Anion-binding exosite of human .alpha.-thrombin and fibrin(ogen) recognition

Fenton, John W.; Olson, T.; Zabrinski, Michael P.; Wilner, George D.

doi:10.1021/bi00418a066

Cited by 182 publications

(109 citation statements)

References 42 publications

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“…Such multifunctional characteristics of thrombin are connected to its ability to interact with various proteins and cell receptors through a number of binding sites on its surface (17). These include the anion-binding exosite, which interacts with various anionic compounds and proteins and is involved in fibrin(ogen) binding (19,20). The crystal structure of thrombin revealed two positively charged patches on its surface (21,22).…”

mentioning

confidence: 99%

Crystal structure of the complex between thrombin and the central “E” region of fibrin

Pechik

Madrazo

Mosesson

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

114

104

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Nonsubstrate interactions of thrombin with fibrin play an important role in modulating its procoagulant activity. To establish the structural basis for these interactions, we crystallized D-Phe-ProArg-chloromethyl ketone-inhibited human thrombin in complex with a fragment, E ht, corresponding to the central region of human fibrin, and solved its structure at 3.65-Å resolution. The structure revealed that the complex consists of two thrombin molecules bound to opposite sides of the central part of E ht in a way that seems to provide proper orientation of their catalytic triads for cleavage of fibrinogen fibrinopeptides. As expected, binding occurs through thrombin's anion-binding exosite I. However, only part of it is involved in forming an interface with the complementary negatively charged surface of E ht. Among residues constituting the interface, Phe-34, Ser-36A, Leu-65, Tyr-76, Arg-77A, Ile-82, and Lys-110 of thrombin and the A␣ chain Trp-33, Phe-35, Asp-38, Glu-39, the B␤ chain Ala-68 and Asp-69, and the ␥ chain Asp-27 and Ser-30 of E ht form a net of polar contacts surrounding a well defined hydrophobic interior. Thus, despite the highly charged nature of the interacting surfaces, hydrophobic contacts make a substantial contribution to the interaction.

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mentioning

confidence: 99%

Crystal structure of the complex between thrombin and the central “E” region of fibrin

Pechik

Madrazo

Mosesson

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

114

104

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“…Exosite 1 was initially recognized as the fibrinogen binding site, but more recent studies indicate that it also binds hirudin, heparin cofactor II, and the thrombin receptor (5)(6)(7)(8)(9). The docking interaction at exosite 1, used by both substrates and inhibitors, precedes the reaction at the active site.…”

mentioning

confidence: 99%

Evidence for Allosteric Linkage between Exosites 1 and 2 of Thrombin

Fredenburgh

Stafford

Weitz

1997

Journal of Biological Chemistry

106

119

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Investigations to date have demonstrated that ligand binding to exosites 1 or 2 on thrombin produces conformational changes at the active site. In this study, we directly compared the effect of ligand binding to exosites 1 and 2 on the structure and function of the active site of thrombin and investigated functional linkage between the two exosites. Binding studies were performed in solution with fluorescein-Phe-Pro-Arg-CH 2 Cl (FPR)-thrombin. Hirudin-(54 -65) and sF2, a synthetic peptide corresponding to residues 63-116 of prothrombin fragment 2, were used as ligands for exosites 1 and 2 of thrombin, respectively. The two ligands produce diametric changes in the fluorescence of fluorescein-FPRthrombin and also have opposing effects on the rate of thrombin hydrolysis of a number of chromogenic substrates. These results indicate that sF2 and hirudin-(54 -65) differentially affect the conformation of the active site. Experiments then were performed to investigate whether both ligands can bind to thrombin simultaneously. When thrombin-bound fluorescein-sF2 is titrated with hirudin-(54 -65), complete displacement of fluorescein-sF2 is observed. Likewise, when thrombinbound fluorescein-hirudin-(54 -65) is titrated with sF2, complete displacement occurs. Additional support for reciprocal binding was obtained in fluorescence experiments where both probes were labeled and in experiments monitoring ligand binding to agarose-immobilized thrombin. This mutually exclusive binding of either ligand can be explained by reciprocal, allosteric modulation of ligand affinity between the two exosites. Thus, not only do the two exosites differentially influence the active site, they also affect the binding properties of the opposing exosite.Thrombin is a trypsin-like enzyme that plays a major role in hemostasis by regulating the procoagulant, anticoagulant, and fibrinolytic pathways (1, 2). A distinguishing feature of thrombin is the presence of two positively charged patches found on opposite sides of the thrombin molecule (3). These regions, termed anion binding exosites 1 and 2, contribute to the specificity of thrombin by serving as binding sites for substrates, cofactors, and other ligands that modulate thrombin activity (4).Exosite 1 was initially recognized as the fibrinogen binding site, but more recent studies indicate that it also binds hirudin, heparin cofactor II, and the thrombin receptor (5-9). The docking interaction at exosite 1, used by both substrates and inhibitors, precedes the reaction at the active site. The importance of exosite 1 is demonstrated by the reduced reactivity of ␥-thrombin, a proteolytic derivative of thrombin lacking exosite 1, with fibrinogen, hirudin, and heparin cofactor II (9, 10). A second role of exosite 1, revealed by crystallographic and fluorescence studies with hirudin or thrombomodulin, is to confer structural changes in the active site environment that facilitate subsequent binding interactions (11)(12)(13)(14)(15). In the case of hirudin, this serves to optimize alignment of the amin...

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“…Thrombin binds to fibrin directly through an interaction at its anion-binding exosite (46). With time, thrombin bound to or trapped within blood clots will leak out and the thrombin content in an aged clot will decrease gradually.…”

Section: Reshuffling Of Disulfide Bonds In Hirudin-e Coil and He-sakk-bmentioning

confidence: 99%

Engineering of a Staphylokinase-based Fibrinolytic Agent with Antithrombotic Activity and Targeting Capability toward Thrombin-rich Fibrin and Plasma Clots

Lian¹,

Szarka²,

et al. 2003

Journal of Biological Chemistry

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Current clinically approved thrombolytic agents have significant drawbacks including reocclusion and bleeding complications. To address these problems, a staphylokinase-based thrombolytic agent equipped with antithrombotic activity from hirudin was engineered. Because the N termini for both staphylokinase and hirudin are required for their activities, a Y-shaped molecule is generated using engineered coiled-coil sequences as the heterodimerization domain. This agent, designated HE-SAKK, was produced and assembled from Bacillus subtilis via secretion using an optimized co-cultivation approach. After a simple in vitro treatment to reshuffle the disulfide bonds of hirudin, both staphylokinase and hirudin in HE-SAKK showed biological activities comparable with their parent molecules. This agent was capable of targeting thrombin-rich fibrin clots and inhibiting clot-bound thrombin activity. The time required for lysing 50% of fibrin clot in the absence or presence of fibrinogen was shortened 21 and 30%, respectively, with HE-SAKK in comparison with staphylokinase. In plasma clot studies, the HE-SAKK concentration required to achieve a comparable 50% clot lysis time was at least 12 times less than that of staphylokinase. Therefore, HE-SAKK is a promising thrombolytic agent with the capability to target thrombin-rich fibrin clots and to minimize clot reformation during fibrinolysis.

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Anion-binding exosite of human .alpha.-thrombin and fibrin(ogen) recognition

Cited by 182 publications

References 42 publications

Crystal structure of the complex between thrombin and the central “E” region of fibrin

Crystal structure of the complex between thrombin and the central “E” region of fibrin

Evidence for Allosteric Linkage between Exosites 1 and 2 of Thrombin

Engineering of a Staphylokinase-based Fibrinolytic Agent with Antithrombotic Activity and Targeting Capability toward Thrombin-rich Fibrin and Plasma Clots

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