Regulation of Thrombin by Antithrombin and Heparin Cofactor II

Olson, Steven T.; Björk, Ingemar

doi:10.1007/978-1-4615-3296-5_5

Cited by 49 publications

(60 citation statements)

References 275 publications

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“…However, upon cleavage, a conformational change in the inhibitor has been proposed to result in the trapping of the enzyme either as a tetrahedral intermediate or as an acyl-enzyme. Unlike the intermediate typically formed with substrates, the intermediate formed between thrombin and ATIII is stable and resistant to hydrolysis perhaps due to the conformational change which may exclude water from the active site (9).…”

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confidence: 99%

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Functional Requirements for Inhibition of Thrombin by Antithrombin III in the Presence and Absence of Heparin

Tsiang

Jain

Gibbs

1997

Journal of Biological Chemistry

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Mutation of 79 highly exposed amino acids that comprise approximately 62% of the solvent accessible surface of thrombin identified residues that modulate the inhibition of thrombin by antithrombin III, the principal physiological inhibitor of thrombin. Mutations that decreased the susceptibility of thrombin to inhibition by antithrombin III in the presence and absence of heparin (W50A, E229A, and R233A) also decreased hydrolysis of a small tripeptidyl substrate. These residues were clustered around the active site cleft of thrombin and were predicted to interact directly with the "substrate loop" of antithrombin III. Despite the large size of antithrombin III (58 kDa), no residues outside of the active cleft were identified that interact directly with antithrombin III. Mutations that decreased the susceptibility of thrombin to inhibition by antithrombin III in the presence but not in the absence of heparin (R89A/R93A/ E94A, R98A, R245A, K248A, K252A/D255A/Q256A) in general did not also affect hydrolysis of the tripeptidyl substrate. These residues were clustered among a patch of basic residues on a surface of thrombin perpendicular to the face containing the active site cleft and were predicted to interact directly with heparin. Three mutations (E25A, R178A/R180A/D183A, and E202A) caused a slight enhancement of inhibition by antithrombin III.Vascular injury or inflammation results in the activation of thrombin (1, 2). Thrombin cleaves and activates multiple substrates to mediate hemostasis through fibrin clot formation and platelet aggregation (3-7) and also regulates blood coagulation by activating the anticoagulant protein C pathway (8). The predominant mechanism for the clearance of thrombin is through inhibition by antithrombin III (ATIII), 1 a member of the family of serine protease inhibitors known as serpins (1, 9 -11). The physiological importance of this process is illustrated by the observation that individuals possessing inherited ATIII deficiency or insufficiency are subject to recurrent thrombosis (12).ATIII is a 58-kDa glycoprotein that inhibits thrombin in two kinetically distinct steps: the formation of a weak initial complex followed by rapid conversion to a stable complex (13). Based on the crystal structures of ATIII (14) and other serpins in cleaved (15, 16) and uncleaved (17) forms, inhibition has been proposed to proceed through the recognition of the Arg 393 -Ser 394 bond in an exposed loop of ATIII as a substrate. However, upon cleavage, a conformational change in the inhibitor has been proposed to result in the trapping of the enzyme either as a tetrahedral intermediate or as an acyl-enzyme. Unlike the intermediate typically formed with substrates, the intermediate formed between thrombin and ATIII is stable and resistant to hydrolysis perhaps due to the conformational change which may exclude water from the active site (9).The rate-limiting step in the inhibition of thrombin by ATIII is the formation of the initial complex (ϳ2 ϫ 10 5 M Ϫ1 min Ϫ1 ) which can be accelerated by 3 orders of...

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confidence: 99%

“…Mutations that affect inhibition of thrombin appear to fall into three functional classes that are spatially distinct (9,10,12,16,18). Mutations that map to the substrate loop of ATIII perturb direct interactions with thrombin and result in diminished potency or altered specificity of inhibition.…”

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confidence: 99%

Functional Requirements for Inhibition of Thrombin by Antithrombin III in the Presence and Absence of Heparin

Tsiang

Jain

Gibbs

1997

Journal of Biological Chemistry

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“…ATIII inhibitory activity can be potentiated, however, by heparin-like molecules (15). The physiological source of cofactor activity is heparan sulfate proteoglycans on and under the endothelium, which serve to localize and concentrate antithrombin on the vessel wall where activated coagulation enzymes are generated (16,17).…”

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confidence: 99%

Identification of the Antithrombin III Heparin Binding Site

Ersdal-Badju

Zuo

et al. 1997

Journal of Biological Chemistry

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The heparin binding site of the anticoagulant protein antithrombin III (ATIII) has been defined at high resolution by alanine scanning mutagenesis of 17 basic residues previously thought to interact with the cofactor based on chemical modification experiments, analysis of naturally occurring dysfunctional antithrombins, and proximity to helix D. The baculovirus expression system employed for this study produces antithrombin which is highly similar to plasma ATIII in its inhibition of thrombin and factor Xa and which resembles the naturally occurring ␤-ATIII isoform in its interactions with high affinity heparin and pentasaccharide (Ersdal-Badju, E., Lu, A., Peng, X., Picard, V., Zendehrouh, P., Turk, B., Bjö rk, I., Olson, S. T., and Bock, S. C. (1995) Biochem. J. 310, 323-330). Relative heparin affinities of basic-to-Ala substitution mutants were determined by NaCl gradient elution from heparin columns. The data show that only a subset of the previously implicated basic residues are critical for binding to heparin. The key heparin binding residues, Lys-11, Arg-13, Arg-24, Arg-47, Lys-125, Arg-129, and Arg-145, line a 50-Å long channel on the surface of ATIII. Comparisons of binding residue positions in the structure of P14-inserted ATIII and models of native antithrombin, derived from the structures of native ovalbumin and native antichymotrypsin, suggest that heparin may activate antithrombin by breaking salt bridges that stabilize its native conformation. Specifically, heparin release of intramolecular helix D-sheet B salt bridges may facilitate s123AhDEF movement and generation of an activated species that is conformationally primed for reactive loop uptake by central ␤-sheet A and for inhibitory complex formation. In addition to providing a structural explanation for the conformational change observed upon heparin binding to antithrombin III, differences in the affinities of native, heparin-bound, complexed, and cleaved ATIII molecules for heparin can be explained based on the identified binding site and suggest why heparin functions catalytically and is released from antithrombin upon inhibitory complex formation.

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“…The proteolytic activity of coagulation proteases of both the intrinsic and extrinsic clotting cascades is primarily regulated by the serpin inhibitor antithrombin (AT) in plasma (1)(2)(3)(4)(5). However, AT is a poor inhibitor of coagulation proteases unless it is bound to heparin-like glycosaminoglycans found on the surface of the endothelium (1,6).…”

Section: Introductionmentioning

confidence: 99%

“…First, the interaction of heparin with a basic region on AT induces a conformational change in the structure of the L. Yang et al www.bjournal.com.br serpin, thereby facilitating its recognition by the coagulation enzymes (allosteric activation mechanism) (9,10). Second, heparin can bind to a basic exosite on coagulation proteases, thus being capable of bridging AT and the target protease in one complex, thereby decreasing the dissociation constant for the initial interaction of two proteins (template mechanism) (3,5). The extent of the contribution of each mechanism to the rateaccelerating effect of heparin in protease inactivation by AT has been extensively studied.…”

Section: Introductionmentioning

confidence: 99%

The role of autolysis loop in determining the specificity of coagulation proteases

Yang

Manithody

Rezaie

2007

Braz J Med Biol Res

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We recently demonstrated that the substitution of the autolysis loop (residues 143 to 154 in the chymotrypsin numbering system) of activated protein C (APC) with the corresponding loop of factor Xa (fXa) renders the APC mutant (APC/fX 143-154 ) susceptible to inhibition by antithrombin (AT) in the presence of pentasaccharide. Our recent results further indicated, that in addition to an improvement in the reactivity of APC/fX 143-154 with AT, both the amidolytic and antifactor Va activities of the mutant APC have also been significantly increased. Since the autolysis loop of APC is five residues longer than the autolysis loop of fXa, it could not be ascertained whether this loop in the mutant APC specifically interacts with the activated conformation of AT or if a shorter autolysis loop is responsible for a global improvement in the catalytic activity of the mutant protease. To answer this question, we prepared another APC mutant in which the autolysis loop of the protease was replaced with the corresponding loop of trypsin (APC/Tryp 143-154 ). Unlike an ~500-fold improvement in the reactivity of APC/fX [143][144][145][146][147][148][149][150][151][152][153][154] with AT in the presence of pentasaccharide, the reactivity of APC/Tryp 143-154 with the serpin was improved ~10-fold. These results suggest that both the length and structure of residues of the autolysis loop are critical for the specificity of the coagulation protease interaction with AT. Further factor Va inactivation studies with the APC mutants revealed a similar role for the autolysis loop of APC in the interaction with its natural substrate. Correspondence

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Regulation of Thrombin by Antithrombin and Heparin Cofactor II

Cited by 49 publications

References 275 publications

Functional Requirements for Inhibition of Thrombin by Antithrombin III in the Presence and Absence of Heparin

Functional Requirements for Inhibition of Thrombin by Antithrombin III in the Presence and Absence of Heparin

Identification of the Antithrombin III Heparin Binding Site

The role of autolysis loop in determining the specificity of coagulation proteases

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