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In light of the complexity of wound tissue, proteomic analysis may not clearly reveal the nature of the wound or the processes involved in healing. However, exudate associated with wounds may provide a "window" on cellular events leading to the development of the wound and/or its healing. In this investigation we performed proteomic analysis on wound exudates from muscular wounds in mice caused by two very different types of snake venom toxins: BaP1, a snake venom metalloproteinase and Mtx-I, a snake venom phospholipase A2. Proteomic analysis of the exudates associated with these wounds clearly differentiated them and offered new perspectives on functional mechanisms by which these toxins cause tissue damage. In the case of wounds caused by the metalloproteinase, there was evidence of degradation of nonfibrillar collagens whereas the phospholipase wound exudate was noted by the presence of fibrillar collagen type I, apolipoproteins A-I, A-IV, and E, and fibronectin. These results suggest that the hemorrhage caused by snake venom metalloproteinases may be associated with the degradation of specific extracellular matrix proteins which play a role in matrix/capillary stabilization and that release of apolipoproteins from their complexes may be involved with the dysfunctional hemostasis observed following snake envenoming.

The historical development of discoveries and conceptual frames for understanding the hemorrhagic activity induced by viperid snake venoms and by hemorrhagic metalloproteinases (SVMPs) present in these venoms is reviewed. Histological and ultrastructural tools allowed the identification of the capillary network as the main site of action of SVMPs. After years of debate, biochemical developments demonstrated that all hemorrhagic toxins in viperid venoms are zinc-dependent metalloproteinases. Hemorrhagic SVMPs act by initially hydrolyzing key substrates at the basement membrane (BM) of capillaries. This degradation results in the weakening of the mechanical stability of the capillary wall, which becomes distended owing of the action of the hemodynamic biophysical forces operating in the circulation. As a consequence, the capillary wall is disrupted and extravasation occurs. SVMPs do not induce rapid toxicity to endothelial cells, and the pathological effects described in these cells in vivo result from the mechanical action of these hemodynamic forces. Experimental evidence suggests that degradation of type IV collagen, and perhaps also perlecan, is the key event in the onset of microvessel damage. It is necessary to study this phenomenon from a holistic, systemic perspective in which the action of other venom components is also taken into consideration.

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Snake venom hemorrhagic metalloproteinases (SVMPs) of the PI, PII and PIII classes were compared in terms of tissue localization and their ability to hydrolyze basement membrane components in vivo, as well as by a proteomics analysis of exudates collected in tissue injected with these enzymes. Immunohistochemical analyses of co-localization of these SVMPs with type IV collagen revealed that PII and PIII enzymes co-localized with type IV collagen in capillaries, arterioles and post-capillary venules to a higher extent than PI SVMP, which showed a more widespread distribution in the tissue. The patterns of hydrolysis by these three SVMPs of laminin, type VI collagen and nidogen in vivo greatly differ, whereas the three enzymes showed a similar pattern of degradation of type IV collagen, supporting the concept that hydrolysis of this component is critical for the destabilization of microvessel structure leading to hemorrhage. Proteomic analysis of wound exudate revealed similarities and differences between the action of the three SVMPs. Higher extent of proteolysis was observed for the PI enzyme regarding several extracellular matrix components and fibrinogen, whereas exudates from mice injected with PII and PIII SVMPs had higher amounts of some intracellular proteins. Our results provide novel clues for understanding the mechanisms by which SVMPs induce damage to the microvasculature and generate hemorrhage.

Hemorrhage is a clinically important manifestation of viperid snakebite envenomings, and is induced by snake venom metalloproteinases (SVMPs). Hemorrhagic and non-hemorrhagic SVMPs hydrolyze some basement membrane (BM) and associated extracellular matrix (ECM) proteins. Nevertheless, only hemorrhagic SVMPs are able to disrupt microvessels; the mechanisms behind this functional difference remain largely unknown. We compared the proteolytic activity of the hemorrhagic P-I SVMP BaP1, from the venom of Bothrops asper, and the non-hemorrhagic P-I SVMP leucurolysin-a (leuc-a), from the venom of Bothrops leucurus, on several substrates in vitro and in vivo, focusing on BM proteins. When incubated with Matrigel, a soluble extract of BM, both enzymes hydrolyzed laminin, nidogen and perlecan, albeit BaP1 did it at a faster rate. Type IV collagen was readily digested by BaP1 while leuc-a only induced a slight hydrolysis. Degradation of BM proteins in vivo was studied in mouse gastrocnemius muscle. Western blot analysis of muscle tissue homogenates showed a similar degradation of laminin chains by both enzymes, whereas nidogen was cleaved to a higher extent by BaP1, and perlecan and type IV collagen were readily digested by BaP1 but not by leuc-a. Immunohistochemistry of muscle tissue samples showed a decrease in the immunostaining of type IV collagen after injection of BaP1, but not by leuc-a. Proteomic analysis by LC/MS/MS of exudates collected from injected muscle revealed higher amounts of perlecan, and types VI and XV collagens, in exudates from BaP1-injected tissue. The differences in the hemorrhagic activity of these SVMPs could be explained by their variable ability to degrade key BM and associated ECM substrates in vivo, particularly perlecan and several non-fibrillar collagens, which play a mechanical stabilizing role in microvessel structure. These results underscore the key role played by these ECM components in the mechanical stability of microvessels.