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

Abstract

The serine proteinase alpha-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 VIII. Thrombomodulin, 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. Activated protein C then deactivates the coagulation cascade by degrading activated factors V and VIII. The thrombin-thrombomodulin complex inhibits fibrinolysis by activating the procarboxypeptidase thrombin-activatable fibrinolysis inhibitor. Here we present the 2.3 A crystal structure of human alpha-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.

NMR resonance assignments of thrombin reveal the conformational and dynamic effects of ligation

“…In contrast, the C-terminal β-barrel appears to be entirely insensitive to the binding of hirugen, with the exception of the stabilization of C-terminal stock of the γ-loop. This observation explains why the crystal structure of thrombin bound to TM in the presence of active-site inhibitor (EGRCK) showed no conformational differences when compared to active-site inhibited thrombin (18). PPACK converts the very flexible and dynamic apo thrombin into a rigid and stable enzyme.…”

“…Binding of inhibitors to the active-site cleft increases the affinity of thrombin for exosite I ligands, confirming the thermodynamic linkage between the two sites (17). In spite of the evidence that TM binding alters the conformation of thrombin, crystallographic structures of the complex did not reveal any significant differences when compared to active-site inhibited thrombin (18,19). The reasons for the lack of conformational change are unclear, but may be due to the occupancy of the activesite cleft by a peptidyl inhibitor in the two structures.…”

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

“…55). Whether other thrombin-binding partners including antithrombin 56 , thrombomodulin, heparin cofactor II and glycosaminoglycans contribute to the modulation of thrombin's proinflammatory functions remains to be explored.…”

Thrombin's faces revealed: cellular effects include induction of neoangiogenesis

“…Thrombomodulin binds to thrombin's anion-binding exosite-I (preventing binding to procoagulant substrates such as ®brino-gen), without inducing allosteric structural recon®guration at the catalytic triad comprising thrombin's active site. Rather, the thrombin/thrombomodulin complex provides an additional substrate binding site for protein C docking, while promoting association (with subsequent activation) of PC's scissile peptide bond within the thrombin active site [4].…”