Snake venom metalloproteinases (SVMPs) in Viperid
venoms primarily function to give rise to local and systemic
hemorrhage following
snake envenomation. Years of research on these toxins, both in vitro and in vivo, indicate that they function by disrupting capillary basement membranes, stromal matrix and cell-cell and cell-matrix contacts to allow escape of capillary contents under pressure. However, most of these studies used either defined substrates in vitro or were limited by relevant
antibodies for detection of sites of action in vivo. In this investigation we use stable
isotope-labeled
amino acids in culture (SILAC) to determine novel proteolytic activities for exogenously added
atrolysin A, a hemorrhagic PIII SVMP isolated from Crotalus atrox
venom. When comparing the solubilized products of SILAC-labeled cultured human fibroblasts treated with
atrolysin A to that of untreated fibroblasts using LC/MS/MS, several
proteins were identified as being released into the
culture media specifically due to
atrolysin A proteolytic activity. These included
collagen VI,
fibronectin,
fibulin 2 and
annexin V. Of particular interest was the observation of
collagen VI and
annexin V in that the release of these substrates could play a role in altering hemostasis and promote
hemorrhage caused by the more typical actions of
atrolysin A. In summary, this study demonstrates the utility of SILAC for exploring sheddase activity with cells in culture and suggests the presence of two novel substrates for SVMPs that may play a pathological role in altering host hemostasis during envenomation.