Molecular weight dependency of the heparin potentiated inhibition of thrombin and activated factor X. Effect of heparin neutralization in plasma

Andersson, Lars‐Olov; Barrowcliffe, Trevor W.; Holmer, E.; Johnson, Edward A.; Söderström, Gunilla

doi:10.1016/0049-3848(79)90159-2

Cited by 179 publications

(66 citation statements)

References 23 publications

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“…A similar molecular-weight-dependence of the anticoagulant activity of high-affinity heparins as that observed in the present work has been shown earlier (Laurent et al, 1978;Andersson et al, 1979). However, these investigators measured the average activity of the whole broad high-affinity peak from affinity chromatography and did not quantitatively characterize the binding of the fractions to antithrombin.…”

Section: Molecular Weightssupporting

confidence: 87%

Binding to antithrombin of heparin fractions with different molecular weights

Danielsson

Björk

1981

Biochemical Journal

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The interaction between bovine antithrombin, a plasma proteinase inhibitor, and heparin species of different molecular weights was studied. A commercial heparin preparation was divided by gel chromatography into a number of fractions with average molecular weights ranging from 6000 to 34 700. Each of these fractions was further fractionated by affinity chromatography on matrix-bound antithrombin. In the latter procedure, those heparin fractions that had molecular weights lower than about 14000 were separated into three peaks. The material in the first of these was not adsorbed on the column, and the other two peaks corresponded to the low-affinity and high-affinity peaks described previously. In contrast, high-molecular-weight heparin samples gave only the low-affinity and high-affinity fractions. U.v. difference absorption studies showed that the non-adsorbed heparin fraction bound to antithrombin in solution with a binding constant at physiological ionic strength only slightly lower than that of low-affinity heparin. The division between the two fractions thus is arbitrary and only dependent on the conditions selected for the affinity-chromatography experiment. Stoicheiometries and binding constants for the binding of several high-affinity heparin species to antithrombin were determined by fluorescence titrations. High-affinity heparin fractions of equal elution positions in the beginning of the peaks of the affinity chromatographies, but with different molecular weights, showed stoicheiometries that were not experimentally distinguishable from 1:1 and also had no appreciable differences in binding constants. However, the anticoagulant activities, calculated on a molar basis, of these fractions increased markedly with molecular weight, a behaviour that thus cannot be explained by differences in the binding of the fractions to antithrombin. In contrast, high-affinity samples of similar molecular weights, which were eluted at increasing ionic strengths from matrix-linked antithrombin, were found to have an increasing proportion of chains with two binding sites for antithrombin and also to have progressively higher binding constants. These binding properties at least partly explain the increasing anticoagulant activities that were observed for these fractions.

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Section: Molecular Weightssupporting

confidence: 87%

Binding to antithrombin of heparin fractions with different molecular weights

Danielsson

Björk

1981

Biochemical Journal

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“…1 is a glycosaminoglycan (GAG) that catalyzes inhibition of the coagulant enzymes factor Xa and thrombin by the serine protease inhibitor (serpin) antithrombin (AT) (1)(2)(3). Reaction occurs via the allosteric activation of AT, due to H binding, followed by attack of the enzyme on the reactive center of the inhibitor (4).…”

Section: Unfractionated Standard Heparin (H)mentioning

confidence: 99%

Mechanisms Responsible for Catalysis of the Inhibition of Factor Xa or Thrombin by Antithrombin Using a Covalent Antithrombin-Heparin Complex

Paredes

Wang

Berry

et al. 2003

Journal of Biological Chemistry

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Covalent antithrombin-heparin (ATH) complexes, formed spontaneously between antithrombin (AT) and unfractionated standard heparin (H), have a potent ability to catalyze the inhibition of factor Xa (or thrombin) by added AT. Although Ϸ30% of ATH molecules contain two AT-binding sites on their heparin chains, the secondary site does not solely account for the increased activity of ATH. We studied the possibility that all pentasaccharide AT-binding sequences in ATH may catalyze factor Xa inhibition. Chromatography of ATH on Sepharose-AT resulted in >80% binding of the load. Similar chromatographies of non-covalent AT ؉ H mixtures lead to a lack of binding for AT and fractionation of H into unbound (separate from AT) or bound material. Gradient elution of ATH from Sepharose-AT gave 2 peaks, a peak containing higher affinity material that had greater anti-factor Xa catalytic activity (708 units/mg heparin) compared with the peak containing lower affinity material (112 units/mg). Sepharose-AT chromatography of the ATH component with short heparin chains (<12 monosaccharides) resulted in active unbound (40%) and bound fractions (190 and 560 units/ mg, respectively). Factor Xa-ATH or thrombin-ATH inhibitor complexes gave chromatograms on Sepharose-AT with more unbound material compared with that of free ATH. Also, ATH did not bind to Sepharoseheparin, and the intrinsic fluorescence due to activation of AT in ATH by its heparin chain was reversed at higher [NaCl] than that required to dissociate non-covalent AT⅐H complexes. Thus, exogenous AT can compete with the AT moiety of ATH for binding to the covalently linked heparin chain, leading to catalytic inhibition of factor Xa or thrombin. These data may suggest that access to pentasaccharide units in non-covalent AT⅐H complexes by free AT may be facile. Unfractionated standard heparin (H)1 is a glycosaminoglycan (GAG) that catalyzes inhibition of the coagulant enzymes factor Xa and thrombin by the serine protease inhibitor (serpin) antithrombin (AT) (1-3). Reaction occurs via the allosteric activation of AT, due to H binding, followed by attack of the enzyme on the reactive center of the inhibitor (4). In the case of thrombin, binding to the heparin chain by the enzyme must also occur for efficient reaction to take place (3). After formation of thrombin-AT or factor Xa-AT inhibitor complexes, affinity of the AT moiety for the heparin chain decreases, leading to release of the catalyst for further reactions with AT and enzyme (5, 6). Although binding to thrombin is through nonselective interaction of negative charges on the GAG with the anion-binding exosite of the enzyme (7, 8), AT binding to H occurs through high affinity to a specific pentasaccharide sequence on the heparin chain (2, 9). Moreover, it has been shown that the rate-determining step for catalysis of thrombin (or factor Xa) inhibition involves the initial binding of AT and H (10).Previously, we produced a covalent AT-heparin complex (ATH) to further study the mechanism of enzyme inhibition by H-activated AT...

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“…The characteristics and homogeneity of the fragments were very similar to those described by Thomas et al (21): h16 contained polysaccharides ranging from 14 to 18 monosaccharide units, and h12 consisted of material with 10 to 14 monosaccharide units. Heparin concentrations were measured using the carbazole-H2SO4 method (22), and heparin activity was determined by Factor Xa inhibition using the chromogenic substrate N-benzoyl-Lisoleucyl-L-glutamyl-glycyl-L-arginine-pNA (S-2222; Kabi AB) (6). Covalent complexes between AT and heparin or heparin fragments were synthesized as previously described (15,16).…”

Section: Methodsmentioning

confidence: 99%

“…Such low-Mr heparins have high anti-Factor Xa activity but do not significantly prolong the activated partial thromboplastin time (6)(7)(8).…”

Section: Introductionmentioning

confidence: 99%

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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Molecular weight dependency of the heparin potentiated inhibition of thrombin and activated factor X. Effect of heparin neutralization in plasma

Cited by 179 publications

References 23 publications

Binding to antithrombin of heparin fractions with different molecular weights

Binding to antithrombin of heparin fractions with different molecular weights

Mechanisms Responsible for Catalysis of the Inhibition of Factor Xa or Thrombin by Antithrombin Using a Covalent Antithrombin-Heparin Complex

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

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