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J Choay

Publications and source records attributed to J Choay.

At least 37 records · Page 2Linked to original sources
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

Crystallization and preliminary crystallographic data for bovine antithrombin III.

Crystals of bovine antithrombin III were obtained in the presence of metal ions with ammonium sulphate as precipitating agent. Crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2 with cell parameters a = b = 91.4 A, c = 383.1 A; there are two molecules per asymmetric unit. Electrophoresis experiments and amino acid sequence analysis of the N-terminal part of redissolved crystals suggest that the protein molecules are cleaved at the active site.

Animals↗
PubMed1989

Free factor Xa is on the main pathway of thrombin generation in clotting plasma.

The effect of a synthetic pentasaccharide that specifically causes the inactivation of factor Xa on the development of prothrombinase activity in human plasma was monitored using four triggers of coagulation: (a) human brain thromboplastin; (b) contact activation; (c) factor X activating enzyme complex; (d) prothrombin activating enzyme complex. Inhibition was similar with the triggers a, b and c. With prothrombinase (d), the inhibition strongly decreased with increasing amounts of factor Va present. This indicates that only free factor Xa is inhibited. Because both the intrinsic pathway (b) and the extrinsic pathway (a) are inhibited by the pentasaccharide, we conclude that free factor Xa plays a rate-limiting role in the pathways, so that there is no reason to postulate the existence of 'supercomplexes' consisting of factors IXa, VIIIa, X(a), Va and prothrombin adsorbed on the same phospholipid particle (intrinsic system) or factor VII(a), X(a), Va and prothrombin adsorbed on tissue thromboplastin (extrinsic system).

Blood Coagulation↗
PubMed1989

The action of a synthetic pentasaccharide on thrombin generation in whole plasma.

We investigated the effect on thrombin generation in plasma of the pentasaccharide that represent the AT III/binding site in heparin. This compound has no effect on the breakdown of thrombin in plasma. It dose-dependently inhibits the formation of thrombin in both the intrinsic and the extrinsic pathway. If coagulation is triggered by the complete prothrombinase complex (phospholipid--factor Va--factor Xa) under conditions in which the large majority of factor Xa is bound to the complex, the inhibition of prothrombinase activity is only minor. If no factor Va is present or if the prothrombinase activity is triggered by adding complete tenase (PL-FVIIIa-FIXa) or incomplete tenase (PL-FIXa) to the plasma the inhibition by pentasaccharide is of the same magnitude as that in the intrinsic or extrinsic system. We conclude that the pentasaccharide inhibits blood coagulation by catalysing the inactivation of free factor Xa. In contrast to classical heparin it does inhibit the peak of thrombin formation in platelet rich plasma, probably because it is less subject to inactivation by heparin binding proteins from platelets than classical heparin is.

Enzyme Activation↗
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

Examination, by 1H-n.m.r. spectroscopy, of the binding of a synthetic, high-affinity heparin pentasaccharide to human antithrombin III.

Binding of a synthetic, high-affinity heparin pentasaccharide and of intact heparin to both native and elastase-modified human antithrombin III have been examined by 1H-n.m.r. spectroscopy. The pentasaccharide perturbs many protein resonances in the same way as does intact heparin. There are, however, differences that seem to arise both from fewer contacts in the heparin binding-site when the pentasaccharide binds and from dissimilar conformational changes in the protein. The resonance of the H-2 atom of the histidine, considered to be the N-terminal residue and to be located in the heparin binding-site, is strongly perturbed by heparin binding both to native and modified antithrombin. The pentasaccharide has little effect on this histidine in either protein. Resonances from two of the remaining four histidine units are sensitive to longer-range conformational changes, and show differences between binding of the two heparin species both in native and modified ATIII. It is concluded that the pentasaccharide only partly fills the heparin binding-site and does not produce a conformational change identical to that caused by intact heparin. This is particularly significant as regards the mechanism of action of heparin, because the synthetic pentasaccharide activates ATIII towards Factor Xa, but not towards thrombin.

Antithrombin III↗
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↗
PubMed1989

Pharmacological properties of dermatan sulfate, of a low molecular weight dermatan sulfate and of two oversulfated derivatives.

The pharmacological properties of unfractionated dermatan sulfate (U-DS, mean MW 25 kd, range 12-45 kd) of a low molecular weight fraction (LMW-DS, mean MW 4 kd range 1.6-8 kd), and of 2 oversulfated derivatives (S-DS1 and S-DS2, 2 and 3.8 sulfate groups per disaccharide units) were investigated. In a purified system, LMW-DS, S-DS1 and S-DS2 were respectively 0.5, 10 and 17 times more potent than U-DS to catalyse thrombin inhibition by heparin cofactor II. Identical differences were observed for the respective anticoagulant activities (activated partial thromboplastin time and thrombin clotting time). After bolus IV injection of increasing doses the pharmacokinetic parameters of U-DS were slightly dose dependent, and the total clearance of LMW-DS was, on the average, 2 times higher. The patterns of disappearance of S-DS1 and S-DS2 were strongly dose dependent and became concave-convex, suggesting different mechanisms of clearance. After SC injection, the bioavailability was less than 50% for U-DS and at least 100% for LMW-DS. The antithrombotic activity (Wessler-thromboplastin model) of LMW-DS was 2 timer lower than that of U-DS. In contrast to their in vitro (and ex vivo) enhanced anticoagulant activities, the antithrombotic potency of S-DS1 was identical to that of U-DS, while, at the same doses S-DS2 was devoid of any activity.

Animals↗
PubMed1988

Importance of a 3-O-sulfate group in a heparin pentasaccharide for antithrombotic activity.

Previous theoretical and experimental evidence led to the formulation of a specific pentasaccharide structure which represents the site in heparin for binding to antithrombin III. This pentasaccharide was subsequently synthesized. A pentasaccharide of the same structure but lacking only the sulfate group on the hydroxyl group of the middle glucosamine (position C-3) was also synthesized to test the structure - activity relationships. Previous biochemical studies showed the 3-O-desulfated pentasaccharide to have a low affinity binding to AT III and to be devoid of the high anti-factor Xa activity characteristic of the pentasaccharide. Our in vivo studies, in a venous stasis thrombosis model proved the 3-O-desulfated pentasaccharide, at equigravimetric dosages, to be devoid of the antithrombotic activity previously reported for the pentasaccharide. These studies confirm the fact that inhibition of factor Xa at a high level of activity produces an antithrombotic effect.

Animals↗
PubMed1988

Influence of heparin fragments on the biological activities of elastase(s) and alpha 1 proteinase inhibitor.

The in vitro and in vivo effects of heparin fragments (CY 216; CY 222) towards elastase(s) and elastase inhibitor (alpha 1 Pi) were studied. Heparin as well as its lower Mr fragments were shown to inhibit rat leucocyte elastase. The interaction between this enzyme and heparins appears to occur via electrostatic forces. Porcine pancreatic elastase is unaffected by heparin(s) but CY 216 and CY 222 could partly abolish the hydrolytic activity of hamster serum on Suc-Ala-Ala-Ala-N-PhNO2. N desulphated N acetylated CY 142 and CY 143 had no effect. CY 216 and CY 222 decreased in vitro the inhibitory potential of alpha 1 proteinase inhibitor (alpha 1 Pi) as well as the elastase inhibitory capacity of hamster serum. Maximum effect (30% decrease) was observed at ng concentrations of CY 216 and CY 222. Their N desulphated N acetylated counterparts (CY 142 and CY 143), but not heparin, exhibited similar effects. CY 216 and CY 222 were administered daily subcutaneously to hamsters and blood was collected 1, 2, 4, 7 and 24 hr after treatment for determining both serum elastase activity (E.A.) and serum elastase inhibitory capacity (E.I.C.). E.A. levels dropped by 30% 2 hr after CY 216 or CY 222 injection but returned to original values 4-7 hr later. This effect is independent of the duration of the treatment. Hamster serum E.I.C. was significantly increased (greater than 30%) after 3-4 weeks of treatment with CY 216 and CY 222. These findings point towards the potential use of these compounds in elastase-related diseases such as emphysema.

Acetylation↗
PubMed1988

Biosynthesis of heparin. O-sulfation of the antithrombin-binding region.

The antithrombin-binding region in heparin is a pentasaccharide sequence with the predominant structure GlcNAc(6-OSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-IdoA -(2-OSO3)-GlcNSO3(6-OSO3) (where GlcA and IdoA represent D-glucuronic and L-iduronic acid, respectively), in which the 3-O-sulfate residue on the internal glucosaminyl unit is a marker group for this particular region of the polysaccharide molecule. A heparin octasaccharide which contained the above pentasaccharide sequence was N/O-desulfated and re-N-sulfated and was then incubated with adenosine 3'-phosphate 5'-phospho[35S]sulfate in the presence of a microsomal fraction from mouse mastocytoma tissue. Fractionation of the resulting 35S-labeled octasaccharide on antithrombin-Sepharose yielded a high affinity fraction that accounted for approximately 2% of the total incorporated label. Structural analysis of this fraction indicated that the internal glucosamine unit of the pentasaccharide sequence was 3-O-35S-sulfated, whereas both adjacent glucosamine units carried 6-O-[35S]sulfate groups. In contrast, the fractions with low affinity for antithrombin (approximately 98% of incorporated 35S) showed no consistent O-35S sulfation pattern and essentially lacked glucosaminyl 3-O-[35S]sulfate groups. It is suggested that the 3-O-sulfation reaction concludes the formation of the antithrombin-binding region. This proposal was corroborated in a similar experiment using a synthetic pentasaccharide with the structure GlcNSO3(6-OSO3)-GlcA-GlcNSO3(6-OSO3)-Id oA (2-OSO3)-GlcNSO3(6-OSO3) as sulfate acceptor. This molecule corresponds to a functional antithrombin-binding region but for the lack of a 3-O-sulfate group at the internal glucosamine unit. The 35S-labeled pentasaccharide recovered after incubation bound with high affinity to antithrombin-Sepharose and contained a 3-O-[35S]sulfate group at the internal glucosamine residue as the only detectable labeled component. The use of this pentasaccharide substrate along with the affinity matrix provides a highly specific assay for the 3-O-sulfotransferase.

Animals↗
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

Binding of heparin to antithrombin III: a chemical proof of the critical role played by a 3-sulfated 2-amino-2-deoxy-D-glucose residue.

Known methyl (prop-1-enyl 2,3-di-O-benzyl-alpha-D-glucopyranosid)uronate was first converted into methyl (prop-1-enyl 2,3-di-O-benzyl-4-O-levulinyl-alpha-D-gluco-pyranosid)uro nat e. Acid hydrolysis, followed by treatment with (bromomethylene)-dimethylammonium bromide, gave methyl (2,3-di-O-benzyl-4-O-levulinyl-alpha-D-glucopyranosyl bromide)uronate. Condensation of this bromide with 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-beta-D-glucopyranose gave 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-4-O-(methyl 2,3-di-O-benzyl-4-O- levulinyl-beta-D-glucopyranosyluronate)-beta-D-glucopyranose. Acetolysis, followed by selective anomeric O-deacetylation and treatment with (bromomethylene)dimethylammonium bromide then gave 6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-(methyl 2,3-di-O-benzyl-4-O-levulinyl -beta-D-glucopyranosyluronate)-alpha-D-glucopyranosyl bromide. Condensation of this bromide with benzyl 6-O-acetyl-3-O-benzyl-2-benzyloxycarbonylamino-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-levulinyl-beta-D- glucopyranosyluronate)-(1----4)-O-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy - alpha-D-glucopyranosyl)- (1----4)-O-(methyl 2-O-acetyl-3-O-benzyl-alpha-L-idopyranosyluronate)-(1----4)- 6-O-acetyl-3-O-benzyl-2-benzyloxycarbonylamino-2-deoxy-alpha-D-glu copyranoside. Removal of the levulinyl group 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-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2,3-di- O-benzyl-beta-D-glucopyranosyluronate)-(1----4)-O-(6-O-acetyl-2-azido-3- O- benzyl-2- deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2-O-acetyl-3-O-benzyl-alpha-L- idopyranosyluronate)-(1----4)-6-O-acetyl-3-O-benzyl-2-benzyloxycarbon ylamino-2- deoxy-alpha-D-glucopyranoside in 78% yield. O-Deacetylation followed by re-esterification, O-sulfation, catalytic hydrogenolysis, saponification, and N-sulfation gave the non-sodium salt of O-(2-deoxy-6-O-sulfo-2-sulfoamino-alpha-D-glucopyranosyl)-(1----4) -O- (beta-D-glucopyranosyluronic acid)-(1----4)O-(2-deoxy-6-O-sulfo-2-sulfoamino- alpha-D-glucopyranosyl)-(1----4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)- (1----4)-2-deoxy-6-O-sulfo-2-sulfoamino-D-glucopyranose. This synthetic pentasaccharide neither binds to antithrombin III nor induces anti-factor Xa activity.

Alcohols↗