Anticoagulant properties of heparin fractionated by affinity chromatography on matrix-bound antithrombin III and by gel filtration

Andersson, Lars‐Olov; Barrowcliffe, T W; Holmer, E.; Johnson, Edward A.; Sims, G.E.C.

doi:10.1016/0049-3848(76)90105-5

Cited by 481 publications

(174 citation statements)

References 8 publications

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“…U.S.A. 77, It is now recognized that the main effect of heparin on blood coagulation is mediated by antithrombin III. Only about one-third of the polysaccharide chains that constitute conventional heparins have high affinity for antithrombin III (Lam et al, 1976;Hook et al, 1976;Andersson et aL, 1976). The binding structures are contained in specific oligosaccharide segments (Hopwood et al, 1978).…”

Section: (Received 23 February 1981/accepted 27 April 1981)mentioning

confidence: 99%

The structure of heparin oligosaccharide fragments with high anti-(factor Xa) activity containing the minimal antithrombin III-binding sequence Chemical and13C nuclear-magnetic-resonance studies

Casu¹,

Oreste²,

Torri³

et al. 1981

Biochemical Journal

336

122

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The chemical composition and the 13C n.m.r. spectra of heparin oligosaccharides (essentially octasaccharides), having high affinity for antithrombin III and high anti-(Factor Xa) activity, prepared by three independent approaches (extraction, partial deaminative cleavage with HNO2 and partial depolymerization with bacterial heparinase), leading to different terminal residues, have been studied and compared with those of the corresponding inactive species. Combined with chemical data, the spectra of the active oligosaccharides and of their fragmentation products afforded information on composition and sequence. The three types of active oligosaccharides were shown to have the common hexasaccharide core

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Section: (Received 23 February 1981/accepted 27 April 1981)mentioning

confidence: 99%

The structure of heparin oligosaccharide fragments with high anti-(factor Xa) activity containing the minimal antithrombin III-binding sequence Chemical and13C nuclear-magnetic-resonance studies

Casu¹,

Oreste²,

Torri³

et al. 1981

Biochemical Journal

336

122

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“…Box 616, 6200 MD Maastricht, The Netherlands. maintain their potency as measured by anti-factor Xa assays (Andersson et al, 1976: Lane et al, 1978: Barrowcliffe et al, 1979. Studies on the structure-function relationship of heparin have learned that a pentasaccharide sequence, necessary for the interaction with ATIII, is sufficient to obtain catalysis of the inactivation of factor Xa (Choay, 1989)' The heparin-catalysed inactivation of thrombin is dependent on the simultaneous binding of both proteins, which requires a heparin chain length of at least 1 8 saccharide units (Lane et aL 7984t Danielsson et aI, 1986).…”

mentioning

confidence: 99%

Ratios of anti‐factor Xa to antithrombin activities of heparins as determined in recalcified human plasma

Schoen¹,

Lindhout

Hemker

1992

Br J Haematol

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Summary. Anti-factor Xa and anti-thrombin activities of unfractionated (UF) and low molecular weight (tMW) heparins have been measured in human plasma and with purified human antithrombin III (ATIII) in the absence and presence of 1'5 mu calcium. The anti-factor Xa and antithrombin activities were measured directly, by assessing the heparin-dependent pseudo-first order rate constants of inactivation of human factor Xa or thrombin. These activities were studied with the 4th International Standard for UF heparin, the lst International Standard for LMW heparin, C'1216, enoxaparin, Cy222, and the synthetic pentasaccharide' In plasma, calcium equally well increased the specific antifactor Xa catalytic activities as compared to purified ATIII' That is, 1 '5 mu calcium stimulated the UF standard heparincatalysed inactivation of factor Xa 2'L-2'4 times' In the presence of the LMW heparins the effect of calcium was smaller (1 '3-1 '7 times), and in plasma there was no effect of calcium on the pentasaccharide-catalysed inactivation of factor Xa. Thus, the largest effects of calcium in the inactivation reaction of factor Xa is seen with UF standard heparin. Calcium reduced the anti-thrombin activities of all the heparin preparations studied about 1'5 times when purified ATIII was used, although in plasma this effect was less clear. Consequently, in the presence of 1'5 mlu calcium the ratio of the anti-factor Xa to the anti-thrombin activities of UF standard heparin approximated those of the LMW heparins. The only exception was CY222, which under all conditions retained anti-factor Xa/anti-thrombin ratios significantly higher than those ofUF standard heparin' One of the regulatory mechanisms of blood coagulation is the inhibition of enzymes of the coagulation system by the plasma protein antithrombin III (ATIII)' its main targets in clotting plasma being thrombin and factor Xa (Harpel' 1987). Inactivation ofthese serine proteases is catalysed by heparin, and thus the heparin-dependent anti-factor Xa and anti-thrombin activities have become main points of interest in studying the mode of action and the standardization of heparin. It should also be noted here, however, that the ATlll-dependent inactivation of free factor fXa can contribute significantly to the heparin effect in clotting plasma (B6guin et al, l99l).Since 1976 it is known that low molecular weight (LMW) heparin fractions have little effect on tests of overall clotting' such as the activated partial thromboplastin time (APTT)' but + Present address:

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“…Commercial heparin preparations are heterogeneous with respect to their biological activity, and can be separated into two functionally distinct fractions by affinity chromatography on matrix-linked antithrombin or analogous methods. One of these fractions has high affinity for antithrombin and high anticoagulant activity, whereas the other has low affinity for the inhibitor and is almost inactive (Lam et al, 1976;Hook et al, 1976;Andersson et al, 1976). Previous reports from our laboratory and by others have dealt with the binding of these two fractions to antithrombin in solution (Nordenman & Bj6rk, 1978a;Danielsson & Bj6rk, 1978;Jordan et al, 1979).…”

mentioning

confidence: 98%

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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Anticoagulant properties of heparin fractionated by affinity chromatography on matrix-bound antithrombin III and by gel filtration

Cited by 481 publications

References 8 publications

The structure of heparin oligosaccharide fragments with high anti-(factor Xa) activity containing the minimal antithrombin III-binding sequence Chemical and13C nuclear-magnetic-resonance studies

The structure of heparin oligosaccharide fragments with high anti-(factor Xa) activity containing the minimal antithrombin III-binding sequence Chemical and13C nuclear-magnetic-resonance studies

Ratios of anti‐factor Xa to antithrombin activities of heparins as determined in recalcified human plasma

Binding to antithrombin of heparin fractions with different molecular weights

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