Thrombosis is one of the leading causes of mortality and morbidity that is associated with myocardial infarction, stroke and pulmonary embolism. Anti-thrombotic agents which intend to reduce the occurrence and severity of thrombosis usually target the enzymes of the coagulation cascade. FXIa, a 160 kDa homodimer is gaining popularity of late as a potential target for anti-thrombotic agents due to its relative safety. A number of inhibitors which target the active site of FXIa have been reported but to our knowledge there have been no inhibitors which act via an allosteric mechanism. The aim of this project was to screen for allosteric inhibitors of FXIa from of pool of sulfated small-molecules.These molecules were primarily designed to act as heparin mimetics; heparin being a natural anti-coagulant. These compounds were then analyzed to determine whether inhibition was via an allosteric mechanism.

Chemoenzymatically synthesized low molecular weight lignin polymers have been previously found to be potent inhibitors of a number of serine proteases via allosteric mechanisms targeting heparin binding sites. Herein, we describe the creation of synthetic sulfated β-O4 lignin (SbO4L) polymer, which is more homogenous compared to previous lignins with respect to its inter-monomeric linkage. SbO4L is a selective inhibitor of thrombin and plasmin. SbO4L was found to act via a unique mechanism targeting thrombin exosite 2 in a manner similar to platelet glycoprotein Ibα (GPIbα). Advanced hemostasis and thrombosis assays demonstrated that SbO4L acts via a dual mechanism: as an anticoagulant, by allosteric inhibition of thrombin catalysis; and as an antiplatelet agent, by competing with platelet GPIbα. These mechanisms are comparable in potency to low molecular weight heparins currently used in the market, indicating that targeting exosite 2 may yield clinically useful drugs in the future. Since the β-O4 type lignin was found to be selective for thrombin and plasmin, we hypothesized that other scaffolds from lignins could be potent inhibitors of other serine proteases. In particular, we screened a library of synthetic sulfated small molecules against factor XIa – an emerging target for prophylactic anticoagulation. Our search identified a sulfated benzofuran trimer (a mimic of β-5 type linkage found in lignins) as a potent inhibitor of factor XIa. Surprisingly, this inhibitor did not compete with heparin. A plausible binding site in the A3 domain of factor XIa was proposed by using molecular modeling techniques. The binding pose demonstrated good correlation with the structure activity data from in vitro studies. Further confirmation that the apple domains were required was proved by testing the trimer against recombinant catalytic domain. A 40-fold decrease in activity was observed. A temperature-dependant perrin plot demonstrated that factor XIa undergoes a large conformational change in the presence of the trimer, which is possibly converting the enzyme back into the zymogen-like shape. In general, the synthetic sulfated lignins can act as a useful foundation to develop anticoagulant, antiplatelet, and anti-inflammatory molecules in the future.