Biologicalactivityofanovel fibrinolyticenzymesecretedbyHornodermoporus martiusLBM224

ACOSTA GABRIELA ALEJANDRA; FONSECA MARIA ISABEL; FARIÑA JULIA INÉS; ZAPATA PEDRO DARÍO

Rosario

Congreso; Congreso de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB). Reunión anual edición LIX; 2023

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Cardiovascular diseases represent global disorders that impact the circulatory system, constituting primary causes of morbidity and mortality. Fungal fibrinolytic enzymes offer promising advantages over conventional therapies, facilitating targeted clot lysis and mitigating hemorrhagic risks.The aim of this study was to characterize the biological activity of a novel fibrinolytic enzyme secreted by Hornodermoporus martius LBM 224. The plasminogen activator activity of the purified enzyme was studied using agarose-fibrin plates (0.5 % w/v) with and without plasminogen. The difference in the area of lytic zones between those plates indicated 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 bovine plasma 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 100 U/mL sodium heparin, 500 µL of 50 mM pH 7.4 tris-HCl buffer, and 500 µL of saline solution were placed in four different sterile tubes. To each tube, 1 mL of blood was added, gently mixed by inversion, and incubated at 37 ± 1 °C. The tubes were tilted once every 30 s, and the time required for blood coagulation was recorded. Heparin, tris-HCl buffer, and saline solution were used as controls.In the evaluation of the plasminogen activator activity, halos of fibrin degradation were observed both in the plates with and without 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 activating plasminogen into plasmin.After incubation, for the enzyme with fibrinogen solution no fibrin clot formation was observed, indicating that it does not exhibit thrombin-like activity. However, the subsequent addition of thrombin prevented the formation of fibrin clots. These results suggest that the enzyme could act, not only as a fibrinolytic agent for direct degradation of the fibrin clot, but also for the prevention of clot formation.The in vitro anticoagulant capacity showed blood clot formation in the same time as negative controls. However, after 1 h of incubation, the blood clots previously formed in the tubes containing the enzyme were degraded indicating that the enzyme has no 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 alternative for clot degradation with potential application as a novel thrombolytic agent.