“Insularin, a disintegrin from Bothrops insularis venom: Inhibition of platelet aggregation and endothelial cell adhesion by the native and recombinant GST-insularin proteins”

• DOI: 10.1016/j.toxicon.2010.10.013
• Publication Date: 2010-11-11T09:21:03Z
• Authors: Maisa Splendore D...; Inácio Junqueira-...; Diego Butera; Patrícia Bianca C...; Daiana S. Lopes; Solange M.T. Serrano; Daniel C. Pimenta; Geraldo S. Magalhães; Paulo Lee Ho; Ana Maria Moura-d...
• Keywords: 0301 basic medicine; Umbilical Veins; Platelet Aggregation; Disintegrins; Recombinant Fusion Proteins; Molecular Sequence Data; Infant, Newborn; Peptide Mapping; 03 medical and health sciences; Crotalid Venoms; Cell Adhesion; Animals; Humans; Bothrops; Amino Acid Sequence; Endothelium, Vascular; Cloning, Molecular; Cells, Cultured; Platelet Aggregation Inhibitors; Glutathione Transferase

Insularin (INS) was obtained from Bothrops insularis venom by reversed-phase high-performance liquid chromatography using a C(18) column and characterized as a disintegrin by peptide mass fingerprint and inhibition of ADP-induced platelet aggregation. A cDNA coding for P-II a metalloproteinase/disintegrin was cloned from a cDNA library from B. insularis venom glands. The deduced protein sequence possesses 73 amino acid residues, including the N-terminal, internal peptides of native insularin, the ARGDNP-sequence and 12 cysteines in a conserved alignment. This cDNA fragment was subcloned in the pGEX-4T-1 vector and expressed in a prokaryotic expression system as a fusion protein with glutathione S-transferase (GST-INS). Both native and recombinant insularin inhibited ADP-induced platelet aggregation and endothelial cells (HUVEC) adhesion with similar activities indicating that GST-INS folded correctly and preserved the integrin-binding loop. Insularin may be a tool in studies that involve platelets and endothelial cell adhesion dependent on alphaIIbeta3 and alphavbeta3 integrins.