485 © Schattauer 2010 Thrombosis and Haemostasis 104.3/2010 Blood Coagulation, Fibrinolysis and Cellular Haemostasis New insights into the structural elements involved in the skin haemorrhage induced by snake venom metalloproteinases Ana K. Oliveira 1 ; Adriana F. Paes Leme 1 ; Amanda F. Asega 1 ; Antonio C. M. Camargo 1 ; Jay W. Fox 2 ; Solange M. T. Serrano 1 1 Laboratório Especial de Toxinologia Aplicada/CAT-CEPID, Instituto Butantan, São Paulo, Brazil; 2 Department of Microbiology, University of Virginia, Charlottesville, Virginia, USA Summary Haemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon resulting in capillary disruption and extrava- sation. This study analysed structural elements important for the inter- action of four Bothrops jararaca SVMPs of different domain organi- sation and glycosylation levels with plasma and extracellular matrix proteins: HF3 (P-III class) is highly glycosylated and ∼80 times more haemorrhagic than bothropasin (P-III class), which has a minor carbo- hydrate moiety; BJ-PI (P-I class) is not haemorrhagic and the DC protein is composed of disintegrin-like/cysteine-rich domains of bothropasin. HF3, bothropasin and BJ-PI showed different degradation profiles of fi- brinogen, fibronectin, vitronectin, von Willebrand factor, collagens IV and VI, laminin and Matrigel™; however, only bothropasin degraded collagen I. In solid-phase binding assays HF3 and bothropasin inter- acted with fibrinogen, fibronectin, laminin, collagens I and VI; the DC protein bound only to collagens I and VI; however, no binding of BJ-PI Correspondence to: S. M. T. Serrano Instituto Butantan Av. Vital Brasil 1500, 05503–900, São Paulo, Brazil Tel.: +55 11 3726 1024 E-mail: solangeserrano@butantan.gov.br to these proteins was detected. N-deglycosylation caused loss of struc- tural stability of bothropasin and BJ-PI but HF3 remained intact, al- though its haemorrhagic and fibrinogenolytic activities were partially impaired. Nevertheless, N-deglycosylated HF3 bound with higher affin- ity to collagens I and VI, although its proteolytic activity upon these col- lagens was not enhanced. This study demonstrates that features of carbohydrate moieties of haemorrhagic SVMPs may play a role in their interaction with substrates of the extracellular matrix, and the ability of SVMPs to degrade proteins in vitro does not correlate to their ability to cause haemorrhage, suggesting that novel, systemic approaches are necessary for understanding the mechanism of haemorrhage gener- ation by SVMPs. Keywords Extracellular matrix, haemorrhage, metalloproteinase, proteolysis, snake venom Financial support: This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (04/15974–1; 98/14307–9) and Instituto Nacional de Ciência e Tecnolo- gia de Toxinas (INCTTox). Received: December 21, 2009 Accepted after major revision: April 12, 2010 Prepublished online: July 20, 2010 doi:10.1160/TH09-12-0855 Thromb Haemost 2010; 104: 485–497 Introduction Metalloproteinases play important roles in the pathological effects of snake venoms including severe local tissue damage, haem- orrhage and coagulopathy (1–5). The haemorrhage and coagulo- pathy induced by snake venom metalloproteinases (SVMPs) are complex phenomena involving their synergic effect upon plasma proteins, platelets and the capillary vessels (4, 6–10). The protei- nase domain of all the haemorrhagic SVMPs is believed to func- tion to degrade capillary basement membranes, endothelial cell surfaces, and stromal matrix ultimately causing extravasation of capillary contents into the surrounding stroma. SVMPs are zinc- dependent enzymes, and members of the M12B subfamily of metalloproteinases along with the A Disintegrin And Metallopro- teinase (ADAM) and the A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) proteins (11–13). Besides the catalytic domain, SVMPs contain additional regulatory do- mains involved in their interactions with plasma components, extracellular matrix (ECM) and integrins (14). SVMPs have been shown to degrade blood coagulation proteins, such as fibrinogen, fibrin, and von Willebrand factor (vWF) (6, 15–18). They are clas- sified in three classes: the mature proteins of class P-I contain only a catalytic domain; the other classes (P-II and P-III) contain addi- tional non-catalytic domains such as disintegrin (or disintegrin- like), cysteine-rich and C-type lectin-like domains (14). Similar structures are found in the ADAMs, which contain an epidermal growth factor-like domain, a transmembrane region and a cyto- plasmic tail (13, 19), and in the ADAMTSs, which contain throm- bospondin-type repeats (20). Despite the structural similarity of their catalytic domains, not all SVMPs are able to induce haemor- rhage. Interestingly some members of the P-III class are signifi- cantly more potent in causing haemorrhage compared with those of the P-I class that lack the disintegrin-like/cysteine-rich domains found in the P-III proteinases. In fact, accumulated evidence in the literature suggests that the disintegrin-like/cysteine-rich domains of the SVMPs as well as those of the ADAMs and ADAMTSs are in- For personal or educational use only. No other uses without permission. All rights reserved. Downloaded from www.thrombosis-online.com on 2018-04-14 | ID: 1001066444 | IP: 54.70.40.11