Revisiting the effect of cholesteryl sulfate on clotting and fibrinolysis: Inhibition of human thrombin and other human blood proteases.

ABSTRACT

Cholesteryl sulfate (CS) was quantitatively synthesized by microwave-assisted sulfonation of cholesterol followed by sodium exchange chromatography. In vitro effects of CS on human thrombin and other serine proteases of the coagulation and fibrinolysis processes were investigated using a series of biochemical and biophysical techniques. CS was found to inhibit thrombin with an IC50 value of 140.8 ± 21.8 µM at pH 7.4 and 25 ○C. Michaelis-Menten kinetics indicated that thrombin inhibition by CS is non-competitive (allosteric) in nature. Fluorescence-based binding studies indicated that CS binds to thrombin with a KD value of 180.9 ± 18.9 µM. Given the lack of competition with heparins and a hirudin peptide in competitive inhibition assays, it appears that CS does not bind to thrombin's exosites 1 or 2 and it rather recognizes a different allosteric exosite. CS was found to partially inhibit thrombin-mediated fibrinogen activation with an IC50 value of 175.5 ± 17.5 µM and efficacy of ∼26.0 ± 6.6%. Likewise, CS selectively doubled the activated partial thromboplastin time with EC2x of 521 µM. Interestingly, CS was found to also inhibit factors Xa and XIa as well as plasmin with IC50 values of ∼85-250 µM and efficacy of 94-100%. Nevertheless, CS most potently inhibited factor XIIa with an IC50 Value of ∼17 µM and efficacy of 60%. Surprisingly, CS did not inhibit factor IXa. These results encourage further in vitro and in vivo investigation of CS to better understand its (patho-) physiological roles in coagulation and hemostasis.