On the molecular-weight-dependence of the anticoagulant activity of heparin

Anticoagulant therapy in general is designed to prevent either the generation or activity of thrombin; however, a cell-based model of coagulation provides a physiologic view of individual phases of the process, allowing more specific targets for attenuating the initiation, priming, or propagation of thrombus formation. Future categorization schemes will consider individual coagulation factors, individual sites on a given coagulation factor, and specific phases of coagulation to better identify an agent’s biochemical and physiologic activity. Unfractionated heparin (UFH) is a heterogeneous, negatively charged mucopolysaccharide consisting of approximately 18 to 50 saccharide units (molecular weight 5000–30,000 Da). Antithrombin (AT), required for the interaction (and subsequent neutralization) of UFH with thrombin and coagulation proteases including factors Xa, IXa, XIa, and XIIa, is bound by one third of administered drug (only molecules containing the critical pentasaccharide sequence can bind AT). Following IV administration, UFH binds to a variety of plasma proteins, endothelial cells, and macrophages, explaining, in part, the wide variability in anticoagulant effects for a given dose. It is cleared from the circulation through both a rapid saturable mechanism and a slower first-order mechanism. As a result, there is a dose-dependent half-life ranging from 60 minutes after a dose of 100 U/kg to 180 minutes for a dose of 400 U/kg (Beguin et al., 1988; Lam et al., 1976). Heparin-induced thrombocytopenia and hemorrhage are the most feared complications of UFH administration (see Chapter 29). Other adverse effects include osteopenia (with long-term administration). Low-molecular-weight heparin (LMWH) is prepared by the depolymerization of porcine UFH. A variety of processes are used, giving distinctive products whose molecular weights range from 4,000 to 6,500 Da (Hirsh and Levine, 1992). Like UFH, approximately one third of LMWH polysaccharide chains contain the pentasaccharide binding site for antithrombin. The LMWH–antithrombin complex (consisting of a predominance of shorter chain polysaccharides) has relatively weak antithrombin activity but retains the ability to inactivate factor Xa. The ratio of anti-Xa activity to anti-IIa (antithrombin) activity varies from 2:1 to 4:1. Similar to UFH, LMWH is not able to inhibit thrombin bound to fibrin (Weitz, 1997).

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The molecular-weight dependence of the rate-enhancing effect of heparin on the inhibition of thrombin, factor Xa, factor IXa, factor XIa, factor XIIa and kallikrein by antithrombin

Biochemical Journal ◽

10.1042/bj1930395 ◽

1981 ◽

Vol 193 (2) ◽

pp. 395-400 ◽

Cited By ~ 180

Author(s):

E Holmer ◽

K Kurachi ◽

G Söderström

Keyword(s):

Molecular Weight ◽

Factor Xa ◽

Low Molecular Weight ◽

Clotting Factors ◽

Factor Ixa ◽

Molecular Weight Dependence ◽

Factor Xiia ◽

Factor Xia ◽

Heparin Fractions ◽

Inhibition Of Thrombin

Heparin fractions of different molecular weight and with high affinity for antithrombin were studied with respect to their ability to potentiate the inhibition of activated clotting factors by antithrombin. Inhibition of thrombin, Factor IXa and Factor XIa showed similarities in the dependence on the molecular weight of heparin and was found to decrease with decreasing molecular weight. Inactivation of Factor Xa, Factor XIIa and kallikrein was, however, less dependent on the size of the polysaccharide and, to a great extent, was potentiated even by low-molecular-weight heparin fractions that had virtually no effect on the inhibition of thrombin, Factor IXa and Factor XIa.

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Binding of Dextran Sulphate to Antithrombin

10.1055/s-0039-1687057 ◽

1979 ◽

Author(s):

B. Nordenman ◽

I. Björk

Keyword(s):

Anticoagulant Activity ◽

Factor Xa ◽

Molecular Size ◽

Spectroscopic Properties ◽

Dextran Sulphate ◽

High Affinity ◽

Fluorescence Measurements ◽

Heparin Fractions ◽

Difference Absorption

The interaction between dextran sulphate (DS) a synthetic sulphated polysaccharide with in vitro anticoagulant activity, and antithrombin (AT) has been studied. About 90% of a commercial, molecular-size-fractionated preparation of DS bound to an AT-Sepharose column. This material was eluted by a salt gradient at a peak ionic strength of about 0.5 and was used for further studies. Difference absorption and fluorescence measurements showed that in solution an average of about 3 molecules of AT bound to each molecule of DS of the size used (~18000 daltons) with an average binding constant of 3 x 106 M-1. The changes of the spectroscopic properties of AT induced by DS were intermediate to those caused by the low-affinity and high-affinity heparin fractions. This was parallelled by the ability of DS to accelerate-the AT-thrombin reaction to a greater extent than low-affinity heparin, but not as pronounced as high-affinity heparin. DS had relatively little effect on the reaction between AT and Factor Xa, while the influence of high-affinity heparin on this reaction was similar to that on the AT-thrombin reaction. These results suggest that DS may induce a conformational change of AT which is less extensive than that caused by high-affinity heparin. This change may be sufficient to accelerate the reaction between AT and thrombin but inadequate to correspondingly affect the reaction between the inhibitor and Factor Xa.

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Binding to antithrombin of heparin fractions with different molecular weights

Biochemical Journal ◽

10.1042/bj1930427 ◽

1981 ◽

Vol 193 (2) ◽

pp. 427-433 ◽

Cited By ~ 33

Author(s):

Ȧke Danielsson ◽

Ingemar Björk

Keyword(s):

Molecular Weight ◽

Affinity Chromatography ◽

Binding Constants ◽

Gel Chromatography ◽

Molecular Weights ◽

High Affinity ◽

Heparin Fractions ◽

Fluorescence Titrations ◽

In The Beginning ◽

Difference Absorption

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 34700. 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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Effects of heparin fractions of different affinities to antithrombin III and thrombin on the inactivation of thrombin and factor Xa by antithrombin III

Canadian Journal of Biochemistry and Cell Biology ◽

10.1139/o84-125 ◽

1984 ◽

Vol 62 (10) ◽

pp. 975-983 ◽

Cited By ~ 7

Author(s):

Andrew L. Cerskus ◽

Kathy J. Birchall ◽

Frederick A. Ofosu ◽

Jack Hirsh ◽

Morris A. Blajchman

Keyword(s):

Molecular Weight ◽

Low Molecular Weight Heparin ◽

Antithrombin Iii ◽

Factor Xa ◽

Similar Rate ◽

Low Molecular Weight ◽

Heparin Binding ◽

High Affinity ◽

Relative Contribution ◽

Heparin Fractions

To investigate the relative contribution of heparin-binding thrombin and antithrombin III to the enhancement of the rate of inactivation of thrombin by antithrombin III, standard heparin was fractionated on matrix-linked thrombin and (or) antithrombin III. There was a good correlation between heparin affinity for antithrombin III and its ability to enhance the inactivation of thrombin and factor Xa. In addition, there was a good correlation between affinity of heparin for thrombin and its catalytic activity on the inactivation of thrombin by antithrombin III. Thus fractions with high affinity to thrombin had similar rate-enhancing activity for thrombin inactivation to that of fractions with high affinity to antithrombin III. Fractions with high affinity to both proteins were more potent than fractions with high affinity to either protein alone. No significant differences in mean molecular weight were observed among the various heparin fractions. A heparin fraction with very low affinity to thrombin and high affinity to antithrombin III was prepared by repeated fractionation of a low molecular weight heparin on the two affinity columns. This fraction had very weak rate-enhancing activity for the inactivation of thrombin by antithrombin III, but retained substantial activity for the inactivation of factor Xa. The results of these studies support the concept that, for both standard and low molecular weight heparin, the enhancement of the inactivation of thrombin by antithrombin III requires the interaction of the heparin with both thrombin and antithrombin III.

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Isolation and Structural Charaderization of a Potent Inhibitor of Coagulation Factor Xa from the Leech Haementeria ghilianii

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646610 ◽

1989 ◽

Vol 61 (03) ◽

pp. 437-441 ◽

Cited By ~ 28

Author(s):

Cindra Condra ◽

Elka Nutt ◽

Christopher J Petroski ◽

Ellen Simpson ◽

P A Friedman ◽

...

Keyword(s):

Molecular Weight ◽

Salivary Gland ◽

Amino Acid ◽

Amino Acid Sequence ◽

Western Blot ◽

Potent Inhibitor ◽

Factor Xa ◽

Coagulation Factor ◽

Sds Page ◽

Bovine Factor

SummaryThe present work reports the discovery and charactenzation of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalrs, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host’s blood.

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Comparison of the Non-Specific Binding of Unfractionated Heparin and Low Molecular Weight Heparin (Enoxaparin) to Plasma Proteins

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649639 ◽

1993 ◽

Vol 70 (04) ◽

pp. 625-630 ◽

Cited By ~ 71

Author(s):

Edward Young ◽

Benilde Cosmi ◽

Jeffrey Weitz ◽

Jack Hirsh

Keyword(s):

Molecular Weight ◽

Unfractionated Heparin ◽

Plasma Proteins ◽

Low Molecular Weight Heparin ◽

Specific Binding ◽

Anticoagulant Activity ◽

Factor Xa ◽

Low Molecular Weight ◽

Heparin Binding ◽

Fixed Amount

SummaryThe non-specific binding of anticoagulantly-active heparin to plasma proteins may influence its anticoagulant effect. We used low affinity heparin (LAH) essentially devoid of anti-factor Xa activity to investigate the extent and possible mechanism of this non-specific binding. The addition of excess LAH to platelet-poor plasma containing a fixed amount of unfractionated heparin doubled the anti-factor Xa activity presumably because it displaces anticoagulantly-active heparin from plasma proteins. Although dextran sulfates of varying molecular weights also increased the anti-factor Xa activity, less sulfated heparin-like polysaccharides had no effect. These findings suggest that the ability to displace active heparin from plasma protein binding sites is related to charge and may be independent of molecular size. In contrast to its effect in plasma containing unfractionated heparin, there was little augmentation in anti-factor Xa activity when LAH was added to plasma containing low molecular weight heparin (LMWH), indicating that LMWH binds less to plasma proteins than unfractionated heparin. This concept is supported by studies comparing the anticoagulant activity of unfractionated heparin and LMWH in plasma with that in buffer containing antithrombin III. The anti-factor Xa activity of unfractionated heparin was 2-fold less in plasma than in the purified system. In contrast, LMWH had identical anti-factor Xa activity in both plasma and buffer, respectively. These findings may be clinically relevant because the recovered anti-factor Xa activity of unfractionated heparin was 33% lower in plasma from patients with suspected venous thrombosis than in plasma from healthy volunteers. The reduced heparin recovery in patient plasma reflects increased heparin binding to plasma proteins because the addition of LAH augmented the anti-factor Xa activity. In contrast to unfractionated heparin, there was complete recovery of LMWH added to patient plasma and little increase of anti-factor Xa activity after the addition of LAH. These findings may explain why LMWH gives a more predictable dose response than unfractionated heparin.

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Toxicity of Heparinoids, with Special Reference to the Precipitation of Fibrinogen

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655587 ◽

1964 ◽

Vol 12 (01) ◽

pp. 232-261 ◽

Cited By ~ 2

Author(s):

S Sasaki ◽

T Takemoto ◽

S Oka

Keyword(s):

Molecular Weight ◽

Dextran Sulfate ◽

Anticoagulant Activity ◽

Molecular Structures ◽

Severe Reaction ◽

Clinical Use ◽

Molecular Weights ◽

Close Relationship

SummaryTo demonstrate whether the intravascular precipitation of fibrinogen is responsible for the toxicity of heparinoid, the relation between the toxicity of heparinoid in vivo and the precipitation of fibrinogen in vitro was investigated, using dextran sulfate of various molecular weights and various heparinoids.1. There are close relationships between the molecular weight of dextran sulfate, its toxicity, and the quantity of fibrinogen precipitated.2. The close relationship between the toxicity and the precipitation of fibrinogen found for dextran sulfate holds good for other heparinoids regardless of their molecular structures.3. Histological findings suggest strongly that the pathological changes produced with dextran sulfate are caused primarily by the intravascular precipitates with occlusion of the capillaries.From these facts, it is concluded that the precipitates of fibrinogen with heparinoid may be the cause or at least the major cause of the toxicity of heparinoid.4. The most suitable molecular weight of dextran sulfate for clinical use was found to be 5,300 ~ 6,700, from the maximum value of the product (LD50 · Anticoagulant activity). This product (LD50 · Anticoagulant activity) can be employed generally to assess the comparative merits of various heparinoids.5. Clinical use of the dextran sulfate prepared on this basis gave satisfactory results. No severe reaction was observed. However, two delayed reactions, alopecia and thrombocytopenia, were observed. These two reactions seem to come from the cause other than intravascular precipitation.

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Covalent Complexes Between Low Molecular Weight Heparin Fragments and Antithrombin III – Inhibition Kinetics and Turnover Parameters

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657333 ◽

1983 ◽

Vol 49 (02) ◽

pp. 109-115 ◽

Cited By ~ 4

Author(s):

M Hoylaerts ◽

E Holmer ◽

M de Mol ◽

D Collen

Keyword(s):

Molecular Weight ◽

Half Life ◽

Low Molecular Weight Heparin ◽

Antithrombin Iii ◽

Factor Xa ◽

Life Time ◽

Low Molecular Weight ◽

High Affinity ◽

Plasma Half Life ◽

Covalent Complex

SummaryTwo high affinity heparin fragments (A/r 4,300 and M, 3,200) were covalently coupled to antithrombin III (J. Biol. Chem. 1982; 257: 3401-3408) with an apparent 1:1 stoichiometry and a 30-35% yield.The purified covalent complexes inhibited factor Xa with second order rate constants very similar to those obtained for antithrombin III saturated with these heparin fragments and to that obtained for the covalent complex between antithrombin III and native high affinity heparin.The disappearance rates from plasma in rabbits of both low molecular weight heparin fragments and their complexes could adequately be represented by two-compartment mammillary models. The plasma half-life (t'/j) of both low Afr-heparin fragments was approximately 2.4 hr. Covalent coupling of the fragments to antithrombin III increased this half-life about 3.5 fold (t1/2 ≃ 7.7 hr), approaching that of free antithrombin III (t1/2 ≃ 11 ± 0.4 hr) and resulting in a 30fold longer life time of factor Xa inhibitory activity in plasma as compared to that of free intact heparin (t1/2 ≃ 0.25 ± 0.04 hr).

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Novel concatameric heparin-binding peptides reverse heparin and low-molecular-weight heparin anticoagulant activities in patient plasma in vitro and in rats in vivo

Blood ◽

10.1182/blood-2003-07-2334 ◽

2004 ◽

Vol 103 (4) ◽

pp. 1356-1363 ◽

Cited By ~ 30

Author(s):

Barbara P. Schick ◽

David Maslow ◽

Adrianna Moshinski ◽

James D. San Antonio

Keyword(s):

Molecular Weight ◽

Low Molecular Weight Heparin ◽

Tandem Repeats ◽

Factor Xa ◽

Low Molecular Weight ◽

Heparin Binding ◽

High Affinity ◽

Binding Peptides

Abstract Patients given unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) for prophylaxis or treatment of thrombosis sometimes suffer serious bleeding. We showed previously that peptides containing 3 or more tandem repeats of heparin-binding consensus sequences have high affinity for LMWH and neutralize LMWH (enoxaparin) in vivo in rats and in vitro in citrate. We have now modified the (ARKKAAKA)n tandem repeat peptides by cyclization or by inclusion of hydrophobic tails or cysteines to promote multimerization. These peptides exhibit high-affinity binding to LMWH (dissociation constant [Kd], ≈ 50 nM), similar potencies in neutralizing anti–Factor Xa activity of UFH and enoxaparin added to normal plasma in vitro, and efficacy equivalent to or greater than protamine. Peptide (ARKKAAKA)3VLVLVLVL was most effective in all plasmas from enoxaparin-treated patients, and was 4- to 20-fold more effective than protamine. Several other peptide structures were effective in some patients' plasmas. All high-affinity peptides reversed inhibition of thrombin-induced clot formation by UFH. These peptides (1 mg/300 g rat) neutralized 1 U/mL anti–Factor Xa activity of enoxaparin in rats within 1 to 2 minutes. Direct blood pressure and heart rate measurements showed little or no hemodynamic effect. These heparin-binding peptides, singly or in combination, are potential candidates for clinical reversal of UFH and LMWH in humans.

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