagulation cascade. The X-ray structure of its cleaved form has been determined and refined to 3.2 A resolution. The overall topology is similar to that of alpha 1-antitrypsin, another member of the serpin (serine protease inhibitor) superfamily. The biological activity of antithrombin III is mediated by a polysaccharide, heparin. The binding site of this effector is described. A possible structural transition from the native to the cleaved structure is discussed.

Amino Acid Sequence↗
PubMed1990

Pharmacologic properties of a low molecular weight dermatan sulfate: comparison with unfractionated dermatan sulfate.

The anticoagulant, pharmacodynamic, and antithrombotic properties of a low molecular weight dermatan sulfate (molecular weight range 1600 to 8000, peak 4000) were compared with those of unfractionated dermatan sulfate (molecular weight range 12,000 to 45,000, peak 25,000). Anticoagulant activities were evaluated as the ability of the compounds to catalyze the inhibition of thrombin in the presence of heparin cofactor II in a purified system and to prolong the activated partial thromboplastin time or the thrombin clotting time of human and rabbit plasmas. On the basis of weight, low molecular weight dermatan sulfate was two times less potent than unfractionated dermatan sulfate. After bolus intravenous injection into rabbits, the volume of distribution of low molecular weight dermatan sulfate was 10 times larger than that of unfractionated compound, and the half-life of disappearance was two to four times longer despite a 1.4 to 2.3 times higher total clearance. The bioavailability of low molecular weight dermatan sulfate from its subcutaneous depot was 100%; it was absorbed faster from that depot than unfractionated dermatan sulfate. The antithrombotic activities of unfractionated and of low molecular weight dermatan sulfate were also examined with a Wessler-type model with tissue factor as the thrombogenic stimulus. When evaluated 3 minutes after a bolus intravenous injection, unfractionated dermatan sulfate was twice as active as low molecular weight dermatan sulfate on the basis of weight. With subcutaneous injection, 10 mg/kg of low molecular weight dermatan sulfate generated an activity in plasma equivalent to 5.6 micrograms/ml 1 hour later. This concentration was associated with a significant antithrombotic effect that lasted for less than 6 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Structural determinants of heparin's growth inhibitory activity. Interdependence of oligosaccharide size and charge.

The glycosaminoglycan heparin inhibits the growth of several cell types in vitro including smooth muscle cells and rat cervical epithelial cells. The commercially available heparin which has antiproliferative activity is a structurally heterogeneous polymer that undergoes extensive modifications during maturation. In this report we have performed structure-function studies on heparin's antiproliferative activity using three different cell types: both rat and calf vascular aortic smooth muscle cells and rat cervical epithelial cells. The minimal oligosaccharide size requirements for antiproliferative activity were determined for the three cell types by using oligosaccharide fragments of defined length prepared by nitrous acid cleavage and gel filtration and a synthetic pentasaccharide. The size requirements are similar but not identical for the different cell types. Hexasaccharide fragments are antiproliferative for all three cell types but the synthetic pentasaccharide inhibits the growth of only the rat and calf vascular aortic smooth muscle cells. The interdependence between size and charge for antiproliferative activity was investigated using chemically modified oligosaccharides as well as oligosaccharides prepared from heparin and separated into fractions of differing charge by ion-exchange chromatography. There is a strong interdependence between size and charge for antiproliferative activity. For example, increasing the charge of inactive tetrasaccharide fragments by O-oversulfation makes them antiproliferative whereas reducing the charge of active larger fragments causes them to loose their antiproliferative activity. Finally the importance of 2-O-sulfate glucuronic acid moieties for antiproliferative activity was investigated using heparin preparations that lack 2-O-sulfate glucuronic acid. These compounds possess antiproliferative activity indicating that 2-O-sulfate glucuronic acid is not required for antiproliferative activity.

Animals↗
PubMed1989

Heparin-derived oligosaccharides: affinity for acidic fibroblast growth factor and effect on its growth-promoting activity for human endothelial cells.

The minimal structural requirements for the interaction of heparin with acidic fibroblast growth factor (aFGF) were investigated. Oligosaccharides (tetra- to decasaccharides) obtained by nitrous acid depolymerisation of standard heparin were separated by affinity chromatography on Sepharose-immobilised aFGF. The shortest fragment retained by the affinity column at 0.2 M NaCl and eluted at 1 M NaCl was a "regular" hexasaccharide, a trimer of the most abundant disaccharide sequence in heparin. More complex octa- and decasaccharides were also retained by the column. The oligosaccharides eluted by 1 M NaCl from the affinity column ("high-affinity" oligosaccharides) and those washed from the column at 0.2 M NaCl ("low-affinity" oligosaccharides) were compared for their capacity to protect aFGF from proteolysis and to potentiate its mitogenic activity. At a low ionic strength, all oligosaccharides tested, except the "regular" disaccharide, protected aFGF against trypsin and collagenase digestion. At higher ionic strength (greater than 0.2 M NaCl), only high-affinity oligosaccharides showed a protective effect. The high-affinity oligosaccharides (hexa- to decasaccharides) potentiated the mitogenic activity of aFGF, as measured by [3H]thymidine incorporation into DNA of human fibroblasts. The effect of the oligosaccharides on human endothelial cell proliferation was more complex: inhibition of proliferation was observed in the presence of serum and low concentrations of aFGF (1-5 ng/ml) and potentiation in the presence of higher concentrations of aFGF. The potentiating effect increased as a function of molecular size of the heparin fragments and, for a given size, as a function of the anionic charge of the oligosaccharide. Our results suggest that inhibition of cell proliferation by heparin may result from interference with an autocrine basic FGF-like activity.

Cell Division↗
PubMed1988

Effects of increased sulfation of dermatan sulfate on its in vitro and in vivo pharmacological properties.

The in vitro and in vivo pharmacological properties of two oversulfated dermatan sulfate (DS) derivatives, S-DS1 and S-DS2, containing 2 and 3.7 sulfate groups/disaccharide unit respectively were compared to those of the parent DS (1 sulfate group/disaccharide unit). In a purified system the ability of S-DS1 and of S-DS2 to catalyse thrombin inhibition by heparin cofactor II was increased by ten- and seventeen-fold respectively. These compounds also had more potent anticoagulant activities in the activated partial thromboplastin time and the thrombin clotting time assays. Plasma immunodepleted in antithrombin III, heparin cofactor II and both cofactors allowed it to be demonstrated that these enhanced anti-coagulant activities were partly (S-DS1) or totally (S-SD2) independent of any plasma cofactors. In spite of these enhanced anticoagulant activities in vitro the oversulfated derivatives did not exhibit an increased antithrombotic activity in a thromboplastin Wessler type model. Moreover, at the doses investigated, S-DS2 had no antithrombotic effect. The influence of oversulfation on the pharmacokinetic pattern of DS was also investigated. As reported for unfractionated heparin, the biological activities generated after IV injection of high doses of S-DS1 and S-DS2 disappeared according to a concave-convex pattern. This may result from the higher affinities of S-DS1 and of S-DS2 toward endothelial cells in comparison with that of DS.

Animals↗
PubMed1988

Interaction of heparin and antithrombin III. The role of O-sulfate groups.

A synthetic pentasaccharide corresponding to the sequence involved in heparin for binding and activation of antithrombin III contains eight sulfate groups. The role of some of them in the interaction with the protein has been demonstrated through the study of fragments obtained from heparin. An approach based on the total chemical synthesis of heparin fragments allows us to provide new information on the O-sulfate groups borne by the iduronic acid and the glucosamine units that constitute the reducing-end disaccharide of the above pentasaccharide sequence. Although not strictly necessary for a weak interaction to take place, these two sulfates co-operate to express maximal activity. This suggests that they belong to a secondary sub-region of interaction with antithrombin III, the primary one being accounted for by other critical parts of the structure and particularly the trisaccharide sequence placed at the non-reducing end of the pentasaccharide.

Antithrombin III↗
PubMed1988

Studies on the structural requirements of heparin for the catalysis of thrombin inhibition by heparin cofactor II.

The structural requirements of heparin for the catalysis of thrombin inhibition by heparin cofactor II (HC II) were investigated. A series of well characterized heparin derivatives were prepared and their activities were measured using human thrombin in the presence of an excess of purified human HC II and, for comparison, antithrombin III (AT III). The 50% inhibitory concentrations of each derivative were calculated and compared with those of unmodified heparin. Heparin activity was strongly dependent on molecular weight (Mr) in a manner grossly comparable for the two inhibitors. High-Mr fractions were the most active. Below 10 kDa, the activity dropped rapidly. A minimum size of 26 residues appeared to be required for HC II activation (against 16-18 for AT III). Below 5 kDa, a residual activity two orders of magnitude lower than that of high-Mr species remained with HC II (but not with AT III). Heparin was selectively desulfated or oversulfated in the O- and/or N-position. When an N-acetyl group was substituted for the original N-sulfate in the glucosamine and the derivative was oversulfated in the O-position, a strong activity with HC activities with both inhibitors decreased when the overall sulfate content (i.e., the charge density) was reduced, and vice-versa. Carboxyl-reduced heparin was also inactive but activity could be restored after O-sulfation. Our results thus suggest that, unlike the case of AT III, no functional group in heparin is critical for optimal thrombin inhibition by HC II. Sulfate and carboxylate are important in as much as they contribute to the global charge of the molecule.

Antithrombin III↗
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

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

[A method of assay of the anti-factor Xa activity of CY 216].

The assay method of the anti-factor Xa activity of an heparin fragment: the CY 216, is described; it consists of an adaptation of the method of Yin et al. to an automatic clot timer. The anti-factor Xa unit of CY 216 is defined in this assay by comparison to the 4th International Standard of Heparin.

Biological Assay↗
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

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↗