Metalloproteases are hydrolytic enzymes that rely on the binding of a metal, primarily zinc (Zn2+), at their catalytic site, which is crucial for their activity, stability, and prevention of conformational changes in their structure [1,2]. These enzymes are found across a wide range of organisms, from bacteria to mammals. They are classified hierarchically (Class, Tribe, Clan, and Families) based on structural similarities in the zinc-binding motif, substrate-binding site, and catalytic motifs [3,4].

Snake venom consists of a complex and diverse mixture of proteins, with snake venom metalloproteases (SVMPs) being among the major components [[5], [6], [7]]. SVMPs are implicated in degrading basement membrane proteins, particularly collagen, surrounding tissues at the site of a snakebite [8]. These proteins typically have a molecular mass ranging from 20 to 100 kDa [1] and are categorized into three classes (P–I to P-III) based on the presence of accessory domains to the proteolytic domain [[9], [10], [11]]. The P–I class (20–30 kDa) comprises solely a metalloproteinase (M) domain characterized by a conserved sequence of amino acid residues (HEXXHXXGXXH), with histidine residues responsible for metal ion binding, where X represents any amino acid residue [12,13]. The P-II class (30–60 kDa) contains both a metalloproteinase and a disintegrin domain, while the P-III class (60–100 kDa) consists of a metalloproteinase, a disintegrin-like domain, and a cysteine-rich domain [14,15].

SVMPs, in general, can induce pain through inflammatory hyperalgesia [16], inflammation via edematogenic activity independent of pro-inflammatory mediators [17], hemorrhage, and coagulopathy by cleaving the basement membrane of capillaries and endothelial cell adhesion proteins [12,18], as well as necrosis due to its dermonecrotic activity dependent on TNF-α signaling [17].

BjussuMP-II is a non-hemorrhagic P–I SVMP isolated from Bothrops jararacussu venom with a molecular mass of 24 kDa. It hydrolyzes the α chain and, to a lesser extent, the β chain of fibrinogen, thus being classified as an α/β-fibrinogenase. BjussuMP-II did not induce myotoxicity, lethality, or hemorrhage at doses up to 200 μg, and its activity was enhanced when incubated with Zn2+ and Ca2+ ions but inhibited when incubated with heparin and EDTA. Additionally, it exhibits inhibitory activity on platelet aggregation induced by ADP (adenosine diphosphate) and collagen [19].

Messenger molecules such as tumor necrosis factor (TNF), interleukin (IL)-1, IL-6, and IL-8 play crucial roles in coordinating the inflammatory response. These cytokines are pleiotropic proteins that modulate vascular endothelial permeability, recruit blood cells to inflammatory tissues, regulate the death of inflammatory tissue cells, and stimulate the synthesis of acute-phase proteins [[20], [21], [22], [23], [24], [25]]. Recent studies emphasize the importance of the simultaneous production of pro- and anti-inflammatory cytokines in immune responses, as they can trigger signaling cascades with potentially detrimental effects even at low protein levels [[26], [27], [28]].

TNF-α, a potent pro-inflammatory cytokine primarily produced by activated monocytes and macrophages, induces various actions, including macrophage differentiation, neutrophil degranulation, leukotriene release, and expression of cell adhesion molecules on endothelial cells, thereby promoting local leukocyte migration [[29], [30], [31], [32], [33]]. TNF-α plays a key role in modulating inflammatory reactions and is implicated in the pathogenesis of several autoimmune and inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis [20]. In this study, human umbilical vein endothelial cells (HUVEC) were utilized as a model to investigate the effects of BjussuMP-II on viability, adhesion, detachment, release of inflammatory cytokines, and cleavage.