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

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 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 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 value of 175.5 ± 17.5 μM and efficacy of ∼26.0 ± 6.6%. Likewise, CS selectively doubled the activated partial thromboplastin time with of 521 μM. Interestingly, CS was found to also inhibit factors Xa and XIa as well as plasmin with values of ∼85-250 μM and efficacy of 94-100%. Nevertheless, CS most potently inhibited factor XIIa with an Value of ∼17 μM and efficacy of 60%. Surprisingly, CS did not inhibit factor IXa. These results encourage further and investigation of CS to better understand its (patho-) physiological roles in coagulation and hemostasis.