Pentamidine is bis-oxybenzamidine-based antiprotozoal drug that is used to prevent and/or treat Pneumocystis jirovecii pneumonia and the early stage of African trypanosomiasis. The parenteral use of this drugs is associated with hematological toxicities including leukopenia, thrombocytopenia, and hemolytic anemia. Rare but potentially life-threatening blood clot formation has also been reported. Pentamidine was also found to cause disseminated intravascular coagulation syndrome. Previous studies indicated that pentamidine affects platelet aggregation, prothrombin time, and thrombin time. To further investigate the potential underlying mechanism(s) of these toxicities, we studied pentamidine isethionate effects on a panel of human serine proteases including thrombin, factor Xa, factor XIa, plasmin, and neutrophil elastase. Chromogenic substrate hydrolysis assays indicated that pentamidine inhibits factor Xa and plasmin with IC50 values of 10.4±0.8 µM and 8.4±0.8 µM, respectively, under the physiological conditions of pH 7.4 and 37 oC. Under similar conditions, it did not inhibit thrombin, factor XIa, or neutrophil elastase at the highest concentration tested of 100 µM. Pentamidine did not significantly inhibit guinea pig transglutaminase, an experimental surrogate for the coagulation transglutaminase factor XIIIa, in fluorescence-based transglutamination assay at 500 µM. Michaelis-Menten kinetics revealed that pentamidine is a mixed inhibitor of factor Xa, but competitive inhibitor of plasmin. Using citrated human plasma, pentamidine isethionate doubled the activated partial thromboplastin clotting time at 27.5±6.3 µM and the prothrombin clotting time at 45.7±1.2 µM. Likewise, pentamidine dose-dependently inhibited plasmin-mediated clot lysis in turbidity assay. Molecular modeling studies further supported the binding of pentamidine to the active sites of factor Xa and plasmin by occupying the corresponding S1 subsite with one amidine moiety engaging in salt bridge interactions with the side chain carboxylic acid group of Asp189 in both enzymes and the other amidine group with the carboxylic acid group of Glu97 in factor Xa or the backbone oxygen of Arg94 in plasmin. Overall, this study provides quantitative mechanistic insights into the in vitro effects of pentamidine isethionate on fibrinolysis and coagulation. It puts forward the disruption of the proteolytic activity of factor Xa and plasmin as a potential attribute to its hematological toxicities.

In Vitro effects of pentamidine isethionate on fibrinolysis and coagulation

2019 Spring National Meeting

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