Caracterização estrutural e funcional de uma metaloproteinase de classe P-I do veneno de Lachesis muta rhombeata

Abstract

Rhomb-I, a 23-kDa metalloproteinase was isolated from L. m. rhombeata venom. Its dimethylcasein proteolysis was abolished by metal chelators, and slightly enhanced by Ca2+ and Mg2+ ions, but inhibited by Co2+

, Zn2+ and α2-macroglobulin. In aqueous solution, rhomb-I autoproteolyzed to a 20- and 11-kDa fragments at 37 oC. The amino acid sequence showed high homology with other SVMPs. Rhomb-I causes hemorrhage that may be ascribed to hydrolysis of essential BM, ECM and plasma proteins. It preferentially cleaves the α-chains of fibrin(ogen). Rhomb-I inhibited convulxin- and vWF-induced aggregation on human platelets without significant effect

on collagen-stimulated aggregation or other effectors. It digests vWF into a low- molecular-mass multimers of vWF and a rvWF-A1 domain to a 27-kDa fragment as

revealed by western blotting with mouse anti-rvWF A1-domain IgG. Incubation of platelets with rhomb-I resulted in adhesion to and cleavage of platelet receptors GPIbα and GPVI to release a 130-kDa and 55-kDa soluble form. Both membrane glycoproteins, GPIbα that binds vWF, together with GPVI which binds collagen, play a key role in mediating platelet adhesion/activation and can initiate (patho)physiological thrombus formation. Conclusions: rhomb-I is implicated in the pathophysiology of Lachesis envenoming by disrupting vasculature, hemostasis and platelet aggregation through impairing vWF-GPIb axis and blocking GPVI-collagen binding.