Partial Activation of Antithrombin without Heparin through Deletion of a Unique Sequence on the Reactive Site Loop of the Serpin

Rezaie, Alireza R.

doi:10.1074/jbc.m108544200

Cited by 14 publications

(18 citation statements)

References 28 publications

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“…These results support the structural data that Arg 399 is a target for the conformational activation of the serpin by heparin (22). This observation is also consistent with our previous mutagenesis data showing that the deletion of this residue results in a decline in the reactivity of the protease with the mutant serpin specifically in the presence of pentasaccharide (42). The observation that the reactivities of both wild-type FIXa and FIXa-fX 39-loop with AT remained essentially identical in the presence of pentasaccharide, which is an order of magnitude lower than the rate of the FXa-AT reaction under identical conditions, suggests that the slower reactivity of FIXa with the activated conformation of the serpin is not due to differences in the residues of the 39-loop.…”

Section: At-wtsupporting

confidence: 93%

Role of the Residues of the 39-Loop in Determining the Substrate and Inhibitor Specificity of Factor IXa

Yang

Manithody

Qureshi

et al. 2010

Journal of Biological Chemistry

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The activation of antithrombin (AT) by heparin facilitates the exosite-dependent interaction of the serpin with factors IXa (FIXa) and Xa (FXa), thereby improving the rate of reactions by 300-to 500-fold. Relative to FXa, AT inhibits FIXa with ϳ40-fold slower rate constant. Structural data suggest that differences in the residues of the 39-loop (residues 31-41) may partly be responsible for the differential reactivity of the two proteases with AT. This loop is highly acidic in FXa, containing three Glu residues at positions 36, 37, and 39. By contrast, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and an Asp at positions 36 and 39, respectively. To determine whether differences in the residues of this loop contribute to the slower reactivity of FIXa with AT, we prepared an FIXa/FXa chimera in which the 39-loop of the protease was replaced with the corresponding loop of FXa. The chimeric mutant cleaved a FIXa-specific chromogenic substrate with normal catalytic efficiency, however, the mutant exhibited ϳ5-fold enhanced reactivity with AT specifically in the absence of the cofactor, heparin. Further studies revealed that the FIXa mutant activates factor X with ϳ4-fold decreased k cat and ϳ2-fold decreased K m , although the mutant interacted normally with factor VIIIa. Based on these results we conclude that residues of the 39-loop regulate the cofactor-independent interaction of FIXa with its physiological inhibitor AT and substrate factor X.Factor IXa (FIXa) 2 is a vitamin K-dependent plasma serine protease that, upon complex formation with factor VIIIa (FVIIIa), on negatively charged membrane surfaces in the presence of Ca 2ϩ (intrinsic Tenase) activates factor X (FX) to factor Xa (FXa) during the blood coagulation process (1-5). FIXa plays an important role in the clotting cascade, because its deficiency is associated with the life-threatening disease, hemophilia B (6). The activity of FIXa toward its physiological substrate FX is very poor in the absence of FVIIIa, however, complex formation with the cofactor improves the catalytic efficiency of FIXa by 4 -5 orders of magnitude in the intrinsic Tenase complex (1-5). The proteolytic activity of FIXa in plasma is regulated by the serpin inhibitor, antithrombin (AT) (7)(8)(9)(10)(11). Surprisingly, in contrast to a dramatic cofactor-mediated improvement in the activity of FIXa toward FX, FVIIIa has a minimal cofactor effect on the reactivity of the protease with AT or its activity toward small synthetic substrates (12, 13), suggesting that FVIIIa-mediated exosite interactions with FX, involving sites remote from the active-site pocket, play dominant roles in the catalytic reaction (14, 15). In the case of the FIXa reaction with AT, the cofactor function of heparin-like glycosaminoglycans, similar to those lining the surface of the endothelium (16), is required to facilitate the protease recognition of the serpin (17-19).Heparin accelerates the reactivity of AT with FIXa by two distinct mechanisms: a conformational ac...

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Section: At-wtsupporting

confidence: 93%

Role of the Residues of the 39-Loop in Determining the Substrate and Inhibitor Specificity of Factor IXa

Yang

Manithody

Qureshi

et al. 2010

Journal of Biological Chemistry

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“…Support for this proposal has recently been provided by a study which noted that antithrombin has a three-residue insertion on the primed side of the reactive loop when aligned with other serpins (34). Progressive deletion of these residues corresponding to the P6Ј-P8Ј positions resulted in partial activation of the inhibitor as judged from an enhanced basal rate of factor Xa inhibition, which is a sensitive indicator of activation.…”

Section: Fig 3 Sds and Nondenaturing Page Of Purified Normal And Vamentioning

confidence: 89%

Deletion of P1 Arginine in a Novel Antithrombin Variant (Antithrombin London) Abolishes Inhibitory Activity but Enhances Heparin Affinity and Is Associated with Early Onset Thrombosis

Raja¹,

Chhablani²,

Swanson³

et al. 2003

Journal of Biological Chemistry

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A novel variant of antithrombin, the major serpin inhibitor of coagulation proteases, has been identified in a patient with early onset thrombosis and abnormal plasma antithrombin activity. Sequencing of the antithrombin genes of the patient revealed that one of the two alleles was abnormal due to an in-frame deletion of the codon for the P1 arginine residue. The abnormal antithrombin was separated from the normal inhibitor by complexing the latter with thrombin followed by heparin-agarose affinity chromatography. The purified variant, antithrombin London, was completely inactive as a thrombin or factor Xa inhibitor even after heparin activation. Surprisingly, the variant bound heparin with a K D reflecting an ϳ10-fold greater affinity than the normal inhibitor. Stopped-flow kinetic analysis showed that this was almost entirely due to a more favorable conformational activation of the variant than the normal inhibitor, as reflected by a decreased rate constant for reversal of the activation. Consistent with its higher than normal heparin affinity, the inactive antithrombin variant was a potent competitive antagonist of the heparin-catalyzed reaction of normal antithrombin with thrombin but did not affect the uncatalyzed reaction. These results suggest that deletion of the antithrombin P1 residue partially activates the serpin by inducing strain in the reactive center loop, which destabilizes the native loop-buried state and favors the activated loopexposed state with high heparin affinity. The unusually severe thrombosis associated with the heterozygous mutation may be explained by the ability of antithrombin London to bind endogenous heparan sulfate or heparin molecules with high affinity and to thereby block activation of the normal inhibitor.

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“…Simultaneous deletion of two residues, P7 0 and P8 0 (Val400 and Thr401), from antithrombin's longer RCL increased the factor Xa inhibition rates nearly tenfold. 62 Yet, these deletions compromised the inhibitor's ability to inactivate thrombin. In combination, the mutational studies suggest that whereas the specificity of factor Xa and thrombin is dependent on the P1-P1 0 recognition site, it is weakly dependent on the flanking RCL residues and possibly more dependent on an unrecognized exosite on the inhibitor.…”

Section: A N T I T H R O M B I N M U T a N T Smentioning

confidence: 99%

New antithrombin‐based anticoagulants

Desai

2003

Medicinal Research Reviews

131

115

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Clinically used anticoagulants are inhibitors of enzymes involved in the coagulation pathway, primarily thrombin and factor Xa. These agents can be either direct or indirect inhibitors of clotting enzymes. Heparin-based anticoagulants are indirect inhibitors that enhance the proteinase inhibitory activity of a natural anticoagulant, antithrombin. Despite its phenomenal success, current anticoagulation therapy suffers from the risk of serious bleeding. The need for safer and more effective antithrombotic agents clearly exists. The past decade has seen enormous effort directed toward discovering and/or designing new molecules with anticoagulant activity. These new molecules can be classified into (a). antithrombin and its mutants, (b). natural polysaccharides, (c). synthetic modified heparins and heparin-mimics, (d). synthetic oligosaccharides, and (e). synthetic non-sugar antithrombin activators. This review focuses on these efforts in designing or discovering new molecules that act through the antithrombin pathway of anticoagulation.

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Partial Activation of Antithrombin without Heparin through Deletion of a Unique Sequence on the Reactive Site Loop of the Serpin

Cited by 14 publications

References 28 publications

Role of the Residues of the 39-Loop in Determining the Substrate and Inhibitor Specificity of Factor IXa

Role of the Residues of the 39-Loop in Determining the Substrate and Inhibitor Specificity of Factor IXa

Deletion of P1 Arginine in a Novel Antithrombin Variant (Antithrombin London) Abolishes Inhibitory Activity but Enhances Heparin Affinity and Is Associated with Early Onset Thrombosis

New antithrombin‐based anticoagulants

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