Arg-129 plays a specific role in the confirmation of antithrombin and in the enhancement of factor Xa inhibition by the pentasaccharode sequence of heparin

Najjam, Saloua; Chadeuf, Gilliane; Gandrille, Sophie; Aiach, Martine

doi:10.1016/0925-4439(94)90070-1

Cited by 7 publications

(9 citation statements)

References 41 publications

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“…More conclusive evidence that the antithrombin re gion approximately from residues 100 to 145 participates in heparin binding has been provided by the characteriza tion of a congenital antithrombin variant with decreased heparin affinity, in which Arg 129 , a residue also impli cated by chemical modification, was substituted by Gln. 211,212 This residue may also be of importance for maintaining the active conformation of antithrombin. 212 Similarly, antithrombin variants with Leu 99 replaced by Phe or Ser 116 replaced with Pro showed decreased hep arin affinity, presumably because of local distortion of the structure of the heparin-binding region.…”

Section: Binding Of Heparin To Antithrombinmentioning

confidence: 99%

“…211,212 This residue may also be of importance for maintaining the active conformation of antithrombin. 212 Similarly, antithrombin variants with Leu 99 replaced by Phe or Ser 116 replaced with Pro showed decreased hep arin affinity, presumably because of local distortion of the structure of the heparin-binding region. 213,214 Further evidence has come from the isolation of heparin-binding antithrombin fragments comprising residues 104-251 215 or 114-156 216 after degradation of the protein with cy anogen bromide or V8 protease, respectively.…”

Section: Binding Of Heparin To Antithrombinmentioning

confidence: 99%

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Regulation of Thrombin Activity by Antithrombin and Heparin

Olson¹,

Björk²

1994

Semin Thromb Hemost

136

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The central role of thrombin in blood clotting is well established. The classic function of thrombin in this pro cess is to cleave fibrinogen, a reaction that leads to for mation of the fibrin gel, the essential constituent of the blood clot. However, thrombin also has important regu latory functions in blood clotting. It thus activates the cofactors, Factor V and Factor VIII, of the clotting sys tem in a positive feedback loop that greatly accelerates the generation of the enzyme and thereby increases the rate of fibrin formation. In addition, thrombin can also retard blood clotting in a negative feedback loop. When bound to a membrane protein, thrombomodulin, on the endothelial cell surface, thrombin thus activates protein C, which in turn inactivates Factors V and VIII, thereby decreasing the rate of generation of thrombin and conse quently also of fibrin. A further function of thrombin is to activate Factor XIII, which then stabilizes the clot by covalent crosslinks. These divergent roles of thrombin in the clotting system suggest that precise control of the activity of the enzyme is of paramount importance for normal hemostasis.The activity of thrombin generated in the clotting process has been shown to be predominantly regulated by inactivation of the enzyme by four different plasma pro teinase inhibitors. Two of these, α 1 -proteinase inhibitor and α 2 -macroglobulin, are general proteinase inhibitors that can inactivate a number of enzymes, whereas the other two, antithrombin and heparin cofactor II, are more specific for thrombin and, in the case of antithrombin, also other coagulation proteinases. The latter two inhibi tors also have in common that the rate of their inactiva tion of thrombin is greatly accelerated by the binding of heparin and certain similar glycosaminoglycans.In this work we will review the structure and func tion of antithrombin, the physiologically most important of the two specific thrombin inhibitors. We will discuss the properties of antithrombin and how it inactivates thrombin and other clotting proteinases. We will also present current knowledge regarding the binding of hep arin to antithrombin and thrombin or other proteinases and the role of these interactions in the mechanism by which heparin accelerates the inhibition of the protein ases by the inhibitor. Finally, we will discuss the physio logical role of antithrombin and heparin-like polysaccha rides and how other proteins in plasma, on the vessel wall, or released by platelets and neutrophils may modu late the effect of heparin in accelerating proteinase inacti vation by antithrombin.

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Section: Binding Of Heparin To Antithrombinmentioning

confidence: 99%

Section: Binding Of Heparin To Antithrombinmentioning

confidence: 99%

Regulation of Thrombin Activity by Antithrombin and Heparin

Olson¹,

Björk²

1994

Semin Thromb Hemost

136

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“…Naturally occurring genetic variants have demonstrated important roles for Arg-24 (16), , and Arg-129 (21) in heparin and pentasaccharide binding. In addition, recombinant ATIII expressed in baby hamster kidney cells has been used to implicate Lys-125 in both pentasaccharide and longchain heparin binding (22).…”

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confidence: 99%

Lysine Residue 114 in Human Antithrombin III Is Required for Heparin Pentasaccharide-mediated Activation

Kridel

Knauer

1997

Journal of Biological Chemistry

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Recombinant native antithrombin III (ATIII) and two genetic variants with glutamine substitutions at lysine residues 114 and 139 were expressed in insect cells using a baculovirus-driven expression system. The purified proteins were used to evaluate the potential role(s) of these residues in the pentasaccharide-mediated activation of ATIII. The second order rate constants for the inhibition of factor Xa by both of the genetic variants were nearly identical to those of recombinant native ATIII, indicating that the glutamine substitutions did not result in serious protein conformational changes. The glutamine substitution at lysine 139 had no effect on the pentasaccharide-mediated activation of ATIII toward factor Xa. In contrast, lysine 114 was found to be critical in the activation of ATIII toward factor Xa. No activation was observed, even at a pentasaccharide concentration 10 times higher than that required to activate recombinant native ATIII. These data are the first to demonstrate a pivotal role for lysine 114 in the pentasaccharide-mediated activation of ATIII.The D-helix and adjacent regions in ATIII 1 are important for the binding of long-chain heparin and heparin pentasaccharide and the subsequent activation of ATIII toward thrombin and factor Xa (1-4). Whereas the heparin pentasaccharide is sufficient to induce the complete activation of ATIII toward factor Xa, longer-chain heparins are required to achieve complete activation of ATIII toward thrombin (5-9). Current models also suggest that the pentasaccharide is sufficient to fully induce the conformational changes in ATIII associated with activation and that the requirement of longer heparin chains for the full activation of ATIII toward thrombin is completely attributable to template activity (10 -15). This point remains controversial and is most directly contradicted by a study that demonstrated the activation of ATIII toward thrombin by a Fab fragment of an antibody that specifically recognized residues 138 -145 in ATIII (4).Naturally occurring genetic variants have demonstrated important roles for Arg-24 (16), , and Arg-129 (21) in heparin and pentasaccharide binding. In addition, recombinant ATIII expressed in baby hamster kidney cells has been used to implicate Lys-125 in both pentasaccharide and longchain heparin binding (22).1 H NMR studies (5-9) and the crystal structure of a dimerized form of ATIII (23), along with the biochemical and genetic evidence, have led to a proposed pentasaccharide binding site that includes Arg-24, Arg-47, Lys-125, and Lys-129 (23). However, the potential roles of other positively charged residues in this region have not been ruled out. Recently a definitive role for Lys-114 and Lys-139 in longchain heparin binding and ATIII activation was demonstrated using recombinant ATIII expressed in insect cells (24). These residues lie at opposite ends of the D-helix, outside of the proposed pentasaccharide binding pocket, precluding simultaneous binding to the pentasaccharide. This suggests the mechanistic role of these two...

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“…Further characterization showed reduced specific heparin-cofactor activity (1.5 U/mL instead of 5 to 10 U/mL for normal AT) and a normal ability to form complexes with tnrombin (not shown), as already described for this mutation. 31 -34 ' 38 The other variant, which did not bind to heparin sepharose, migrated as a high-molecular-mass species in the absence of reducing agent but had the normal molecular mass of AT after reduction. As in the other two cases described here, this suggests that the mutated AT has a free Cys available for disulfide bonding with other molecules.…”

Section: Discussionmentioning

confidence: 99%

Three novel mutations of antithrombin inducing high-molecular-mass compounds.

Emmerich

Vidaud

Alhenc‐Gelas

et al. 1994

Arterioscler Thromb

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We have identified three novel mutations of the antithrombin (AT) gene in patients with thrombotic complications: a Cys 128 -» Tyr mutation, a G -» A mutation in the intervening sequence 4 (FVS4) 14 nucleotide 5' to exon 5, and a 9 bp deletion in the 3' end of exon 6 resulting in a short aberrant sequence after Arg 425. The latter mutation was associated with an Arg 47 -• His mutation in two compound heterozygous brothers. These three mutations led to the expression in the circulation of small amounts of inactive molecules with a high molecular mass in immunoblot analysis. In reducing conditions, these variant molecules had a normal molecular mass, which led us to postulate that these mutations prevent the formation of one intramolecular disulfidc bond and allow the formation of intermolecular disulfide bonds. Plasma from a heterozygous patient bearing the Cys 128 -• A ntithrombin (AT) is the main natural inhibitor of /\ thrombin and other coagulation proteases, and X A. its physiological importance was first shown in 1965 by the description of a hereditary AT deficiency in a family with unexplained thrombosis.1 The inhibitory effect of AT, due to the almost irreversible trapping of target proteases, is strongly enhanced by natural glycosaminoglycans such as heparin.The AT gene, the nucleotide sequence of which has recently been elucidated, 2 maps more than 13 480 bp, comprises 7 exons (1, 2, 3a, 3b, 4, 5, and 6), and encodes a polypeptide of 464 amino acids. Several intracellular modifications occur before the secretion of the folded protein, such as the formation of three intramolecular disulfide bonds in positions 8-128, 21-95, and 247-430 of the amino-acid sequence, glycosylation at four sites, and cleavage of a 32-amino-acid signal peptide (for a review see Reference 3). The structure of the mature protein presents large homologies with other serine protease inhibitors, forming a superfamily called serpins. Crystallographic study of cleaved a-1-antitrypsin provided a working model of three-dimensional serpin structure, 4 the validity of which was recently confirmed by crystal

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Arg-129 plays a specific role in the confirmation of antithrombin and in the enhancement of factor Xa inhibition by the pentasaccharode sequence of heparin

Cited by 7 publications

References 41 publications

Regulation of Thrombin Activity by Antithrombin and Heparin

Regulation of Thrombin Activity by Antithrombin and Heparin

Lysine Residue 114 in Human Antithrombin III Is Required for Heparin Pentasaccharide-mediated Activation

Three novel mutations of antithrombin inducing high-molecular-mass compounds.

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