EXPRESSION AND BIOLOGICAL ACTIVITY OF THE DISINTEGRIN DOMAIN OF BOTHROSTATIN (D-BTT) FROM THE VENOM GLAND OF Bothrops jararaca 

SILVA, C.A.(1), MENTELE, R.(2), CAMARGO, A.C.M.(1), SERRANO, S.M.T.(1)

(1)Laboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo, Brasil. (2)Department of Clinical Chemistry and Clinical Biochemistry, University Hospital of Surgery, Ludwig-Maximilians University, Munich, Germany.

Disintegrins form a group of RGD containing peptides isolated from the venom of Viperidae and Crotalidae snakes. A large number of disintegrins has been isolated and characterized because of their clinical potential as antithrombotic agents. They possess both a high sequence homology and a notable variability in potency and selectivity in their interactions with integrin receptors. Venom disintegrins bind to the fibrinogen receptor aIIbb3 on the surface of platelets resulting in the inhibition of fibrinogen-dependent platelet aggregation, and more recently they have been shown to competitively inhibit the adhesive functions of a variety of integrins on cell surfaces. Bothrostatin represents a precursor of the N-II class of the Reprolysins, isolated from a cDNA library derived from the venom glands of B. jararaca. Its cDNA has 2045 base pairs in length and encodes a pro-domain, a metalloproteinase domain with the characteristic zinc binding sequence and a disintegrin domain with the RGD sequence. The objective of this work was to express and analyze the biological activity of the disintegrin domain on human platelets. For this purpose the disintegrin domain was subcloned in pGEX-4T1 and expressed as a fusion protein with glutathione S-transferase (GST) in E. coli BL21. The recombinant disintegrin domain of bothrostatin (D-BTT) was obtained after cleavage of the fusion protein with thrombin and its authenticity was confirmed by N-terminal protein sequencing. Both D-BTT and GST/D-BTT proteins inhibited collagen-induced platelet aggregation in a dose-dependent fashion with estimated IC50 values of 12 nM and ~1mM, respectively.

Financial support: CAT, Cepid, FAPESP.

CORRESPONDENCE TO:

Carlos Alberto da Silva, Alameda Barros, 150, Apto. 105^A, São Paulo, SP, CEP: 01232-000, Brasil, Email: lesco_var@hotmail.com