Degradation of Aα and Bβ chains from bovine fibrinogen by serine proteases of the Amazonian scorpion Brotheas amazonicus

Background

Proteolytic enzymes within venoms from different scorpion species belong to serine and metalloproteases class, and some isoforms of these enzymes show proteolytic activity over Aα and Bβ subunits of fibrinogen [1–3]. Brotheas amazonicus is a non-lethal amazonian scorpion, belonging to a different group from Tityus genus, and there are scarce studies about its venom in literature [4]. Enzymes acting over fibrinogen can lead to the creation of more efficient antithrombotic drugs, and the low lethality of B. amazonicus venom is a potencial factor for developing a better drug.

Material and methods

Venon was early incubated with metallo or serine proteases inhibitors (PMSF and EDTA), and proteolytic activity was evaluated by zymogram in SDS-PAGE using as substrate bovine fibrinogen. After electrophoresis, gel was rinsed with 2.5% Triton X-100. Gel was incubated in humid chamber for 24 hours at 37ºC, in 0.1M Glycine pH 8.3 solution. Dye was performed using 0.02% Comassie Blue R-250 solution. Proteolytic activity of B. amazonicus venom over bovine fibrinogen was tested, by mixing 200 µL of fibrinogen solution (2µg/µL) in 0.01M PBS pH 7.4 with 50 µg of B. amazonicus venom, for 12 hours at 37ºC. After this process, no clots were observed, so 5µg of Bothrops atrox venom [5] was incorporated to the system, but also no clotting was observed, suggesting B. amazonicus venom had activity over fibrinogen, but with no clotting formation. Changes induced by B. amazonicus venom on bovine fibrinogen were evaluated by 12% SDS-PAGE electrophoresis stained with silver nitrate. Inhibition efficacy of fibrinogenolytic activity of B. amazonicus venom by anti-scorpionic serum was tested, by adding different concentrations of anti-venom in 200µL of bovine fibrinogen (2µg/µL) plus 5µg of B. amazonicus venom. This system was incubated for 24 hours at 37ºC, and after this process clotting induction by 5µg of B. atrox venom was performed.

Results and conclusion

Proteolytic activity of B. amazonicus venom over bovine fibrinogen was only inhibited by PMSF - specific inhibitor for serine proteases. B. amazonicus venom degraded bovine fibrinogen without fibrin clots formation, confirmed by clots absence when B. atrox venom was incorporated to the system. In SDS-PAGE electrophoresis of degraded fibrinogen, it was possible to detect that B. amazonicus venom degraded Aα and Bβ subunits of fibrinogen, and anti-scorpionic serum specific for Tityus species shows great neutralizing efficacy when 1:1 proportion, suggesting that B. amazonicus toxins show similar antigenic properties of serine proteases from Tityus genus venom. Results suggest a serine protease with bovine fibrinogen affinity, able to degrade different regions from this molecule unlike thronbin and with a high similarity of proteases from Tityus sp. Such characteristics, plus the fact that this venom has a low toxicity, make these proteases inside B. amazonicus venom as candidates for antithrombotic drugs or even vaccines against scorpionic accidents.