The Size and Shape of Human and Bovine Antithrombin III

Nordenman, Birgitta; Nyström, Curt; Björk, Ingemar

doi:10.1111/j.1432-1033.1977.tb11730.x

Cited by 276 publications

(144 citation statements)

References 45 publications

(28 reference statements)

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“…Protein concentrations were measured spectrophotometrically with the use of specific absorption coefficients (A~.&"") of 6.7 for both intact and modified bovine antithrombin [22], 17.5 for bovine thrombin [4] and 9.4 for bovine factor X, [23]. Values of 11.0 and 7.9 were assumed for the antithrombin-thrombin and the antithrombin-factor X, complexes, respectively ; these values were calculated from the weight fractions and the absorption coefficients of the two proteins comprising each complex.…”

Section: Materials a N D Methodsmentioning

confidence: 99%

Immunological Evidence for a Proteolytic Cleavage at the Active Site of Antithrombin in the Mechanism of Inhibition of Coagulation Serine Proteases

Wallgren¹,

Nordling²,

Björk³

1981

European Journal of Biochemistry

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Previous studies have shown that a modified form of antithrombin, cleaved at a single Arg-Ser bond near the carboxy-terminal end of the chain, is formed during the reaction with thrombin concurrent with the formation of the inactive enzyme-inhibitor complex. A variety of evidence suggests that this cleavage site is the active site of antithrombin. In this work, antisera against intact antithrombin, the modified form of antithrombin and the antithrombin-thrombin complex were used in immunodiffusion analyses to probe the state of the inhibitor in its complexes with coagulation serine proteases. The results show that new antigenic determinants not present in intact antithrombin are created in modified antithrombin by the single peptide-bond cleavage. The same antigenic determinants are found also in complexes between antithrombin and thrombin or factor X,. No evidence for the exposure of other new determinants in the complexes was obtained. The most likely conclusion from these results is that antithrombin exists in its complexes with the serine proteases as the modified, two-chain form of the inhibitor. This suggests that the mechanism of inhibition involves proteolytic cleavage of the active site of antithrombin by the protease.Antithrombin inhibits all the serine proteases of the intrinsic coagulation system by forming inactive, extremely stable, equimolar complexes with the enzymes (for reviews, see [I -31). A recent report from this laboratory showed that the formation of the antithrombin-thrombin complex in vitro is accompanied by the appearance ofa non-complexed, proteolytically modified, two-chain form of the inhibitor [4]. Heparin increases the production of this modified antithrombin (Bjork, I. and Fish, W. W., unpublished results; Lavine, K. K. and Jackson, C. M., personal communication) in addition to dramatically accelerating the formation of the antithrombin-thrombin complex [5]. The modified inhibitor is produced by a single cleavage by thrombin of an Arg-Ser bond near the carboxy-terminal end of the antithrombin polypeptide chain [6,7]. The two peptide chain segments thus formed are held together both by a disulfide bridge and by non-covalent interactions. A protein which is identical to the free thrombin-modified form of antithrombin can be released from the antithrombin-thrombin complex by various procedures [6,8 -101 and also dissociates spontaneously from the complex [Ill. Together, these findings have led to the suggestion that the observed cleavage site in antithrombin is the active site of the inhibitor [6,9]. This proposal has been supported by recent demonstrations of a homologous active site in the carboxy-terminal region of human al-protease inhibitor [12,13].The state of the active-site bond of antithrombin in the complexes between the inhibitor and serine proteases has n o t yet been characterized. Circumstantial evidence obtained in earlier investigations has suggested that actual cleavage of antithrombin by thrombin at the active site of the inhibitor may be involved in the formation ...

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Section: Materials a N D Methodsmentioning

confidence: 99%

Immunological Evidence for a Proteolytic Cleavage at the Active Site of Antithrombin in the Mechanism of Inhibition of Coagulation Serine Proteases

Wallgren¹,

Nordling²,

Björk³

1981

European Journal of Biochemistry

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“…Concentrations of both plasma and recombinant antithrombin were obtained by absorption measurements at 280 nm with the use of a specific absorption coefficient of 0.65 litres * g-1 cm-' and an Mr of 58000 (Nordenman et al, 1977), unless otherwise indicated.…”

Section: Vol 286 793mentioning

confidence: 99%

“…Immunodiffusion was done in a 1 % agarose gel in 0.02 Msodium phosphate/0.1 M-NaCl, pH 7.4 (Nordenman et al, 1977) with a commercial antiserum against plasma antithrombin (Dakopatts, Glostrup, Denmark).…”

Section: Protein Analysesmentioning

confidence: 99%

Decreased affinity of recombinant antithrombin for heparin due to increased glycosylation

Björk

Ylinenjärvi

Olson

et al. 1992

Biochemical Journal

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Recombinant antithrombin produced by baby hamster kidney (BHK) or Chinese hamster ovary (CHO) cells was separated into two fractions, containing comparable amounts of protein, by affinity chromatography on matrix-linked heparin. Fluorescence titrations showed that the more tightly binding fraction had a heparin affinity similar to that of plasma antithrombin (Kd 20 nM), whereas the affinity of the more weakly binding fraction was nearly 10-fold lower (Kd 175 nM). Analyses of the heparin-catalysed rate of inhibition of thrombin further showed that the fractions differed only in their affinity for heparin and not in the intrinsic rate constant of either the uncatalysed or the heparin-catalysed inactivation of thrombin. The recombinant antithrombin fraction with lower heparin affinity migrated more slowly than both the fraction with higher affinity and plasma antithrombin in SDS/PAGE under reducing conditions, consistent with a slightly higher apparent relative molecular mass. This apparent size difference was abolished by the enzymic removal of the carbohydrate side chains from the proteins. Such removal also increased the heparin affinity of the weakly binding fraction, so that it eluted from matrix-linked heparin at a similar position to the deglycosylated tightly binding fraction or plasma antithrombin. Analyses of N-linked carbohydrate side chains showed that the weakly binding fraction from CHO cells had a higher proportion of tetra-antennary and a lower proportion of biantennary oligosaccharides than the tightly binding fraction. We conclude that the recombinant antithrombin produced by the two cell lines is heterogeneously glycosylated and that the increased carbohydrate content of a large proporti6n of the molecules results in a substantial decrease in the affinity of these molecules for heparin. These findings are of particular relevance for studies aimed at characterizing the heparin-binding site of recombinant antithrombin by site-directed mutagenesis.

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“…A molecular weight for antithrombin of 56000 (Nordenman et al, 1977) was used in all calculations. Heparin from pig intestinal mucosa (stage 14; Inolex Pharmaceutical Division, Park Forest South, IL, U.S.A.) was purified as described by Lindahl et al (1965).…”

Section: Methodsmentioning

confidence: 99%

“…Protein concentrations were measured spectrophotometrically, by using a previously measured specific absorption coefficient (A,lcm) of 6.7 for bovine antithrombin (Nordenman et al, 1977).…”

Section: Methodsmentioning

confidence: 99%

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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The Size and Shape of Human and Bovine Antithrombin III

Cited by 276 publications

References 45 publications

Immunological Evidence for a Proteolytic Cleavage at the Active Site of Antithrombin in the Mechanism of Inhibition of Coagulation Serine Proteases

Immunological Evidence for a Proteolytic Cleavage at the Active Site of Antithrombin in the Mechanism of Inhibition of Coagulation Serine Proteases

Decreased affinity of recombinant antithrombin for heparin due to increased glycosylation

Binding to antithrombin of heparin fractions with different molecular weights

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