The Structure of a Complex of Recombinant Hirudin and Human α-Thrombin

Rydel, T.J.; Ravichandran, K. G.; Tulinsky, A.; Bode, Wolfram; Huber, Robert; Roitsch, C.; Fenton, John W.

doi:10.1126/science.2374926

Cited by 779 publications

(684 citation statements)

References 37 publications

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“…The C-terminal hirudin residues bind to thrombin in the fibrinogen exosite (Griitter et al, 1990;Rydel et al, 1990Rydel et al, , 1991Stubbs et al, 1992). This exosite, extending from the active-site cleft ( Fig.…”

Section: The Ternary Complexmentioning

confidence: 99%

Changes in interactions in complexes of hirudin derivatives and human α‐thrombin due to different crystal forms

et al. 1993

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The three-dimensional structures of D-Phe-Pro-Arg-chloromethyl ketone-inhibited thrombin in complex with Tyr-63-sulfated hirudin"-65 (ternary complex) and of thrombin in complex with the bifunctional inhibitor D-PhePro-Arg-Pr~-(Gly)~-hirudin~~-~' (CGP 50,856, binary complex) have been determined by X-ray crystallography in crystal forms different from those described by Skrzypczak-Jankun et al. (Skrzypczak-Jankun, E., Carperos, V.E., Ravichandran, K.G., & Tulinsky, A., 1991, J. Mol. Biol. 221, 1379-1393). In both complexes, the interactions of the C-terminal hirudin segments of the inhibitors binding to the fibrinogen-binding exosite of thrombin are clearly established, including residues 60-64, which are disordered in the earlier crystal form. The interactions of the sulfate group of Tyr-63 in the ternary complex structure explain why natural sulfated hirudin binds with a 10-fold lower Ki than the desulfated recombinant material. In this new crystal form, the autolysis loop of thrombin (residues 146-150), which is disordered in the earlier crystal form, is ordered due to crystal contacts. Interactions between the C-terminal fragment of hirudin and thrombin are not influenced by crystal contacts in this new crystal form, in contrast to the earlier form. In the bifunctional inhibitor-thrombin complex, the peptide bond between Arg-Pro (Pl-Pl') seems to be cleaved.

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Section: The Ternary Complexmentioning

confidence: 99%

Changes in interactions in complexes of hirudin derivatives and human α‐thrombin due to different crystal forms

et al. 1993

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“…Thrombin has two basic anion-binding exosites (exosite I and II) that bind to all natural cofactors and substrates of thrombin. Exosite I binds fibrin, hirudin 18 , Heparin Cofactor II, PARs, thrombomodulin and various coagulation factors 16,19 . Exosite II is the major heparin-binding site 20 , but can also interact with highly sulfated polysaccharides and g 0 -fibrinogen 21 .…”

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confidence: 99%

Thrombin-dependent intravascular leukocyte trafficking regulated by fibrin and the platelet receptors GPIb and PAR4

et al. 2015

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Thrombin is a central regulator of leukocyte recruitment and inflammation at sites of vascular injury, a function thought to involve primarily endothelial PAR cleavage. Here we demonstrate the existence of a distinct leukocyte-trafficking mechanism regulated by components of the haemostatic system, including platelet PAR4, GPIba and fibrin. Utilizing a mouse endothelial injury model we show that thrombin cleavage of platelet PAR4 promotes leukocyte recruitment to sites of vascular injury. This process is negatively regulated by GPIba, as seen in mice with abrogated thrombin-platelet GPIba binding (hGPIba D277N ). In addition, we demonstrate that fibrin limits leukocyte trafficking by forming a physical barrier to intravascular leukocyte migration. These studies demonstrate a distinct 'checkpoint' mechanism of leukocyte trafficking involving balanced thrombin interactions with PAR4, GPIba and fibrin. Dysregulation of this checkpoint mechanism is likely to contribute to the development of thromboinflammatory disorders.

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“…Phes6 of hirudin binds into a lipophilic pocket on the surface of thrombin and is involved in an interaction with Phe34 of thrombin [4]. Phe" of hirullin P18 may interact with thrombin in a similar fashion particularly since it is flanked by Asp and Glu residues which are also involved in specific thrombin interactions in the hirudin-thrombin complex [4].…”

Section: Resultsmentioning

confidence: 99%

“…1). These differences are significant because the C-terminus of hirudin represents a highly conserved domain which binds to thrombin at a non-catalytic site [3,4]. This domain accounts for a significant portion of hirudin's overall binding energy to thrombin.…”

Section: Introductionmentioning

confidence: 99%

The C‐terminal binding domain of hirullin P18

Krstenansky¹,

Owen²,

Yates³

et al. 1990

FEBS Letters

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Hirullin P18 is a 61-amino acid hirudin-related protein having potent antithrombin activity. Similar to hirudin, it contains a highly acidic C-terminus, but has a significantly different sequence from any other known hirudin variant. The present study demonstrates that the C-terminal fragment acetyl-hirullin Pl8,,,,, p assesses an antithrombin potency similar to that of acetyl-desulfatohirududirL,,,. Additionally, like the hirudin fragment analog, it inhibits fibrin-clot formation by binding to a non-catalytic site on thrombin. Sequential shortening of the hirullin P18 C-terminal fragment demonstrates the critical nature of PheSi, which corresponds to the important Phd6 residue of hirudin. Although the sequences of hirullin Pl8,,, and hirudin,,, have substantial differences, the C-terminal functional domain represented by hirullin Pl8,,,, appears to be comparable to hirudins,,, in terms of its functional role in antithrombin activity.Hirullin P 18; Hirudin; Synthetic peptide; Thrombin

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The Structure of a Complex of Recombinant Hirudin and Human α-Thrombin

Cited by 779 publications

References 37 publications

Changes in interactions in complexes of hirudin derivatives and human α‐thrombin due to different crystal forms

Changes in interactions in complexes of hirudin derivatives and human α‐thrombin due to different crystal forms

Thrombin-dependent intravascular leukocyte trafficking regulated by fibrin and the platelet receptors GPIb and PAR4

The C‐terminal binding domain of hirullin P18

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