Purification and Biochemical Characterization of a Probable P-IIId Class Metalloprotease from Bothrops atrox Venom Active in Platelet Aggregation

Introduction: Snake venom represents a relevant source of bioactive molecules with therapeutical potential, particularly for modulating complex pathways such as the coagulation cascade. Snake venom metalloproteinases (SVMPs) are enzymatic proteins classified according to their structural domains: (1) class P-I SVMPs, which exclusively contain the metalloproteinase catalytic domain; (2) class P-II SVMPs, which possess, in addition to the catalytic domain, a disintegrin domain; and (3) class P-III SVMPs, which contain metalloproteinase, disintegrin-like, and cysteine-rich domains. Class P-III SVMPs may also associate with C-type lectin-like domains, which are then classified as P-IIId. Snake venom C-type lectin-like proteins (snaclecs) are non-enzymatic proteins that are structurally organized as heterodimers (α and β subunits). They play a fundamental role in hemostasis through receptor-mediated interactions despite lacking carbohydrate-binding properties. In previous work from our group, a proteolytically active fraction from Bothrops atrox venom showed reactivity with anti-C-type lectin (CTL) antibodies. The present study aimed to characterize the biochemical and functional properties.

Methods: The identified fraction from B. atrox venom was evaluated using SDS-PAGE, plasminogen activation assays, hemagglutinating-activity assays, platelet aggregation assays using washed platelets and platelet-rich plasma (PRP), Western blot analysis for reactivity with anti-SVMP-P-I and -P-III antibodies, and proteolytic-activity assays on extracellular matrix (ECM) components.

Results: The selected fraction induced aggregation in PRP, but only weakly in washed platelets. However, it strongly promoted the aggregation of washed platelets in the presence of human fibrinogen. Western blot analysis revealed a ~26 kDa band recognized by anti-CTL and anti-SVMP-P-I antibodies, while the anti-SVMP-P-III antibody identified bands of ~26 kDa and between 43 and 72 kDa. The fraction showed no hemagglutinating activity, did not activate plasminogen, but effectively degraded ECM components.

Conclusion: These results suggest the presence of a P-IIId in B. atrox venom. Further studies are necessary to fully elucidate its structure and biological activities.