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.

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.

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.

Selective and Sustained Inhibition of Surface-bound Thrombin Activity by Intimatan/Heparin Cofactor II and Its Relevance to Assessing Systemic Anticoagulation In Vivo, Ex Vivo and In Vitro

2001 ◽  
Vol 86 (09) ◽  
pp. 909-913 ◽  
Author(s):  
Glenn Maclean ◽  
Stephanie Brister ◽  
Michael Buchanan
Keyword(s):  
Vessel Wall ◽  
Specific Activity ◽  
Ex Vivo ◽  
Fluid Phase ◽  
Inhibitory Effects ◽  
Heparin Cofactor Ii ◽  
Thrombin Activity ◽  
Sustained Inhibition

SummaryWe compare the relative activities of surface-bound and fluid-phase thrombin and their inhibition by heparin and Intimatan, a novel heparin cofactor II (HCII) agonist. In vitro, we compared the observed amidolytic activities of fluid-phase and surface-bound thrombin with the expected activities based upon 125I-specific activity. In vivo, we compared the inhibitory effects of heparin and Intimatan on thrombin activity bound to injured vessel walls. In vitro, the correlations between observed and expected activities of fluid-phase and surface-bound thrombin, were: r = 0.9974, p < 0.001; and r = 0.9678, p < 0.001; respectively. In vivo, injured vessel wall surface-bound thrombin activity persisted for > 24 h. This activity was not inhibited by heparin, but was inhibited by Intimatan, p < 0.001.We conclude that surface-bound thrombin is as active as fluid-phase thrombin and remains protected from inhibition by heparin, thereby contributing to vessel wall thrombogenicity following injury. In contrast, surface-bound thrombin is inhibited by Intimatan, thereby effectively decreasing vessel wall thrombogenicity following injury in vivo.

TISSUE THROMBOPLASTIN-INDUCED REVERSIBLE DIC AS AN IN VIVO MODEL OF THROMBIN GENERATION AND INHIBITION

1987 ◽  
Author(s):  
G A Marbet ◽  
B Zbinden ◽  
P Satiropas ◽  
F Duckert
Keyword(s):  
Thrombin Generation ◽  
Dermatan Sulfate ◽  
Order Rate Constant ◽  
Thrombin Inhibitors ◽  
In Vivo Model ◽  
Heparin Cofactor Ii ◽  
First Order ◽  
Thrombin Inhibition ◽  
Tissue Thromboplastin

We have tried a kinetical approach to characterize the dynamics of thrombin generation, thrombin action and heparin-enhanced inhibitors in vivo. Integrals (φ) of free thrombin concentration over time were calculated from fibrinogen decrease to characterize tissue thromboplastin-induced DIC in the dog. DIC with φ < 8nMmin was reversible. Dynamic thrombin inhibition (DTI), measured as pseudo-first order rate constant of thrombin inactivation by plasma (baseline DTI = 6.31± 0.79/min, n=30) increased to 68.69±59.98/min (n=7) with heparin (H) and to 22.48±14.91/min (n=5 with pentosan polysulfate (PPS). DTI correlated significantly with heparin doses (p< 0.002), and with the prolongation of APTT (p<0.0 2) and of prothrombin time (p < 0.05). The efficiency β of tissue thromboplastin (Tp) to trigger DIC ( φ per ml TP) was reduced from β =3.81±3.16nMmin/ml to 0.74±0.52nMmin/ml by H (p < 0.01) and to 1.16±1.10nMmin/ml (n.s.) by PPS. As expected from the product DTI.φ = 23.4 ± 13.8nM there was no detectable decrease of prothrombin, of the combined activity of antithrombin III + heparin cofactor II (ATHC) or of heparin cofactor II (HC, specific assay by dermatan sulfate activation) in reversible DIC without glycosaminoglycan protection. However, increasing doses of TP at constant PPS protection induced a statistically significant and persistent decrease of prothrombin by 17.6±9.91 and of HC by 20.4±8.81 indicating HC enhancement by PPS in vivo. The model is suitable for the study of glycosa-minoglycans and thrombin inhibitors.

Unbalanced Effects of Dermatan Sulfates with Different Sulfation Patterns on Coagulation, Thrombosis and Bleeding

2001 ◽  
Vol 86 (11) ◽  
pp. 1215-1220 ◽  
Author(s):  
C. P. Vicente ◽  
P. Zancan ◽  
L. L. Peixoto ◽  
R. Alves-Sá ◽  
F. S. Araújo ◽  
...  
Keyword(s):  
Dermatan Sulfate ◽  
Thrombus Formation ◽  
Anticoagulant Activity ◽  
Sulfated Polysaccharides ◽  
Heparin Cofactor Ii ◽  
Iduronic Acid ◽  
Bleeding Effect ◽  
Mammalian Counterpart

SummaryWe compared the anticoagulant, antithrombotic and bleeding effects of highly sulfated dermatan sulfates from invertebrates and their mammalian counterpart. An invertebrate dermatan sulfate containing 2-O-sulfated α-L-iduronic acid and 4-O-sulfated N-acetyl-β-D-galactosamine residues is a potent anticoagulant due to a high heparin cofactor II activity. It inhibits thrombin due to the formation of a covalent complex with heparin cofactor II, as in the case of mammalian dermatan sulfate, but the effect occurs at lower concentrations for the invertebrate polysaccharide. Surprisingly, the invertebrate dermatan sulfate has a lower potency to prevent thrombus formation on an experimental model and a lower bleeding effect in rats than the mammalian dermatan sulfate. In contrast, another invertebrate dermatan sulfate, also enriched in 2-O-sulfated α-L-iduronic acid, but in this case sulfated at O-6 position of the N-acetyl-β-D-galactosamine units, has no in vitro or in vivo anticoagulant activity, does not prevent thrombus formation but shows a bleeding effect similar to the mammalian glycosaminoglycan. Overall, these results demonstrate unbalanced effects of dermatan sulfates with different sulfation patterns on coagulation, thrombosis and bleeding, and raise interesting questions concerning the relationship among these three biological actions of sulfated polysaccharides.

Antithrombin III-Independent Effect of Depolymerized Holothurian Glycosaminoglycan (DHG) on Acute Thromboembolism in Mice

1997 ◽  
Vol 77 (02) ◽  
pp. 399-402 ◽  
Author(s):  
Hideki Nagase ◽  
Keiko T Kitazato ◽  
Eiji Sasaki ◽  
Masahiko Hattori ◽  
Kenji Kitazato ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Dermatan Sulfate ◽  
Coagulation System ◽  
Independent Effect ◽  
Low Molecular Weight ◽  
Antithrombotic Effect ◽  
Antithrombotic Effects

SummaryA previous study in this laboratory showed that depolymerized holothurian glycosaminoglycan (DHG) has two different antithrombin III (ATIII)-independent inhibitory effects on the in vitro blood coagulation system: heparin cofactor II (HCII)-dependent inhibition of thrombin, and ATIII- and HCII-independent inhibition of factor X activation by factor IXa-factor Villa complex (Nagase et al. Blood 85, 1527-1534, 1995). In the present study, we compared the antithrombotic effects of DHG in normal and in ATIII-deficient mice with those of unfractionated heparin (UFH) and low molecular weight heparin (LMWH). DHG, unlike UFH and LMWH, exerted an in vivo antithrombotic effect even in mice with decreased plasma ATIII activity (about 30% of normal). We then compared the anticoagulant and antithrombotic effects of DHG in mice with those of high molecular weight (HMW)-DHG, low molecular weight (LMW)-DHG, and dermatan sulfate (DS). In terms of in vitro anticoagulant activity assessed by use of purified human components, DHGs (DHG, HMW-DHG, and LMW-DHG) had different anti-thrombin activity in the presence of HCII and anti-factor Xase activities, which differences were dependent on the molecular weight. With respect to in vivo antithrombotic activity, DHG, HMW-DHG, and LMW-DHG showed almost the same inhibitory effect on acute thromboembolism in mice (minimum effective dose [MED]: >0.3 mg/kg). Since the antithrombotic activities of DHGs were not correlated with the anticoagulant-specific activities, the contribution of the two anticoagulant activities to the in vivo antithrombotic effect of DHGs remains unknown. However, DHG was more effective against acute thromboembolism in mice than DS (MED >1 or >3 mg/kg), which showed no inhibitory activity toward factor Xase. Therefore, it seems that factor Xase inhibition contributes greatly to the antithrombotic effect of DHG and that DHG exerts this effect in mice mainly by inhibiting factor Xase.

Pharmacological Effects of a Novel Recombinant Hirudin, CX-397, In Vivo and In Vitro: Comparison with Recombinant Hirudin Variant-1, Heparin, and Argatroban

1999 ◽  
Vol 81 (02) ◽  
pp. 250-255 ◽  
Author(s):  
Yoshifumi Inoue ◽  
Yuso Goto ◽  
Tominaga Fukazawa ◽  
Hideya Hayashi ◽  
Yasuhiko Komatsu
Keyword(s):  
Arterial Thrombosis ◽  
Recombinant Hirudin ◽  
Dominant Type ◽  
Thrombin Inhibition ◽  
Venous Shunt ◽  
Antithrombotic Effects ◽  
Hemorrhagic Risk ◽  
Effective Doses

SummaryThe novel recombinant hirudin analog CX-397 was investigated with respect to its pharmacological activity and antithrombin profiles in vivo and in vitro. In three different types of thrombosis models in rats, including stasis and thrombin-induced venous, glass surface-activated arterio-venous shunt, and ferric chloride-induced arterial thrombosis models, CX-397 and rHV-1 elicited potent antithrombotic effects, where the minimum effective doses of rHV-1 tended to be higher than those of CX-397 in the arterio-venous shunt and arterial thrombosis models. The hemorrhagic risk of CX-397 in template bleeding in rats was not higher than that of rHV-1, indicating that CX-397 is superior to rHV-1 for treating the platelet-dominant type of thrombosis. However, no differences were detected between CX-397 and rHV-1 in their effects on in vitro coagulation times and thrombin-induced platelet aggregation, suggesting the possibility that some unknown mechanisms other than simple thrombin inhibition are also involved in their anti-thrombotic actions.

scholarly journals Vascular dermatan sulfate regulates the antithrombotic activity of heparin cofactor II

Blood ◽  
2008 ◽  
Vol 111 (8) ◽  
pp. 4118-4125 ◽  
Author(s):  
Li He ◽  
Tusar K. Giri ◽  
Cristina P. Vicente ◽  
Douglas M. Tollefsen
Keyword(s):  
Carotid Artery ◽  
Dermatan Sulfate ◽  
Thrombus Formation ◽  
Wild Type ◽  
Heparin Cofactor Ii ◽  
Arterial Endothelium ◽  
Carotid Arterial ◽  
Deficient Mice

AbstractHeparin cofactor II (HCII)–deficient mice form occlusive thrombi more rapidly than do wild-type mice following injury to the carotid arterial endothelium. Dermatan sulfate (DS) and heparan sulfate (HS) increase the rate of inhibition of thrombin by HCII in vitro, but it is unknown whether vascular glycosaminoglycans play a role in the antithrombotic effect of HCII in vivo. In this study, we found that intravenous injection of either wild-type recombinant HCII or a variant with low affinity for HS (K173H) corrected the abnormally short thrombosis time of HCII-deficient mice, while a variant with low affinity for DS (R189H) had no effect. When HCII was incubated with frozen sections of the mouse carotid artery, it bound specifically to DS in the adventitia. HCII was undetectable in the wall of the uninjured carotid artery, but it became concentrated in the adventitia following endothelial injury. These results support the hypothesis that HCII interacts with DS in the vessel wall after disruption of the endothelium and that this interaction regulates thrombus formation in vivo.

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