Investigation of the anticoagulant activity of cyclic sulfated glycosaminoglycan mimetics

Abstract

Thrombotic disorders are among the leading causes of deaths worldwide. Anticoagulants are frequently prescribed for their prevention and/or treatment. Current anticoagulants, which target either thrombin or factor Xa, are plagued with a number of drawbacks, the most important of which is the increased risk of internal bleeding. To develop better antithrombotic agents, the anticoagulant activity of cyclic glycosaminoglycan mimetics was evaluated. Human plasma clotting assays and enzyme inhibition assays were exploited to evaluate the anticoagulant activity of sulfated β-cyclodextrin (SBCD) and its three analogs: sulfated α-cyclodextrin, β-cyclodextrin, and methylated β-cyclodextrin. In normal human plasma, SBCD selectively doubled the activated partial thromboplastin time (APTT) at ∼9 μg/mL, with no effect on prothrombin time (PT) at the same concentration. Likewise, SBCD doubled APTT at ∼9 μg/mL and at ∼8 μg/mL in antithrombin-deficient plasma and heparin cofactor II-deficient plasma, respectively. Interestingly, the three SBCD derivatives were inactive at the highest concentrations tested which highlighted the importance of the sulfate groups and the size of the molecule. Enzyme assays revealed that SBCD inhibits factor XIa (FXIa) with an IC50 value of ∼20 μg/mL and efficacy of near 100%. SBCD did not inhibit other related proteins including thrombin, factor IXa, factor Xa, factor XIIa, factor XIIIa, plasmin, chymotrypsin, or trypsin at the highest concentrations tested demonstrating a significant selectivity. In Michaelis-Menten kinetics, SBCD decreased the VMAX and increased the KM of FXIa hydrolysis of a tripeptide chromogenic substrate indicating a mixed inhibition mechanism. Together, it appears that SBCD is a potent and selective inhibitor of human FXIa with substantial anticoagulant activity in human plasma. Overall, this study introduces SBCD as a promising lead for further development as a safer anticoagulant.