title: Role of collagens and perlecan in microvascular stability: exploring the mechanism of capillary vessel damage by snake venom metalloproteinases.
abstract: 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.
authors.affiliation: Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.
authors.forename: Teresa
authors.initials: T
authors.lastname: Escalante
authors.forename: Natalia
authors.initials: N
authors.lastname: Ortiz
authors.forename: Alexandra
authors.initials: A
authors.lastname: Rucavado
authors.forename: Eladio F
authors.initials: EF
authors.lastname: Sanchez
authors.forename: Michael
authors.initials: M
authors.lastname: Richardson
authors.forename: Jay W
authors.initials: JW
authors.lastname: Fox
authors.forename: José María
authors.initials: JM
authors.lastname: Gutiérrez
mesh_terms: D000510:Alpha-Globulins / Q000378:metabolism; D000818:Animals; D001485:Basement Membrane / Q000187:drug effects / Q000378:metabolism; D015153:Blotting, Western; D017837:Bothrops; D002196:Capillaries / Q000187:drug effects* / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D003094:Collagen / Q000378:metabolism*; D003435:Crotalid Venoms / Q000201:enzymology*; D004338:Drug Combinations; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005122:Exudates and Transudates; D006470:Hemorrhage / Q000473:pathology*; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism*; D006868:Hydrolysis / Q000187:drug effects; D007150:Immunohistochemistry; D007328:Insulin / Q000378:metabolism; D007797:Laminin / Q000378:metabolism; D008666:Metalloendopeptidases / Q000494:pharmacology; D045726:Metalloproteases / Q000494:pharmacology*; D051379:Mice; D008970:Molecular Weight; D009132:Muscles / Q000187:drug effects / Q000473:pathology; D011509:Proteoglycans / Q000378:metabolism; D059748:Proteolysis / Q000187:drug effects; D040901:Proteomics; D014947:Wounds and Injuries / Q000473:pathology
chemical_list: D000510:Alpha-Globulins; D002364:Caseins; D003435:Crotalid Venoms; D004338:Drug Combinations; D016326:Extracellular Matrix Proteins; D019812:Heparan Sulfate Proteoglycans; D007328:Insulin; D007797:Laminin; D011509:Proteoglycans; C025500:alpha(2)-microglobulin; C026018:azocasein; C059485:matrigel; C071927:perlecan; D003094:Collagen; D045726:Metalloproteases; C104933:BaP1 metalloproteinase; D008666:Metalloendopeptidases