The In Vitro Effects of Pentamidine Isethionate on Coagulation and Fibrinolysis

Al‐Horani, Rami A.; Clemons, Daytriona; Mottamal, Madhusoodanan

doi:10.3390/molecules24112146

Cited by 13 publications

(55 citation statements)

References 27 publications

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“…EA Inhibits the Trans-Glutamination Activity of Human FXIIIa. Inhibition of human FXIIIa by EA was evaluated by using a modi ed bi-substrate, uorescence-based trans-glutamination assay, as described earlier [18][19][20][21]. Dansylcadaverine and N,N-dimethyl-casein were used as two substrates, which upon FXIIIa-dependence conjugation show a marked increase in uorescence at 490-550 nm (λ EX = 360 nm).…”

Section: Resultsmentioning

confidence: 99%

“…Direct Inhibition of Human FXIIIa by EA. To evaluate the effect of EA on human FXIIIa, a bi-substrate, uorescencebased trans-glutamination assay was performed as we reported previously [18][19][20]. Generally, 1 µL of EA was diluted with 87 µL of pH 7.4 buffer (50 mM Tris-HCl, 1mM CaCl 2 , 100 mM NaCl, and 2 mg/mL N,Ndimethylcasein) and 5 µL dithiothreitol (20 mM) at 37°C followed by the addition of 2 µL of human FXIIIa (0.3 µM) and incubation for 10 min.…”

Section: Experimental Partmentioning

confidence: 99%

“…Effect of EA on FXIIIa-Mediated Fibrin(ogen) Polymerization. The effect of EA on FXIIIa-mediated brin polymerization was further investigated by gel electrophoresis, as reported earlier [18][19][20]. A solution containing 1.75 mg/ml brinogen and 0.9 µg/mL FXIIIa in 50 mM Tris HCl buffer of pH 7.4 containing 10 mM CaCl 2 was incubated with different concentrations of EA (5 -5000 µM), and then clotted in the presence of human αthrombin (1.25 µg/mL).…”

Section: Experimental Partmentioning

confidence: 99%

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Ethacrynic Acid Is an Inhibitor of Human Factor XIIIa

Kar¹,

Vu²,

Mottamal³

et al. 2021

Preprint

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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. 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 experiment, 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 activated partial thromboplastin time (APTT) or prothrombin time (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. 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.

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Section: Resultsmentioning

confidence: 99%

Section: Experimental Partmentioning

confidence: 99%

Section: Experimental Partmentioning

confidence: 99%

See 1 more Smart Citation

Ethacrynic Acid Is an Inhibitor of Human Factor XIIIa

Kar¹,

Vu²,

Mottamal³

et al. 2021

Preprint

Self Cite

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“…The effect of different concentrations of inhibitor 1 on APTT and PT of normal human plasma was measured ( Table 3 and Figure 4), as described in earlier studies. [54,55,60,61] Results indicated that inhibitor 1 concentrationdependently prolonged APTT but not PT over the concentration range of 0-470 μM. Figure 4A shows the effect of anti-FXIa monoclonal antibody on the clotting times.…”

Section: Effect Of Inhibitor 1 On Clotting Times Of Normal and Fxi Dementioning

confidence: 97%

Discovery of Benzyl Tetraphosphonate Derivative as Inhibitor of Human Factor Xia

2020

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The inhibition of factor XIa (FXIa) is a trending paradigm for the development of new generations of anticoagulants without a substantial risk of bleeding. In this report, we present the discovery of a benzyl tetra-phosphonate derivative as a potent and selective inhibitor of human FXIa. Biochemical screening of four phosphonate/phosphate derivatives has led to the identification of the molecule that inhibited human FXIa with an IC 50 value of~7.4 μM and a submaximal efficacy of~68 %. The inhibitor was at least 14-fold more selective to FXIa over thrombin, factor IXa, factor Xa, and factor XIIIa. It also inhibited FXIa-mediated activation of factor IX and prolonged the activated partial thromboplastin time of human plasma. In Michaelis-Menten kinetics experiment, inhibitor 1 reduced the V MAX of FXIa hydrolysis of a chromogenic substrate without significantly affecting its K M suggesting an allosteric mechanism of inhibition. The inhibitor also disrupted the formation of FXIa-antithrombin complex and inhibited thrombin-mediated and factor XIIa-mediated formation of FXIa from its zymogen factor XI. Inhibitor 1 has been proposed to bind to or near the heparin/polyphosphate-binding site in the catalytic domain of FXIa. Overall, inhibitor 1 is the first benzyl tetraphosphonate small molecule that allosterically inhibits human FXIa, blocks its physiological function, and prevents its zymogen activation by other clotting factors under in vitro conditions. Thus, we put forward benzyl tetra-phosphonate 1 as a novel lead inhibitor of human FXIa to guide future efforts in the development of allosteric anticoagulants.

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“…Pentamidine, an aromatic diamidine, is used to treat first-stage (haemolymphatic stage) T. gambiense HAT (Denise and Barrett, 2001; Barrett et al ., 2007; Baker et al ., 2013). This compound binds nucleic acids with high affinity, leading to accumulation by, and ultimately destruction of, the kinetoplast (Mathis et al ., 2006; Baker et al ., 2013; Gould and Schnaufer, 2014; Al-Horani et al ., 2019; Kennedy and Rodgers, 2019). However, pentamidine is unable to reach the central nervous system (CNS), in part due to its high affinity interactions with serum proteins, charge and relatively high retention in tissues and is therefore ineffective for the treatment of second-stage meningoencephalic HAT (Barrett et al ., 2007; Maclean et al ., 2012).…”

Section: Tbaqp2 and Multidrug Resistancementioning

confidence: 99%