Ethacrynic acid is an inhibitor of human factor XIIIa

Abstract

Background: Ethacrynic acid (EA) is a loop diuretic that is approved orally and parenterally to manage edema-associated diseases. Nevertheless, it was earlier reported that it is also associated with bleeding upon its parenteral administration. In this report, we investigated the effects of EA on human factor XIIIa (FXIIIa) of the coagulation process using a variety of techniques.

Methods: A series of biochemical and computational methods have been used in this study. The potency and efficacy of human FXIIIa inhibition by EA was evaluated using a bisubstrate-based fluorescence trans-glutamination assay under near physiological conditions. To establish the physiological relevance of FXIIIa inhibition by EA, the effect on FXIIIa-mediated polymerization of fibrin(ogen) as well as the formation of fibrin(ogen) - α₂-antiplasmin complex was evaluated using SDS-PAGE experiments. The selectivity profile of EA against other coagulation proteins was assessed by evaluating EA's effect on human clotting times in the activated partial thromboplastin time (APTT) and the prothrombin time (PT) assays. We also used molecular modeling studies to put forward a putative binding mode for EA in the active site of FXIIIa. Results involving EA were the average of at least three experiments and the standard error ± 1 was provided. In determining the inhibition parameters, we used non-linear regression analysis.

Results: FXIIIa is a transglutaminase that works at the end of the coagulation process to form an insoluble, rigid, and cross-linked fibrin rich blood clot. In fact, inhibition of FXIIIa-mediated biological processes has been reported to result in a bleeding diathesis. Inhibition of FXIIIa by EA was investigated given the nucleophilic nature of the thiol-containing active site of the enzyme and the Michael acceptor-based electrophilicity of EA. In a bisubstrate-based fluorescence trans-glutamination assay, EA inhibited FXIIIa with a moderate potency (IC₅₀ ~ 105 µM) and efficacy (∆Y ~ 66%). In SDS-PAGE experiments, EA appears to significantly inhibit the FXIIIa-mediated polymerization of fibrin(ogen) as well as the formation of fibrin(ogen) - α₂-antiplasmin complex which indicates that EA affects the physiological functions of FXIIIa. Interestingly, EA did not affect the clotting times of human plasma in the APTT and the PT assays at the highest concentration tested of 2.5 mM suggesting the lack of effects on the coagulation serine proteases and potentially the functional selectivity of EA with respect to the clotting process. Molecular modeling studies demonstrated that the Michael acceptor of EA forms a covalent bond with catalytic residue of Cys314 in the active site of FXIIIa.

Conclusions: Overall, our studies indicate that EA inhibits the physiological function of human FXIIIa in vitro which may potentially contribute to the bleeding complications that were reported with the association of the parenteral administration of EA.

Keywords: Anticoagulant; Bleeding; Ethacrynic acid; Factor XIIIa; Irreversible inhibitor.

Fig. 1

A The chemical structure of ethacrynic acid (EA; 1). B The inhibition of FXIIIa by EA (●) was measured spectro-fluorometrically through a bisubstrate, fluorescence-based trans-glutamination assay (λ_Ex. = 360 nm and λ_Em. = 490 nm) at pH 7.4 and 37 °C. Solid lines represent sigmoidal fits to the data to obtain IC₅₀, HS, Y_M, and Y_O using Eq. 1. See details in Methods part. Assay was repeated three times

Fig. 2

Effect of EA on FXIIIa-mediated fibrin(ogen) polymerization. A The effect of EA on FXIIIa-mediated fibrin polymerization was investigated by SDS-PAGE. Different concentrations of EA (5 – 5000 µM) were used. The figure shows that EA concentration dependently inhibited the formation of γ–γ (dimers; about 117 kDa) as well as α–α (larger polymers; > 250 kDa). B The dose–response curves for the formation of dimers and polymers from which the inhibition parameters were deduced using Eq. 1. EA inhibited the formation of the γ–γ dimers with an IC₅₀ value of ~ 1176.8 µM and an efficacy of ~ 94.7%, yet it was more potent inhibiting the formation of the larger polymers of α – α with and an IC₅₀ value of ~ 120.1 µM and an efficacy of ~ 81.6%. This shows that EA not only inhibiting FXIIIa activity using non-physiological substrates (N,N-dimethylcasein and dansylcadaverine) but also using its physiological substrates (α-, β-, and γ- fibrin(ogen)) monomers. Original gels are provided in the supplementary information. The edges were removed to provide better clarity. Experiment was repeated three times

Fig. 3

The effect of EA on FXIIIa-mediated formation of fibrin(ogen) – α₂-AP complex. This effect was investigated by western blot assay in which different concentrations of EA (100, 500, 1000, 3000, and 5000 µM) were used. Evidently, EA inhibited the formation of fibrin(ogen) – α₂-AP complex at a concentration as low as 100 µM supporting the physiological relevance of the action of EA. In theory, this would mean that the blood clot becomes more susceptible to hydrolysis by the fibrinolytic enzyme plasmin. Original blot is provided in the supplementary information. The edges were removed to provide better clarity. Experiment was repeated three times

Fig. 4

Plasma clotting assays: Activated partial thromboplastin time (APTT) (●) and prothrombin time (♦). A The effect of EA (0 – 2500 µM) on APTT and PT. B The effect of dabigatran, thrombin inhibitor, (0 – 0.75 µM) on APTT and PT. C The effect of rivaroxaban, FXa inhibitor, (0 – 0.5 µM) on APTT and PT. D The effect of AntiF11, FXIa inhibitor, (0 – 5 µg/mL) on APTT and PT. Thrombin and FXa inhibitors affect the two times i.e. APTT and PT because they affect the common coagulation pathway, whereas FXIa inhibitor affects only APTT but not PT, which is indicative of an effect on the intrinsic pathway. FXIIIa inhibitors demonstrate similar phenomenon of human FXIIIa deficiency in which APTT and PT are not affected. See details in Methods

Fig. 5

The catalytic domain of FXIIIa is presented as a potential binding site for EA showing the covalent bond between the α,β-unsaturated ketone the catalytic Cys324. Other potential important interactions are H-bonds between the ketone group of EA and the NH-groups of the side chains of Gln313 and Trp279. The carboxylic acid also potentially establishes H-bond with the side chain of the Asn371 residue. The 2-Cl substituent also forms a halogen-H interaction with the side chain of the Asn371 residue. EA is represented as stick and balls. Atoms are represented with the following colors: carbon = cyan, chlorine = green, oxygen = red, nitrogen = blue, and sulfur = yellow. The protein backbone cartoon is represented in green-yellowish color