Sulfated, low molecular weight lignins (LMWLs), designed recently as macromolecular mimetics of the low molecular weight heparins (LMWHs), were found to exhibit a novel allosteric mechanism of inhibition of human thrombin, factor Xa and plasmin, which translates into potent human blood anticoagulation potential. To identify the site of binding of sulfated LMWLs, a panel of site-directed thrombin mutants was studied. Substitution of alanine for Arg(93) or Arg(175) induced a 7-8-fold decrease in inhibition potency, while Arg(165)Ala, Lys(169)Ala, Arg(173)Ala and Arg(223)Ala thrombin mutants displayed a 2-4-fold decrease. Other exosite 2 residues including those that play an important role in heparin binding, such as Arg(101), Lys(235), Lys(236) and Lys(240), did not induce any deficiency in sulfated LMWL activity. Thrombin mutants with multiple alanine substitution of basic residues showed a progressively greater defect in inhibition potency. Comparison of thrombin, factor Xa, factor IXa and factor Vila primary sequences reiterated Arg(93) and Arg(175) as residues likely to be targeted by sulfated LMWLs. The identification of a novel site on thrombin with capability of allosteric modulation is expected to greatly assist the design of new regulators based on the sulfated LMWL scaffold. (C) 2011 Elsevier Inc. All rights reserved.