Role of Collagens and Perlecan in Microvascular Stability: Exploring the Mechanism of Capillary Vessel Damage by Snake Venom Metalloproteinases

et al. (2011) Role of Collagens and Perlecan in Microvascular Stability: Exploring the Mechanism of Capillary Vessel Damage by Snake Venom Metalloproteinases. PLoS ONE 6(12): e28017. doi:10.1371/journal.pone.0028017 Role of Collagens and Perlecan in Microvascular Stability: Exploring the Mechanism of Capillary Vessel Damage by Snake Venom Metalloproteinases Teresa Escalante 0 Natalia Ortiz 0 Alexandra Rucavado 0 Eladio F. Sanchez 0 Michael Richardson 0 Jay W. 0 Fox 0 Jose Mara Gutie rrez 0 Rory Edward Morty, University of Giessen Lung Center, Germany 0 1 Instituto Clodomiro Picado, Facultad de Microbiolog a, Universidad de Costa Rica , San Jose , Costa Rica , 2 Departamento de Bioqu mica, Escuela de Medicina, Universidad de Costa Rica , San Jose , Costa Rica , 3 Centro de Pesquisa e Desenvolvimento, Fundac ao Ezequiel Dias (FUNED) , Belo Horizonte, Minas Gerais , Brazil , 4 Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine , Charlottesville, Virginia , United States of America 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. - Funding: This study was supported by Vicerrectora de Investigacio n, Universidad de Costa Rica (projects 741-A7-502 and 741-B0-606), the International Foundation for Science (IFS) and the Organisation for the Prohibition of Chemical Weapons (OPCW) (projectF/4096-1), NeTropica (projects 2-N-2008 and 01N2010), Brazilian agencies CNPq and FAPEMIG, and the University of Virginia School of Medicine (J.W.F.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Zinc-dependent metalloproteinases are abundant components in the proteomes of many snake venoms, especially in those of species of the family Viperidae [1,2]. Snake venom metalloproteinases (SVMPs) are multi-domain proteins which have been classified in various classes on the basis of their domain composition [1]. Class P-I SVMPs comprise enzymes having, in their mature protein, only the metalloproteinase domain, whereas class P-II SVMPs present, in addition to the catalytic domain, a disintegrin domain, which may be cleaved to generate disintegrins. Enzymes of the P-III class have disintegrin-like and cysteine-rich domains following the metalloproteinase domain [1]. SVMPs play key roles in envenomations by snakes of the family Viperidae, and probably also in the case of some species of the family Colubridae (sensu lato) [37]. One of the most notorious effects of SVMPs is their ability to disrupt microvessels, provoking local and systemic hemorrhage [3,8]. It has been proposed that this effect is the consequence of the hydrolysis of proteins forming the basement membrane (BM) of capillary blood vessels, a phenomenon that has been consistently demonstrated in vitro [917]. Although studies on BM damage in vivo have been scarce, a number of observations support the concept that capillary vessel BM is indeed affected by SVMPs when injected in tissues [16,1820]. A unified hypothesis, based on a two-step mechanism, has been proposed to explain the pathogenesis of hemorrhage by SVMPs [8,21]. Initially, SVMPs hydrolyze key peptide bonds in BM and supporting extracellular matrix (ECM) components, promoting the weakening of the mechanical stability of BM. As a consequence, the hemodynamic biophysical forces normally operating in the vasculature, such as microvessel wall tension and shear stress, provoke the distention of the weakened capillary, which leads to microvessel disruption and extravasation [8]. Despite sharing a highly similar structure in their catalytic domain, SVMPs greatly differ in their capacity to induce hemorrhage [8,22]. In general, P-III SVMPs are more potent hemorrhagic toxins than P-I SVMPs. This is likely to depend on the presence, in the former, of extra domains in addition to the catalytic one, since exosites in disintegrin-like and cysteine-rich domains enable these enzymes to bind to relevant targets in the extracellular matrix or in endothelial cells [1,20,2326]. Moreover, P-III SVMPs are highly resistant to inhibition by the plasma proteinase inhibitor a2-macroglobulin (a2M), whereas P-I SVMPs are readily inhibited [2732]. On the other hand, an intriguing observation is that a significant variation in hemorrhagic potency occurs also within the class P-I SVMPs [33,34]. Since these enzymes comprise the metalloproteinase domain only, such difference in hemorrhagic activity depends on variations within this domain. Various proposals have been presented for identifying key structural determinants for hemorrhagic activity in P-I SVMPs [3439], although this issue remains largely unsolved. The functional differences between hemorrhagic and nonhemorrhagic P-I SVMPs have not been clarified either, as both groups are able to hydrolyze a variety of ECM components in vitro [1113,1518,22,40,41]. The cleavage patterns of several SVMPs on BM and plasma components in vitro ha (...truncated)