Insights into the Mechanisms Involved in Strong Hemorrhage and Dermonecrosis Induced by Atroxlysin-Ia, a PI-Class Snake Venom Metalloproteinase

Abstract

Hemorrhage is the most prominent effect of snake venom metalloproteinases (SVMPs) in human envenomation. The capillary injury is a multifactorial effect caused by hydrolysis of the components of the basement membrane (BM). The PI and PIII classes of SVMPs are abundant in viperid venoms and hydrolyze BM components. However, hemorrhage is associated mostly with PIII-class SVMPs that contain non-catalytic domains responsible for the binding of SVMPs to BM proteins, facilitating enzyme accumulation in the tissue and enhancing its catalytic efficiency. Here we report on Atroxlysin-Ia, a PI-class SVMP that induces hemorrhagic lesions in levels comparable to those induced by Batroxrhagin (PIII-class), and a unique SVMP effect characterized by the rapid onset of dermonecrotic lesions. Atroxlysin-Ia was purified from B. atrox venom, and sequence analyses indicated that it is devoid of non-catalytic domains and unable to bind to BM proteins as collagen IV and laminin in vitro or in vivo. The presence of Atroxlysin-Ia was diffuse in mice skin, and localized mainly in the epidermis with no co-localization with BM components. Nevertheless, the skin lesions induced by Atroxlysin-Ia were comparable to those induced by Batroxrhagin, with induction of leukocyte infiltrates and hemorrhagic areas soon after toxin injection. Detachment of the epidermis was more intense in skin injected with Atroxlysin-Ia. Comparing the catalytic activity of both toxins, Batroxrhagin was more active in the hydrolysis of a peptide substrate while Atroxlysin-Ia hydrolyzed more efficiently fibrin, laminin, collagen IV and nidogen. Thus, the results suggest that Atroxlysin-Ia bypasses the binding step to BM proteins, essential for hemorrhagic lesions induced by PII-and P-III class SVMPs, causing a significantly fast onset of hemorrhage and dermonecrosis, due to its higher proteolytic capacity on BM components.

Inflammatory Reaction Induced by Two Metalloproteinases Isolated from Bothrops atrox Venom and by Fragments Generated from the Hydrolysis of Basement Membrane Components

“…Escalante et al [28] demonstrated the extensive hydrolysis of laminin α and γ chains by BaPI (PI-class) and in the case of Jararhagin (PIII-class) by most of hydrolysis was of the nidogen and only the α-chain of laminin. In in vivo experiments, Freitas-De-Sousa et al [20] observed that ATXL and BATXH degraded collagen IV and laminin, with total collagen digestion by ATXL, which would explain the proteinase ability to induce hemorrhage, since these BM components play important role in the stability of capillaries.…”

“…The differences among doses from 1 to 5 μg, within and between each toxin group, were not statistically significant, indicating that the two-times-lower molecular mass of ATXL than BATXH would not impact the comparisons in this range of doses. The intermediate dose of 2 μg/mouse was then selected to evaluate and compare the time course of the edema induced by both toxins, since this dose was capable of inducing edema without causing hemorrhage in the animals [20]. Mice (n = 6) were injected on the left paw with different protein doses (0.5, 1, 2 and 5 μg/animal) or with saline as negative control.…”

“…For this purpose, ATXL or BATXH were incubated with Matrigel for 1 h, and the peptide fraction was isolated by filtering on 10 kDa cut-off membranes, as described in the Methods section. The doses injected in each mouse corresponded to the total amount of peptides released from Matrigel by 10 µg of SVMPs, which is the dose that induced hemorrhage in mice models [20]. Figure 6A,B shows that the products resulting from the hydrolysis of Matrigel by BATXH and ATXL were able to induce edema significantly higher than those of peptides present in Matrigel control samples.…”

“…Recently, our group isolated two hemorrhagic SVMPs from the venom of B. atrox that were named Atroxlysin-Ia [20] and Batroxrhagin [21]. Batroxrhagin (BATXH) is a PIII-class SVMP structurally and functionally similar to Jararhagin, isolated from B. jararaca venom [21].…”

“…However, unlike the previously isolated toxins, ATXL presents a dermonecrotic activity and is capable of inducing an intense hemorrhage, in levels comparable to the PIII-class SVMP. The mechanism suggested for ATXL higher hemorrhagic and dermonecrotic action than other PI-class SVMPs was its higher efficiency to cleave Basement Membrane (BM) components as collagen IV and laminin, important structural elements that guarantees stability to BM [20]. Laminin is a glycoprotein that plays important roles in cell biology, such as cell adhesion, migration and proliferation, among other biological activities [24].…”