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Structural basis for the anticoagulant activity of the thrombin–thrombomodulin complex

Abstract

The serine proteinase α-thrombin causes blood clotting through proteolytic cleavage of fibrinogen and protease-activated receptors and amplifies its own generation by activating the essential clotting factors V and VIII1. Thrombomodulin2, a transmembrane thrombin receptor with six contiguous epidermal growth factor-like domains (TME1–6), profoundly alters the substrate specificity of thrombin from pro- to anticoagulant by activating protein C (see, for example, reference 2). Activated protein C then deactivates the coagulation cascade by degrading activated factors V and VIII2. The thrombin–thrombomodulin complex inhibits fibrinolysis by activating the procarboxypeptidase thrombin-activatable fibrinolysis inhibitor3. Here we present the 2.3 Å crystal structure of human α-thrombin bound to the smallest thrombomodulin fragment required for full protein-C co-factor activity, TME456. The Y-shaped thrombomodulin fragment binds to thrombin's anion-binding exosite-I, preventing binding of procoagulant substrates. Thrombomodulin binding does not seem to induce marked allosteric structural rearrangements at the thrombin active site. Rather, docking of a protein C model to thrombin–TME456 indicates that TME45 may bind substrates in such a manner that their zymogen-activation cleavage sites are presented optimally to the unaltered thrombin active site.

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Figure 1: The protein C anticoagulant pathway.
Figure 2: Thrombomodulin is not an allosteric modulator of thrombin.
Figure 3: TME456 exhibits a novel arrangement of EGF-like repeats.
Figure 4: Sequence and secondary structure of the human TME456 fragment.
Figure 5: The thrombin–thrombomodulin interaction interface is dominated by hydrophobic contacts.
Figure 6: Hypothetical structure of the thrombin–thrombomodulin–protein C complex.

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Acknowledgements

We thank J. McRobbie, Berlex Biosciences, for help and advice on the production of the medium containing TME456, G. Bourenkov for help during data collection at DESY, R. Mentele for sequencing the crystallized material, and T. Mather and M. Bauer for initial crystallization attempts. W.B. acknowledges the financial support of the EU research programs ‘Biomed’, ‘Training and Mobility’ and ‘Biotechnology’, HSFP, the Fonds der Chemischen Industrie and the Sonderforschungsbereich 469.

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Correspondence to Wolfram Bode.

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Fuentes-Prior, P., Iwanaga, Y., Huber, R. et al. Structural basis for the anticoagulant activity of the thrombin–thrombomodulin complex. Nature 404, 518–525 (2000). https://doi.org/10.1038/35006683

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