The Dynamic Functional Consequences of the Thrombin-Thrombomodulin Interaction

Abstract

The coagulation cascade is critical to wound healing, but must be well regulated to prevent pathological clot formation. We previously investigated the backbone dynamics of a key component in this cascade, thrombin, and found that the loops surrounding the thrombin active site show dynamics on the ps-ns timescale when the active site is inhibitor-bound, but have dynamics on the μs-ms timescale when the active site is unoccupied. Thrombomodulin (TM) plays a key role in the regulation of the coagulation cascade by switching the function of thrombin from procoagulant to anticoagulant. A current molecular dynamics study suggests TM does this by influencing the slow timescale dynamics of thrombin, but the actual timescale of these motions is currently unknown. NMR measurements of chemical shift differences (CSDs) can be used to probe changes in the conformational ensemble upon TM binding. Other NMR experiments probe the timescales of motion including R1 and R2 relaxation experiments, which probe the ps-ns time regime, and TROSY Hahn Echo and CPMG experiments, which probe slow (μs-ms) timescale motions. To perform these NMR studies, our lab has developed a truncation of human TM (TM456m) with complete thrombin-altering activity. We have produced 15N-labeled human thrombin using an E. coli expression system and have collected HSQCs of unbound 15N-thrombin and 15N-thrombin in 1:1 complex with unlabeled TM456m. Binding of TM456m induces significant CSDs and differences in peak intensities throughout thrombin. Several residues in the TM binding site, including S36A, L65, I82, M84, and K110, become significantly perturbed upon binding. These perturbations propagate to several residues near the active site, including H57 and D102 of the catalytic triad. We hope that a complete understanding of how TM alters the dynamics and function of thrombin will aid in the design of safer anticoagulant drugs.