Novel heparin mimetics reveal cooperativity between exosite 2 and sodium-binding site of thrombin

Thrombin is a primary target of most anticoagulants. Yet, thrombin's dual and opposing role in pro- as well as anti- coagulant processes imposes considerable challenges in discovering finely tuned regulators that maintain homeostasis, rather than disproportionately changing the equilibrium to one side. In this connection, we have been studying exosite 2-mediated allosteric modulation of thrombin activity using synthetic agents called low molecular weight lignins (LMWLs). Although the aromatic scaffold of LMWLs is completely different from the polysaccharidic scaffold of heparin, the presence of multiple negatively charged groups on both ligands induces binding to exosite 2 of thrombin. This work characterizes the nature of interactions between LMWLs and thrombin to understand the energetic cooperativity between exosite 2 and active site of thrombin.

The thermodynamics of thrombin-LMWL complexes was studied using spectrofluorimetric titrations as a function of ionic strength and temperature of the buffer. The contributions of enthalpy and entropy to binding were evaluated using classic thermodynamic equations. Label-free surface plasmon resonance was used to assess the role of sodium ion in LMWL binding to thrombin at a fixed ionic strength.

Exosite 2-induced conformational change in thrombin's active site is strongly dependent on the structure of the ligand, which has consequences with respect to regulation of thrombin. The ionic and non-ionic contributions to binding affinity and the thermodynamic signature were highly ligand specific. Interestingly, LMWLs display preference for the sodium-bound form of thrombin, which supports the existence of an energetic coupling between exosite 2 and sodium-binding site of thrombin.