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

Thomas, Duncan P.; Merton, R E; Barrowcliffe, Trevor W.; Thunberg, Lennart; Lindahl, Ulf

doi:10.1055/s-0038-1657178

Cited by 103 publications

(66 citation statements)

References 14 publications

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“…The specific antithrombotic activity of H, however, was more than an order of magnitude greater than that of h16 or h12, as expressed in both molar concentration and anti-Factor Xa activity, indicating that the antithrombotic effect of heparin in plasma does not only depend on its antiFactor Xa activity. This lack of correlation between the antithrombotic and anti-Factor Xa activity of heparin fragments confirms previous reports (21,27).…”

Section: Discussionsupporting

confidence: 92%

Antithrombotic properties in rabbits of heparin and heparin fragments covalently coupled to human antithrombin III.

Mattsson¹,

Hoylaerts²,

Holmer³

et al. 1985

J. Clin. Invest.

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Clinical grade heparin is a very heterogeneous mucopolysaccharide, containing molecules with M, ranging from 6,000 to 30,000 that have either a high affinity or a low affinity for antithrombin III (AT). In this study, the antithrombotic properties of intact high-affinity heparin (M, = 15,000) and of two heparin fragments (h16, a 16-monosaccharide fragment, with M, = 4,300, and h12, a 12-monosaccharide fragment, with M, = 3,200) and of their functional covalent stoichiometric complexes with human AT were compared in a venous thrombosis stasis model in rabbits. Thrombosis was induced by injection of glass-activated human plasma and measured in a segment of the jugular vein that was isolated between two vascular clamps for 10 min.Injections of 55 Mg/kg resulted in a clear antithrombotic effect for intact heparin, but not for the two fragments. Equivalent amounts (carbohydrate moiety) of covalent complexes of heparin or of both heparin fragments with human AT resulted in an antithrombotic effect lasting for 45-60 min. Injection of 110 Mtg/kg of heparin and of the heparin fragments yielded an antithrombotic effect, lasting 45-60 min; the corresponding amounts of covalent complexes caused an antithrombotic effect for 60-120 min. The free and conjugated framents produced equal antithrombotic effects at equal plasma levels of anti-Factor Xa activity, but the specific antithrombotic activities of free and complexed intact heparin, on a molar basis, were 10-20-fold greater than those of the free and complexed heparin fragments. The plasma half-life of the covalent complexes of the heparin fragments with AT is, however, 10 times longer than that of the complex between intact heparin and AT and 30 times longer than that of free intact heparin. Covalent complexes between AT and heparin fragments could, therefore, be useful to maintain more stable levels of antithrombotic activity in plasma.

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Section: Discussionsupporting

confidence: 92%

Antithrombotic properties in rabbits of heparin and heparin fragments covalently coupled to human antithrombin III.

Mattsson¹,

Hoylaerts²,

Holmer³

et al. 1985

J. Clin. Invest.

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“…Subsequent studies using purified antithrombin confirmed these trends, and it became clear that acceleration of inhibition of thrombin and FXa by heparin had different MW requirements (14,15). This difference was emphasised in studies by Holmer (16) and in our laboratory (17,18 ); high-affinity (for antithrombin) oligosaccharides with chain lengths mostly below 16 units, had very high anti-Xa activity, over 1,000 IU/mg, but their activity in thrombin inhibition and APTT assays was less than 10 IU/ mg.…”

Section: Mw Dependencementioning

confidence: 73%

Heparin and low-molecular-weight heparin

Mulloy,

Barrowcliffe,

Gray

2008

Thromb Haemost

236

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SummaryHeparin is one of the oldest biological medicines, and has an established place in the prevention and treatment of venous thrombosis. Low-molecular-weight heparins (LMWH) have been developed by several manufacturers and have advantages in terms of pharmacokinetics and convenience of adminisKeywords Heparin, low-molecular-weight heparin tration. They have been shown to be at least as effective and safe as unfractionated heparin and have replaced the latter in many indications. In this article the chemistry, mechanisms of action, measurement of anticoagulant activities, and clinical status of heparin and LMWH are reviewed.

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“…The difference in the magnitude of the cofactor effect between heparin and the pentasaccharide was ϳ5-and ϳ60-fold for FXa in the absence and presence of Ca 2ϩ , and ϳ89-and ϳ74-fold for GDFXa in the absence and presence of Ca 2ϩ , respectively. These findings can explain why the anti-FXa activity of heparin exhibits a minimal chain length dependence in ex vivo and in vitro assays but significant chain length dependence in in vivo assays (16,17). …”

mentioning

confidence: 89%

Calcium Enhances Heparin Catalysis of the Antithrombin-Factor Xa Reaction by a Template Mechanism

Rezaie

1998

Journal of Biological Chemistry

109

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It is believed that heparin accelerates factor Xa (FXa) inactivation by antithrombin (AT) by conformationally activating the inhibitor rather than by bridging AT and FXa in a ternary complex (template effect). This is derived from kinetic studies done in the absence of Ca 2؉ or in the presence of EDTA. To test the possibility that the anionic Gla domain of FXa, when not neutralized by Ca 2؉ ions, prevents heparin binding to FXa, the heparin and pentasaccharide dependence of FXa inactivation by AT in both the absence (100 M EDTA) and presence of Ca 2؉ (2.5 mM) was studied using wild-type FXa and a FXa derivative that lacks the Gla domain (GDFXa). AT inactivated both FXa derivatives similarly in both the absence and presence of Ca 2؉ (k 2 ‫؍‬ 1.7-2. ). The additional cofactor effect of heparin with all FXa derivatives was a bell-shaped curve, which disappeared if the ionic strength of the reaction was increased to ϳ0.4. These results suggest that although the major effect of heparin in acceleration of FXa inactivation is through a heparin-induced conformational change in the reactive site loop of AT, the template effect of heparin, nevertheless, contributes significantly to rapid FXa inactivation at physiological Ca 2؉ .The mechanism by which the therapeutic heparin catalyzes the inactivation of thrombin and factor Xa (FXa) 1 by antithrombin (AT) has been extensively studied in the past (1-4). The consensus view is that heparin accelerates thrombin inactivation by AT by facilitating an initial interaction between the two proteins by a bridging mechanism (4 -6). In FXa inactivation, however, it is believed that a template mechanism has a minimal role or no role at all in the acceleration and that a heparin-induced conformational change in the reactive site loop of AT accounts for all of the accelerating effect of heparin in the reaction (7-10). These distinct inactivation mechanisms are supported by the observation that a unique pentasaccharide fragment of heparin, which can bind and activate AT but is not long enough to bridge the inhibitor and enzyme, specifically promotes FXa but not thrombin inhibition (9, 10). For thrombin inhibition, heparin chains containing the pentasaccharide plus at least 13 additional saccharides are required to accelerate the reaction (2, 4). All the previous heparin-catalyzed protease inactivation studies were conducted under experimental conditions that either lacked Ca 2ϩ (11,12) or contained EDTA in the reaction buffers (9,13,14). Under these conditions heparin-catalyzed thrombin inactivation by AT is at least 10 -20-fold faster than the inactivation of FXa (9, 11-13). Unlike thrombin, however, FXa contains a highly anionic N-terminal domain, which is rich in ␥-carboxyglutamic acid (Gla) residues that are involved in Ca 2ϩ -dependent membrane binding. This domain of FXa contains 11 ␥-carboxylated Glu residues, which are in a random disordered conformation in the absence of Ca 2ϩ (15). To test the possibility that in the absence of Ca 2ϩ the Gla domain of FXa antagonizes heparin bind...

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

Cited by 103 publications

References 14 publications

Antithrombotic properties in rabbits of heparin and heparin fragments covalently coupled to human antithrombin III.

Antithrombotic properties in rabbits of heparin and heparin fragments covalently coupled to human antithrombin III.

Heparin and low-molecular-weight heparin

Calcium Enhances Heparin Catalysis of the Antithrombin-Factor Xa Reaction by a Template Mechanism

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