Sulfated low molecular weight lignins, allosteric inhibitors of coagulation proteinases via the heparin binding site, significantly alter the active site of thrombin and factor xa compared to heparin.

Sulfated low molecular weight lignins (LMWLs) have been found to bind in the heparin binding sites of coagulation proteinases. LMWLs represent a library of diverse non-carbohydrate, aromatic molecules which are structures different from heparin, but still potently inhibit thrombin and factor Xa. To better understand their mechanism of action, we studied the effects of three sulfated LMWLs (CDSO3, FDSO3, and SDSO3) on the active sites of thrombin and factor Xa. LMWLs were found to uniformly inhibit the catalytic activity of thrombin and factor Xa, regardless of the substrate used. Michaelis-Menten kinetic studies indicate that maximal velocity of hydrolysis of each chromogenic substrate decreases significantly in the presence of sulfated LMWLs, while the effect on Michaelis constant is dependent on the nature of the substrate. These studies indicate that LMWLs inhibit thrombin and factor Xa through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. As opposed to heparin, LMWLs significantly alter the binding of the active site fluorescent ligand p-aminobenzamidine. LMWLs also had a greater effect on the molecular orientation of fluorescein-labeled His 57 than heparin. The molecular geometry surrounding the most important catalytic amino acid, Ser 195, was significantly altered by the binding of LMWLs while heparin had no measurable effect on Ser 195. These results further advance the concept of sulfated LMWLs as heparin mimics and will aid the design of anticoagulants based on their novel scaffold.