Characterization of the proinflammatory activity of a new serine protease (cdtsp-2) purified from the total venom of Crotalus durissus terrificus

Abstract

Recently our group isolated a new serine protease from the total venom of Crotalus durissus terrificus (cdtsp-2) as a molecular mass of 27 kDa, and its N-terminal sequence showed structural differences when compared to the gyroxin of Crotalus durissus terrificus, besides not presenting glycosylation such as gyroxin. Venom serine proteases (SVSP) interfere in the regulation and control of important biological reactions of blood homeostasis and can be classified as activators of the fibrinolytic system, procoagulant, anticoagulant and platelet aggregating enzymes. However, our preliminary results show that cdtsp-2 also presents significant edematogenic and myotoxic activity, which can be considered an unpublished fact and may introduce SVSPs as important agents capable of inducing pharmacological events at the cellular level as with sPLA2. In this project, we intend to characterize the pro-inflammatory mechanism and myotoxic activity induced by cdtsp-2 using the paw edema model in mice as the study model and using a combination of inhibitors and agonists related to the protease receptor signaling pathway (PARs ). On the other hand, two events can occur concomitantly, one related to the increase of reactive oxygen / nitrogen species that initiate an intracellular signaling cascade that may increase the expression of protein inflammatory mediators and the other is related to the activation of the acid cascade arachidonic. Thus, we also intend to establish a relationship between the activation of the PAR (s) receptors with the activation of the arachidonic acid cascade, monitoring the activity of cytosolic Fosfolipase A2, COX-2, Prostaglandin E2, TNFalpha, IL-6, IL- NFkb p65 and the relative concentration of Malonaldehyde (MDA).In addition to investigating the mechanism of CDT-2-induced mydynamic action of the venom of Crotalus durissus terrificus, this project will establish comparative bases using in silico analysis of cdtsp-2 of Crotalus durissus terrificus compared to gyroxin and thrombin, identifying structural differences that may be considered important for the differences between cdtsp-2 and gyroxin. The results obtained in this project can expand the action of the SVSPs to an agent of fundamental importance to the side of sPLA2 as agents inducing cellular and systemic events such as inflammation and myotoxicity and not only associated with coagulative changes. These results can help in the development of new therapeutic approaches and consider the contribution of antioxidant elements as enzymes, reassess some pain and inflammation drugs, focusing mainly on their antioxidant activity and the contribution of plant extracts. (AU)