Biological activity of a novel fibrinolytic enzyme secreted by Hornodermoporus martius LBM 224

ACOSTA GA; FONSECA, MI.; FARIÑA, JULIA INES; ZAPATA,P.D.

Congreso; LIX Annual Meeting SAIB 2023; 2023

Cardiovascular diseases represent global disorders that impact the circulatory system, constituting primary causes of morbidityand mortality. Fungal fibrinolytic enzymes offer promising advantages over conventional therapies, facilitating targeted clot lysisand mitigating hemorrhagic risks.The aim of this study was to characterize the biological activity of a novel fibrinolytic enzymesecreted by Hornodermoporus martius LBM 224. The plasminogen activator activity of the purified enzyme was studied usingagarose-fibrin plates (0.5 % w/v) with and without plasminogen. The difference in the area of lytic zones between those platesindicated plasminogen activation activity.The thrombin-like activity and the ability to degrade fibrinogen in vitro was evaluated. To do this, 1 mL of 10 mg/mL bovineplasma fibrinogen, 500 μL of enzyme in 50 mM tris-HCl buffer pH 7.4 were added and incubated at 37 ± 1 °C examining the formation of fibrin clot. After 1 h, 500 μL of 500 U/mL thrombin were added and the formation of the fibrin clot was observed.The in vitro anticoagulant effect was studied using human whole blood. To this end, 500 μL of purified enzyme, 500 μL of 100U/mL sodium heparin, 500 μL of 50 mM pH 7.4 tris-HCl buffer, and 500 μL of saline solution were placed in four differentsterile tubes. To each tube, 1 mL of blood was added, gently mixed by inversion, and incubated at 37 ± 1 °C. The tubes weretilted once every 30 s, and the time required for blood coagulation was recorded. Heparin, tris-HCl buffer, and saline solutionwere used as controls.In the evaluation of the plasminogen activator activity, halos of fibrin degradation were observed both in the plates with andwithout plasminogen. However, the plates with plasminogen showed larger areas (66.36 ± 4.36 mm² and 50.10 ± 1.21 mm²,respectively; p < 0.05), suggesting that the enzyme can degrade the fibrin clot both directly and indirectly by activatingplasminogen into plasmin.After incubation, for the enzyme with fibrinogen solution no fibrin clot formation was observed, indicating that it does notexhibit thrombin-like activity. However, the subsequent addition of thrombin prevented the formation of fibrin clots. Theseresults suggest that the enzyme could act, not only as a fibrinolytic agent for direct degradation of the fibrin clot, but also for theprevention of clot formation.The in vitro anticoagulant capacity showed blood clot formation in the same time as negative controls. However, after 1 h ofincubation, the blood clots previously formed in the tubes containing the enzyme were degraded indicating that the enzyme hasno anticoagulant activity but has the ability to degrade blood clots in vitro.The results obtained demonstrate that the enzyme secreted by H. martius LBM 224 is a promising non-conventional alternativefor clot degradation with potential application as a novel thrombolytic agent.Keywords: Fibrinolytic enzyme, Hornodermoporus martius, Biological activity.Methods: Plasminogen activation on agarose-fibrin plates, Evaluation of thrombin-like activity and fibrinogen degradation, Invitro anticoagulant effect study using human blood