Antithrombins Southport (Leu 99 to Val) and Vienna (Cln 118 to Pro): two novel antithrombin variants with abnormal heparin binding

Chowdhury, Vijoy; Mille, B.; Olds, R.J.; Lane, David A.; Watton, Jane; Barrowcliffe, T W; Pabinger, Ingrid; Woodcock, B. E.; Thein, SL

doi:10.1111/j.1365-2141.1995.tb08369.x

Cited by 13 publications

(4 citation statements)

References 43 publications

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“…5) (34). Two naturally occurring mutants disrupt P-helix formation due to substitutions of serine 116 or glutamine 118 with the imino acid proline (35,36). S116P and Q118P variant antithrombins exhibit heparin binding defects and cause thrombosis, suggesting that P-helix formation is important for heparin binding and for the physiological expression of antithrombin activity.…”

Section: Phe-121 and Phe-122 Contributions To The Non-ionic Componentmentioning

confidence: 99%

Antithrombin III Phenylalanines 122 and 121 Contribute to Its High Affinity for Heparin and Its Conformational Activation

Jairajpuri

Desai

et al. 2003

Journal of Biological Chemistry

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The dissociation equilibrium constant for heparin binding to antithrombin III (ATIII) is a measure of the cofactor's binding to and activation of the proteinase inhibitor, and its salt dependence indicates that ionic and non-ionic interactions contribute ϳ40 and ϳ60% of the binding free energy, respectively. We now report that phenylalanines 121 and 122 (Phe-121 and Phe-122) together contribute 43% of the total binding free energy and 77% of the energy of non-ionic binding interactions. The large contribution of these hydrophobic residues to the binding energy is mediated not by direct interactions with heparin, but indirectly, through contacts between their phenyl rings and the non-polar stems of positively charged heparin binding residues, whose terminal amino and guanidinium groups are thereby organized to form extensive and specific ionic and non-ionic contacts with the pentasaccharide. Investigation of the kinetics of heparin binding demonstrated that Phe-122 is critical for promoting a normal rate of conformational change and stabilizing AT*H, the high affinity-activated binary complex. Kinetic and structural considerations suggest that Phe-122 and Lys-114 act cooperatively through non-ionic interactions to promote P-helix formation and ATIII binding to the pentasaccharide. In summary, although hydrophobic residues Phe-122 and Phe-121 make minimal contact with the pentasaccharide, they play a critical role in heparin binding and activation of antithrombin by coordinating the P-helixmediated conformational change and organizing an extensive network of ionic and non-ionic interactions between positively charged heparin binding site residues and the cofactor.The sulfated polysaccharide heparin functions as an anticoagulant by binding to antithrombin III (ATIII) 1 and greatly accelerating its rates of thrombin and factor Xa inhibition.Heparin binding to ATIII is a two-step process consisting of an initial weak interaction that induces a protein conformational change leading to the formation of a high affinity binary complex with the cofactor (AT*H) and ATIII activation (1, 2). Functional investigations of chemically modified, naturally occurring mutant and recombinant antithrombins have identified Arg-47, Lys-114, Lys-125, and Arg-129 as the most important positively charged amino acid residues in the heparin binding site of . Direct interactions of these basic residues with negatively charged groups of heparin are observed in the crystal structure of an ATIII-pentasaccharide complex (13). Studies of heparin binding kinetics indicate that Lys-125 is the most important amino acid in the initial docking with heparin and that Arg-129, Lys-114, and Arg-47 are critical for the protein conformational change step leading to the high affinity, activated AT*H complex. Heparin binding leads to elongation of ATIII helix D by 1.5 turns at its carboxyl-terminal end as well as the formation of a new alpha helix, the P-helix, at its amino-terminal end (13,14). These structural changes are associated with expulsion of the P14...

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Section: Phe-121 and Phe-122 Contributions To The Non-ionic Componentmentioning

confidence: 99%

Antithrombin III Phenylalanines 122 and 121 Contribute to Its High Affinity for Heparin and Its Conformational Activation

Jairajpuri

Desai

et al. 2003

Journal of Biological Chemistry

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“…Reproduced with permission from Chowdhury et al 1995. 49 apparent type I deficiency with moderately reduced activity and antigen levels due to cumulative loss rather than a deficiency of reduced synthesis. 56 The index patient presented with an ileofemoral thrombosis at 10 years of age in association with a preceding pyrexial respiratory infection thought to have induced sudden, rapid confirmational change.…”

Section: Unusual Antithrombinsmentioning

confidence: 99%

“…The gel is then rotated by 90 degrees and the AT is electrophoresed into a second agarose gel containing a precipitating antibody to AT, the AT mobility being visualized by drying and staining the gel. 40 49 AT bound to heparin has increased electrophoretic mobility and migrates further in the first dimension than AT with an HBS defect, thereby forming two distinct peaks in the second dimension ( Fig. 2 ).…”

Section: Antithrombinmentioning

confidence: 99%

Thrombophilia Screening: Not So Straightforward

Moore

2024

Semin Thromb Hemost

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Although inherited thrombophilias are lifelong risk factors for a first thrombotic episode, progression to thrombosis is multifactorial and not all individuals with inherited thrombophilia develop thrombosis in their lifetimes. Consequently, indiscriminate screening in patients with idiopathic thrombosis is not recommended, since presence of a thrombophilia does not necessarily predict recurrence or influence management, and testing should be selective. It follows that a decision to undertake laboratory detection of thrombophilia should be aligned with a concerted effort to identify any significant abnormalities, because it will inform patient management. Deficiencies of antithrombin and protein C are rare and usually determined using phenotypic assays assessing biological activities, whereas protein S deficiency (also rare) is commonly detected with antigenic assays for the free form of protein S since available activity assays are considered to lack specificity. In each case, no single phenotypic assay is capable of detecting every deficiency, because the various mutations express different molecular characteristics, rendering thrombophilia screening repertoires employing one assay per potential deficiency, of limited effectiveness. Activated protein C resistance (APCR) is more common than discrete deficiencies of antithrombin, protein C, and protein S and also often detected initially with phenotypic assays; however, some centres perform only genetic analysis for factor V Leiden, as this is responsible for most cases of hereditary APCR, accepting that acquired APCR and rare F5 mutations conferring APCR will go undetected if only factor V Leiden is evaluated. All phenotypic assays have interferences and limitations, which must be factored into decisions about if, and when, to test, and be given consideration in the laboratory during assay performance and interpretation. This review looks in detail at performance and limitations of routine phenotypic thrombophilia assays.

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“…Detailed biochemical studies show that, although the heparin−antithrombin interaction appears to be primarily electrostatic, a significant component, ∼60%, of the binding energy arises from nonionic forces (). Several nonionic residues, including Pro41, Trp49, Leu99, Ser116, and Gln118, present near the binding site are known to introduce a heparin-binding defect when mutated ( − ). Tryptophan 49 was one of the first residues suggested to play an important role in heparin binding to antithrombin based on the effects of chemical modification of this residue ( , ).…”

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confidence: 99%

Importance of Tryptophan 49 of Antithrombin in Heparin Binding and Conformational Activation

et al. 2005

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Tryptophan 49 of antithrombin, the primary inhibitor of blood clotting proteinases, has previously been implicated in binding the allosteric activator, heparin, by chemical modification and mutagenesis studies. However, the X-ray cocrystal structure of the antithrombin-pentasaccharide complex shows that Trp49 does not contact the bound saccharide. Here, we provide a detailed thermodynamic and kinetic characterization of heparin binding to a Trp49 to Lys variant of antithrombin and suggest a model for how Trp49 participates in heparin binding and activation. Mutation of Trp49 to Lys resulted in substantial losses of 16-24% in heparin-binding energy at pH 7.4, I 0.15, and 25 degrees C. These losses were due to both the loss of one ionic interaction ( approximately 30%) and the loss of nonionic interactions ( approximately 70%). Rapid kinetics analyses showed that the mutation minimally affected the initial weak binding of heparin to antithrombin or the rate constant for the subsequent conformational activation of the serpin. Rather, the principal effect of the mutation was to increase the rate constant for reversal of the conformational activation step by 70-100-fold, thereby destabilizing the activated conformation. This destabilization could be accounted for by the disruption of a network of interactions involving Trp49, Glu50, and Lys53 of helix A and Ser112 of helix P, which stabilizes the activated conformation.

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Antithrombins Southport (Leu 99 to Val) and Vienna (Cln 118 to Pro): two novel antithrombin variants with abnormal heparin binding

Cited by 13 publications

References 43 publications

Antithrombin III Phenylalanines 122 and 121 Contribute to Its High Affinity for Heparin and Its Conformational Activation

Antithrombin III Phenylalanines 122 and 121 Contribute to Its High Affinity for Heparin and Its Conformational Activation

Thrombophilia Screening: Not So Straightforward

Importance of Tryptophan 49 of Antithrombin in Heparin Binding and Conformational Activation

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