Functional and biological insights of rCollinein-1, a recombinant serine protease from Crotalus durissus collilineatus.

Abstract

ABSTRACTBackground:The prevalent class of snake venom serine proteases (SVSP) in Viperidae venoms is the thrombin-like enzymes, which, similarly to human thrombin, convert fibrinogen into insoluble fibrin monomers. However, thrombin-like serine proteases differ from thrombin by being unable to activate factor XIII, thus leading to the formation of loose clots and fibrinogen consumption. We report the functional and biological characterization of a recombinant thrombin-like serine protease from Crotalus durissus collilineatus, named rCollinein-1.Methods:Heterologous expression of rCollinein-1 was performed in Pichia pastoris system according to a previously standardized protocol, with some modifications. rCollinein-1 was purified from the culture medium by a combination of three chromatographic steps. The recombinant toxin was tested in vitro for its thrombolytic activity and in mice for its edematogenicity, blood incoagulability and effect on plasma proteins.Results:When tested for the ability to induce mouse paw edema, rCollinein-1 demonstrated low edematogenic effect, indicating little involvement of this enzyme in the inflammatory processes resulting from ophidian accidents. The rCollinein-1 did not degrade blood clots in vitro, which suggests that this toxin lacks fibrinolytic activity and is not able to directly or indirectly activate the fibrinolytic system. The minimal dose of rCollinein-1 that turns the blood incoagulable in experimental mice is 7.5 mg/kg. The toxin also led to a significant increase in activated partial thromboplastin time at the dose of 1 mg/kg in the animals. Other parameters such as plasma fibrinogen concentration and prothrombin time were not significantly affected by treatment with rCollinein-1 at this dose. The toxin was also able to alter plasma proteins in mouse after 3 h of injection at a dose of 1 mg/kg, leading to a decrease in the intensity of beta zone and an increase in gamma zone in agarose gel electrophoresis Conclusion:These results suggest that the recombinant enzyme has no potential as a thrombolytic agent but can be applied in the prevention of thrombus formation in some pathological processes and as molecular tools in studies related to hemostasis.