Further Studies on the Mechanism for the Antithrombotic Effects of Naroparcil, an Orally Active Thioxyloside Compound

1999 ◽  
Vol 81 (06) ◽  
pp. 945-950 ◽  
Author(s):  
J. Theveniau ◽  
D. Coup ◽  
T. Grégoire ◽  
M. Vaillot ◽  
D. Dupouy ◽  
...  
Keyword(s):  
Specific Activity ◽  
Heparin Cofactor Ii ◽  
Antithrombotic Activity ◽  
Thrombin Inhibition ◽  
High Affinity ◽  
Antithrombotic Effects ◽  
Orally Active ◽  
The Relationship ◽  
In Vitro Specific Activity

SummaryThe antithrombotic β-D-xyloside, naroparcil, has previously been shown to induce a dose-related increase of circulating glycosaminoglycans (GAGs) together with an antithrombin activity (anti-IIa) via heparin cofactor II (HCII) in the rabbit. In order to go further in the mechanisms, the relationship between the antithrombotic activity, the HCII-mediated anti-IIa activity and the plasma GAG content was investigated. We showed that the in vitro specific activity on the inhibition of thrombin by HCII of the plasma GAG extract from naroparcil-treated rabbits was increased by a factor of 60 when compared to controls. In addition, the fractionation of the plasma GAG extract by affinity chromatography on immobilized HCII led to a more potent material whereas the low-affinity fraction was shown to be inactive in thrombin inhibition by HCII.The qualitative analysis of GAGs showed the presence of the ΔDi-4S DS disaccharide, undetectable in control, which accounted for 22% in the unfractionated GAG extract and for 60% in the high affinity fraction. In vitro experiments using immuno-depleted plasma in antithrombin III (ATIII), HCII or both, indicated that the anti-IIa activity of the plasma GAG extract from naroparcil-treated rabbits was mainly due to HCII potentialisation. The unfractionated GAG extract and the high affinity fraction were shown to be antithrombotic in a Wessler-based model in the rat, giving ED80 values of 610 UA/kg and 56 UA/kg respectively whereas the low-affinity fraction was devoid of any antithrombotic activity. These results show that the antithrombotic activity of naroparcil is dependent on modification in the plasma GAG profile which inactivates thrombin via the HCII.

INCREASED SULFATION IMPROVES THE ABILITY OF VESSEL WALL GLYCOSA-MINOGLYCANS TO REGULATE THROMBIN ACTIVITY AND PROTHROMBIN ACTIVATION IN PLASMA

1987 ◽  
Author(s):  
F A Ofosu ◽  
G J Modi ◽  
M A Blajchman ◽  
M R Buchanan ◽  
E A Johnson
Keyword(s):  
Vessel Wall ◽  
Dermatan Sulfate ◽  
Heparin Cofactor Ii ◽  
Thrombin Inhibition ◽  
Antithrombotic Effects ◽  
The Relationship ◽  
Functional Attribute ◽  
Prothrombin Activation

Studies have shown that dermatan sulfate (DS), heparan sulfate (HS) and chondroitin-4-sulfate (C4S), have antithrombotic properties. The sulfate to carboxylate ratios of these three glycosaminoglycans (GAGs) are approximately half that of heparin (HEP) and the gravimetric dose of each of the three GAGs required to achieve antithrombotic effects in vivo comparable to HEP can be 10 times or more than that of HEPT Since antithrombotic effects depend on the ability of a GAG to catalyse thrombin inhibition and/or to inhibit prothrombin activation, we determined the relationship between the extent of sulfation of various GAGs and their effects on these two reactions in normal plasma. In addition to the three GAGs, DS, HS and C4S were resulfated in vitro to yield DS-S, HS-S and C4S-S, each with a sulfate to carboxylate ratio comparable to that of heparin. As summarized below, increased sulfation improved the ability of a GAG to catalyse thrombin inhibition and to inhibit prothrombin activation. Increasing the degree of sulfation primarily improved the ability of a GAG to accelerate the inhibition of thrombin by heparin cofactor II. The degree of sulfation, therefore, appears to be an important functional attribute of the ability of vessel wall GAGs to regulate the formation and activity of thrombin in plasma.

scholarly journals Antithrombotic properties of a dermatan sulfate hexadecasaccharide fractionated by affinity for heparin cofactor II

Blood ◽  
1993 ◽  
Vol 81 (7) ◽  
pp. 1771-1777 ◽  
Author(s):  
P Sie ◽  
D Dupouy ◽  
C Caranobe ◽  
M Petitou ◽  
B Boneu
Keyword(s):  
Catalytic Activity ◽  
Dermatan Sulfate ◽  
Anticoagulant Activity ◽  
Gel Filtration ◽  
Heparin Cofactor Ii ◽  
High Affinity ◽  
Suitable Target ◽  
The Relationship

Abstract The relationship between the antithrombotic activity of dermatan sulfate (DS) in vivo and its catalytic effect on the inhibition of thrombin by heparin cofactor II (HC II) in vitro was investigated. DS was depolymerized by Smith degradation and the fragments obtained were separated by gel filtration. The fragment of minimal size with full catalytic activity was a hexadecasaccharide, which was further fractionated by affinity for immobilized HC II. Only a small proportion by weight (6.7%) was recovered in the high-affinity fraction, which had about 10 times more catalytic activity than the unfractionated oligosaccharide; the change in activity was primarily caused by the removal of inert materials, recovered in the low-affinity fraction. 1H- NMR spectra indicated strengthening of the signal given by Ido A (2S04) in the high-affinity fraction compared with that of the low-affinity fraction. The anticoagulant activity of the high-affinity fraction was exclusively HC II-dependent. The antithrombotic potency was evaluated in rabbits using the Wessler-thromboplastin model. Half-maximal prevention of thrombosis was obtained after injection of 250 micrograms/kg DS, of 500 micrograms/kg hexadecasaccharide, or of 60 micrograms/kg of its high-affinity fraction. The low-affinity fraction was ineffective at the highest dose tested (1,200 micrograms/kg) and did not potentiate the effect of the high-affinity fraction. These results show that the antithrombotic effect of DS is essentially dependent on HC II binding and activation and that HC II is therefore a suitable target for antithrombotic drugs.

scholarly journals Antithrombotic properties of a dermatan sulfate hexadecasaccharide fractionated by affinity for heparin cofactor II

Blood ◽  
1993 ◽  
Vol 81 (7) ◽  
pp. 1771-1777
Author(s):  
P Sie ◽  
D Dupouy ◽  
C Caranobe ◽  
M Petitou ◽  
B Boneu
Keyword(s):  
Catalytic Activity ◽  
Dermatan Sulfate ◽  
Anticoagulant Activity ◽  
Gel Filtration ◽  
Heparin Cofactor Ii ◽  
High Affinity ◽  
Suitable Target ◽  
The Relationship

The relationship between the antithrombotic activity of dermatan sulfate (DS) in vivo and its catalytic effect on the inhibition of thrombin by heparin cofactor II (HC II) in vitro was investigated. DS was depolymerized by Smith degradation and the fragments obtained were separated by gel filtration. The fragment of minimal size with full catalytic activity was a hexadecasaccharide, which was further fractionated by affinity for immobilized HC II. Only a small proportion by weight (6.7%) was recovered in the high-affinity fraction, which had about 10 times more catalytic activity than the unfractionated oligosaccharide; the change in activity was primarily caused by the removal of inert materials, recovered in the low-affinity fraction. 1H- NMR spectra indicated strengthening of the signal given by Ido A (2S04) in the high-affinity fraction compared with that of the low-affinity fraction. The anticoagulant activity of the high-affinity fraction was exclusively HC II-dependent. The antithrombotic potency was evaluated in rabbits using the Wessler-thromboplastin model. Half-maximal prevention of thrombosis was obtained after injection of 250 micrograms/kg DS, of 500 micrograms/kg hexadecasaccharide, or of 60 micrograms/kg of its high-affinity fraction. The low-affinity fraction was ineffective at the highest dose tested (1,200 micrograms/kg) and did not potentiate the effect of the high-affinity fraction. These results show that the antithrombotic effect of DS is essentially dependent on HC II binding and activation and that HC II is therefore a suitable target for antithrombotic drugs.

Further Studies on the Mechanisms for the Antithrombotic Effects of Sulfated Polysaccharides in Rabbits

1988 ◽  
Vol 60 (02) ◽  
pp. 188-192 ◽  
Author(s):  
F A Ofosu ◽  
F Fernandez ◽  
N Anvari ◽  
C Caranobe ◽  
F Dol ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Minimum Weight ◽  
Sulfated Polysaccharides ◽  
Pentosan Polysulfate ◽  
Heparin Cofactor Ii ◽  
Thrombin Inhibition ◽  
The Relationship ◽  
Prothrombin Activation

SummaryA recent study (Fernandez et al., Thromb. Haemostas. 1987; 57: 286-93) demonstrated that when rabbits were injected with the minimum weight of a variety of glycosaminoglycans required to inhibit tissue factor-induced thrombus formation by —80%, exogenous thrombin was inactivated —twice as fast in the post-treatment plasmas as the pre-treatment plasmas. In this study, we investigated the relationship between inhibition of thrombus formation and the extent of thrombin inhibition ex vivo. We also investigated the relationship between inhibition of thrombus formation and inhibition of prothrombin activation ex vivo. Four sulfated polysaccharides (SPS) which influence coagulation in a variety of ways were used in this study. Unfractionated heparin and the fraction of heparin with high affinity to antithrombin III potentiate the antiproteinase activity of antithrombin III. Pentosan polysulfate potentiates the activity of heparin cofactor II. At less than 10 pg/ml of plasma, all three SPS also inhibit intrinsic prothrombin activation. The fourth agent, dermatan sulfate, potentiates the activity of heparin cofactor II but fails to inhibit intrinsic prothrombin activation even at concentrations which exceed 60 pg/ml of plasma. Inhibition of thrombus formation by each sulfated polysaccharides was linearly related to the extent of thrombin inhibition achieved ex vivo. These observations confirm the utility of catalysis of thrombin inhibition as an index for assessing antithrombotic potential of glycosaminoglycans and other sulfated polysaccharides in rabbits. With the exception of pentosan polysulfate, there was no clear relationship between inhibition of thrombus formation and inhibition of prothrombin activation ex vivo.

The Venous Antithrombotic Profile of Naroparcil in the Rabbit

1994 ◽  
Vol 72 (06) ◽  
pp. 874-879 ◽  
Author(s):  
Jean Millet ◽  
Jocelyne Theveniaux ◽  
Neil L Brown
Keyword(s):  
Oral Administration ◽  
Bleeding Time ◽  
Dermatan Sulfate ◽  
Factor Xa ◽  
Bleeding Risk ◽  
Thrombin Time ◽  
Heparin Cofactor Ii ◽  
Antithrombotic Activity ◽  
Thrombin Inhibition ◽  
Maximal Reduction

SummaryThe venous antithrombotic profile of naroparcil or (4-[4-cyanoben-zoyl]-phenyl)-1.5-dithio-β-D-xylopyranoside was investigated in the rabbit following single i. v. and oral administration. Naroparcil attenuated thrombus development in a Wessler stasis model of venous thrombosis (jugular vein) employing bovine factor Xa as a thrombogenic stimulus giving ED50 values of 21.9 mg/kg and 36.0 mg/kg after respectively i. v. and oral administration. Venous antithrombotic activity was maximal 2-3 h after i. v. administration and 4-8 h after oral administration. Four hours after the oral administration of maximal antithrombotic (Wessler model, factor Xa) doses (100 and 400 mg/kg), naroparcil had no significant effect on bleeding time. In platelet poor plasma obtained from animals treated 4 h previously with various doses (25 to 400 mg/kg) of naroparcil, there was no detectable anti-factor Xa nor antithrombin activity. Similarly, naroparcil had no effect on APTT nor on thrombin time. A sensitized thrombin time (to about 35 s) was modestly but significantly increased following oral administration of the compound at 400 mg/kg. However, thrombin generation by the intrinsic pathway was reduced in a dose-related manner, maximal reduction being 65% at 400 mg/kg. The same doses of naroparcil enhanced the formation of thrombin/heparin cofactor II complexes at the expense of thrombin/antithrombin III complexes in plasma incubated with (125I)-human a-thrombin and induced the appearance of dermatan sulfate-like material in the plasma of treated rabbits, as measured by a heparin cofactor II-mediated thrombin inhibition assay. The results suggest that naroparcil could have a safe venous antithrombotic profile following oral administration (antithrombotic effect compared to bleeding risk). It is probable that part of the mechanism of action of the β-D-xyloside, naroparcil, is due to the induction of chondroitin sulfate-like glycosaminoglycan biosynthesis, this material being detectable in the plasma.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

STUDIES ON THE ANTITHROMBOTIC ACTION OF A LOW MOLECULARWEIGHT HEPARIN OBTAINED FROM BEEF MUCOSAL ORIGIN AND PEROXIDATIVE CLEAVAGE

1987 ◽  
Author(s):  
P Bianchini ◽  
R Nonn ◽  
J Fareed ◽  
J M Walenga ◽  
A Kumar
Keyword(s):  
Molecular Weight ◽  
Structural Integrity ◽  
Factor Xa ◽  
Inhibitory Effects ◽  
Heparin Cofactor Ii ◽  
At Iii ◽  
Antithrombotic Effects ◽  
In Vitro Systems ◽  
Mean Molecular Weight

We have studied a low molecular weight heparin (LMWH) obtained by acontrolled peroxidative depolymerization of beef mucosal heparin (OP 2123, Opocrin, Corlo, Italy). This product was found to be significantly different from other LMWHs in that it exhibits the same COO-/SO2- ratios as unfractionated heparin, contains reducing end groups composed of 2-sulfated iduronic acid or 6-disulfated glucosamine and retains an identical structural integrity as that of native heparin. As opposed to most other LMWHs the oligosaccharide components of OP 2123 consist of homogeneous progressive units. In addition, the relative amount of AT-IIIaffinity components in OP 2123 were 20-30% less than other LMWHs. OP 2123 has a mean molecular weight of 6200 daltons with a potency of 90 anti-factor Xa U/mg and 68 USP U/mg. This agent produced strong antithrombotic actions in a rabbit stasis model against both an activated prothrombin complex and a prothrombin complex concentrate/Russell's viper venomcombination (ED50:(IV) 30-70 ug/kg;(SC) 0.6-1.5 mg/kg). The antithrombotic effects were comparable to other LMWHs in normal rabbits: however, in AT III depleted rabbits (immunodepleted and y thrombin depleted), OP 2123 produced stronger antithrombotic effects than most other LMWHs. The in vitro systems in contrast to other LMWHs, CP 2123 produced stronger inhibitory effects in AT III depleted plasma as measured by fibrinopeptide A generation and amidolytic anti-factor Xa and anti-factor Ila methods. The relative heparin cofactor II activity as measured by amidolytic method was also found to be higher than with most LMWHs. These results suggest that OP 2123, unlike most LMWHs, non AT III mediated actions play a major role in themendiation of the antithrombotic actions.

STRUCTURE ACTIVITY RELATIONSHIPS OF DERMATAN SULFATE : EFFECT OF MOLECULAR SIZE AND CARBOXYL GROUP ESTERIFICATION ON HEPARIN C0FACT0R-II ACTIVATION

1987 ◽  
Author(s):  
J Mardiguian ◽  
M Corgier ◽  
M Jouany
Keyword(s):  
Molecular Weight ◽  
Dermatan Sulfate ◽  
Methyl Esters ◽  
Molecular Size ◽  
Carboxyl Groups ◽  
Gel Chromatography ◽  
Carboxyl Group ◽  
Heparin Cofactor Ii ◽  
High Affinity

Dermatan is a high molecular weight glycosaminoglycan which has been shown to enhance the inhibition of thrombin by heparin-cofactor II. The aim of this study was to establish the influence of the molecular size and the role of the carboxyl group on the in vitro activity of Dermatan Sulfate. Pig skin Dermatan Sulfate was fractionated according to molecular size by gel-chromatography on Ultrogel Ac 44. Each fraction was characterized by its sulfur content and by its mean molecular weight measured on a TSK - 4000 column in reference to standard heparin fractions. Methyl esters of the unfractionated Dermatan Sulfate with varying degree of esterification, where prepared via activation of the carboxyl groups with a carbodiimide and reaction with methanol. The results of this study show that the heparin - cofactor II mediated anti-thrombin activity of Dermatan Sulfate is increasing with the molecular weight and is abolished by esterification of the carboxyl groups. Moreover, it can be speculated that each fraction contains the same amount of high affinity fraction and that, like heparin, the potency of the high affinity component is increasing with the molecular weight.

Impact of Antithrombin Deficiency on Efficacies of DU-176b, a Novel Orally Active Direct Factor Xa Inhibitor, and Antithrombin Dependent Anticoagulants, Fondaparinux and Heparin.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1874-1874 ◽  
Author(s):  
Toshio Fukuda ◽  
Yuko Honda ◽  
Chikako Matsumoto ◽  
Nobutoshi Sugiyama ◽  
Tadashi Matsushita ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Factor Xa ◽  
In Vitro Study ◽  
Antithrombotic Effects ◽  
At Deficiency ◽  
Fxa Inhibitor ◽  
Orally Active ◽  
Relative Potencies

Abstract Antithrombin (AT) is a major physiological inhibitor of coagulation factors, primarily inhibiting thrombin and factor Xa (FXa). Binding of heparin and its related pentasaccharides, fondaparinux, to AT dramatically accelerates inhibition of thrombin and FXa. Entire AT-dependency of heparins may result in decreased anticoagulant effects in patients with inherited or acquired AT deficiencies. Objectives: We have developed an orally active direct (i.e. AT-independent) FXa inhibitor, DU-176b. The objectives of this study were to examine the anticoagulant and antithrombotic effects of DU-176b, fondaparinux, and heparin in heterozygous AT deficient (AT+/−) mice (Refs 1, 2), and to determine the impact of AT deficiency on the efficacies of these anticoagulants. Methods: [In vitro study] Plasma obtained from wild type (AT+/+, C57BL/6J) and AT+/− mice were subjected to measurement of levels of AT antigen and activity. The anticoagulant effects on prothrombin time (PT) and activated partial thromboplastin time (APTT) was measured and the drug concentrations were calculated required to double the clotting time (CT2). [In vivo study] Male AT+/+ and AT+/− mice were fasted over night. Thrombosis was induced in the inferior vena cava by applying filter paper (1 x 5 mm) presoaked in 15% FeCl3 for 10 min. Thrombus was removed 60 min after FeCl3 treatment and its protein content was assessed by Bradford method. DU-176b was orally administered 60 min before, fondaparinux was given s.c. 30 min before, and heparin was injected into the jugular vein 3 min before thrombus induction. Relative potencies of antithrombotic effects in AT+/− mice to those in AT+/+ mice were analyzed by parallel line assay. Results: [In vitro study] Plasma levels of AT antigen and activity in AT+/− mice were deceased to 40% compared with AT+/+ plasma. PT-CT2 of DU-176b was 0.72 μM in AT+/+ plasma and 0.74 μM in AT+/− plasma, respectively, indicating that anticoagulant activity of the direct FXa inhibitor was not affected by heterozygous AT deficiency. APTT-CT2 of fondaparinux and heparin in AT+/+ plasma was 3.8 μM and 14 mU/mL, respectively, whereas APTT-CT2 in AT+/− plasma was 9.2 μM and 20 mU/mL, respectively. Therefore, anticoagulant activities of such AT-dependent inhibitors were attenuated in AT+/− plasma. [In vivo study] All three anticoagulants inhibited venous thrombus formation of AT+/+ mice in dose-dependent manners. In AT+/− mice, the antithrombotic effects of fondaparinux and heparin were less potent than those in AT+/+ mice. In contrast, DU-176b prevented thrombus formation equipotently in both mice. Relative potencies of DU-176b, fondaparinux and heparin were 0.84, 0.40, and 0.70, respectively. Conclusion: DU-176b exerts a comparable antithrombotic effect even in individuals with low plasma AT antigens and activities. Thus, DU-176b may be prioritized over AT-dependent agents for use at the fixed dose in patients with lower plasma AT concentrations.

THE RELATIONSHIP BETWEEN CORTICOSTERONE SYNTHESIS FROM ENDOGENOUS PRECURSORS AND FROM ADDED RADIOACTIVE PRECURSORS BY RAT ADRENAL TISSUE IN VITRO

1966 ◽  
Vol 36 (3) ◽  
pp. 231-238 ◽  
Author(s):  
G. P. VINSON
Keyword(s):  
Specific Activity ◽  
Single Point ◽  
Biosynthetic Pathways ◽  
Activity Change ◽  
Adrenal Tissue ◽  
Specific Activities ◽  
The Relationship ◽  
Synthesis Of Hormones ◽  
Venous Plasma

SUMMARY A kinetic study of corticosterone recovered during incubation of rat adrenal tissue with [4-14C]progesterone and [16-3H]pregnenolone shows that both its 3H: 14C ratio and its specific activity change with time throughout incubation. Corticosterone is itself metabolized, and hypotheses based on the rate of production of corticosterone may be therefore deceptive when the amount is measured at a single point in time. Thus disparities between the specific activities of products and intermediates may be expected which alone do not necessarily indicate differences between the biosynthetic pathways involved in production from endogenous precursors and those from added radioactive precursors. Other experiments suggest that steroids in incubation media, in concentrations comparable with those found in adrenal venous plasma, do not inhibit the continued synthesis of hormones.

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