Bothrops protease A (BPA): a new tool for the design of therapeutic agents for thrombosis and haemostasis.

• Grant number: 11/05055-2
• Support Opportunities: Scholarships in Brazil - Master
• Start date: March 01, 2012
• End date: March 31, 2013
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Solange Maria de Toledo Serrano
• Grantee: Edson Takeshi Yamashiro
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Associated research grant: 98/14307-9 - Center for Applied Toxinology, AP.CEPID

Abstract

Bothrops protease A (BPA) is a serine proteinase isolated from the venom of Bothrops jararaca that shows trypsin-like enzymatic specificity. Unlike many venom enzymes, it is stable at pHs between 3 and 9 and resists heating at 90°C for 10 min. BPA is highly glycosylated protein and migrates on SDS-PAGE as an unique band of 67 kDa. However, the cDNA that encodes BPA shows that it is composed of 234 amino acid residues with a calculated molecular mass of 25.409 Da which indicates that 60% of de molecular mass assessed by electrophoresis corresponds to carbohydrates. Its amino acid sequence shows eight putative sites of N-glycosylation and two sites of O-glycosylation. Complete enzymatic N- and O-deglycosylation is only possible under denaturant conditions and the main generated products present 25 kDa and 55 kDa, respectively, which are enzymaticaly inactive. Recently we showed that BPA possesses a potent proteolytic activity on fibrinogen and fibrin in vitro. Fibrinogen alpha and beta chains are rapidly degraded by BPA while in the case of fibrin just the alpha chain is hydrolyzed. Partial deglycosylation of BPA does not affect its proteolytc activity on these proteins. In vivo, BPA prevented thrombus formation in rats caused by stasis of vena cava or by jugular vein injury. Thus, BPA is a proteinase with potential therapeutical application because of its high stability and high fibrinogenolytic activity. In the present study we intend to obtain recombinant BPA in Pichia pastoris and perform site directed mutations to evaluate the role of the carbohydrate moiety in its proteolytic activity on fibrinogen in vitro and in vivo.