Alternagin-C (ALT-C), a disintegrin-like protein purified from the venom of the Brazilian snake Bothrops alternatus, interacts with the major collagen I receptor, the ␣ 2 ␤ 1 integrin, inhibiting collagen binding. Here we show that ALT-C also inhibits the adhesion of a mouse fibroblast cell line (NIH-3T3) to collagen I (IC 50 2.2 M). In addition, when immobilized on plate wells, ALT-C supports the adhesion of this cell line as well as of human vein endothelial cell (HUVEC). ALT-C (3 M) does not detach cells that were previously bound to collagen I. ALT-C (5 nM) induces HUVEC proliferation in vitro, and it inhibits the positive effect of vascular endothelial growth factor (VEGF) or FGF-2 on the proliferation of these cells, thus suggesting a common mechanism for these proteins. Gene expression analysis of human fibroblasts growing on ALT-C-or collagen-coated plates showed that ALT-C and collagen I induce a very similar pattern of gene expression. When compared with cells growing on plastic only, ALT-C up-regulates the expression of 45 genes including the VEGF gene and downregulates the expression of 30 genes. Fibroblast VEGF expression was confirmed by RT-PCR and ELISA assay. Up-regulation of the VEGF gene and other growth factors could explain the positive effect on HUVEC proliferation. ALT-C also strongly activates Akt/PKB phosphorylation, a signaling event involved in endothelial survival and angiogenesis. In conclusion, ALT-C acts as a survival factor, promoting adhesion and endothelial cell proliferation.Cell attachment to the extracellular matrix depends mostly on the integrins, a large family of glycoproteins expressed at the cell surface (1). Integrins are heterodimers formed of noncovalently associated ␣-and ␤-subunits (2). In many cells in culture, integrin-mediated adhesion results in specialized adhesion sites, named focal contacts (3). In these sites, structural and signaling proteins such as integrins, cytoskeletal proteins, and kinases are concentrated and initiate signal transduction pathways (4). Aggregation of integrin receptors, ligand occupancy, and tyrosine kinase-mediated phosphorylation are the key events that results in diverse processes such as cell migration and differentiation, tissue remodeling, cell proliferation, angiogenesis, and tumor cell invasion and metastasis (1, 5).Antagonists of integrins have been developed in order to provide powerful therapeutic approaches for the treatment of several types of cancer, such as antibodies to the ␣ v integrin (6). Synthetic peptides with the sequence Arg-Gly-Asp (RGD) can competitively block the binding of several integrins to their ligands and efficiently reduce platelet aggregation and the number of experimental metastasis (7). RGD peptides induce the disassembly of focal contacts in melanoma cells and disrupt the actin cytoskeleton (8). Disintegrins are small peptides derived from viperidae snake venoms with an internal RGD or KGD motif (9). Disintegrins can bind to integrins and interfere with integrin function. In platelets, disintegrins...

Recently, a new protein containing a disintegrin domain, alternagin-C (Alt-C), was purified from Bothrops alternatus venom. Unlike other disintegrins, in Alt-C an ECD amino acid motif takes the place of the RGD sequence. Most disintegrins contain an RGD/KGD sequence and are very potent inhibitors of platelet aggregation, as well as other cell interactions with the extracellular matrix, including tumor cell metastasis and angiogenesis. The present study investigated the effects of Alt-C on human neutrophil chemotaxis in vitro and the activation of integrin-mediated pathways. Alt-C showed a potent chemotactic effect for human neutrophils when compared to N-formyl-methionyl-leucylphenylalanine peptide (fMLP), a classic chemotactic agent. Moreover, preincubation of neutrophils with Alt-C significantly inhibited chemotaxis toward fMLP and itself. In addition, a peptide containing an ECD sequence presented a chemotactic activity and significantly inhibited chemotaxis induced by Alt-C and fMLP. A significant increase of F-actin content was observed in cells treated with Alt-C, showing that the chemotactic activity of Alt-C on neutrophils is driven by actin cytoskeleton dynamic changes. Futhermore, this protein was able to induce an increase of phosphotyrosine content triggering focal adhesion kinase activation and its association with phosphatidylinositol 3-kinase. Alt-C was also able to induce a significant increase in extracellular signal-regulated kinase 2 1 nuclear translocation. The chemotactic activity of Alt-C was partially inhibited by LY294002, a specific phosphatidylinositol 3-kinase inhibitor, and by PD98056, a Map kinase kinase 2 inhibitor. These findings suggest that Alt-C can trigger human neutrophil chemotaxis modulated by intracellular signals characteristic of integrin-activated pathways and that these effects could be related to the ECD motif present in disintegrin-like domain.Keywords: neutrophil; chemotaxis; integrin signaling.The recruitment of polymorphonuclear neutrophils to sites of inflammation and tissue injury requires rolling on the vessel walls and subsequent migration through the vascular endothelium. Migration involves multiple neutrophil adhesion receptors, such as L-selectin for rolling and integrins for adherence and locomotion [1,2]. These adhesion receptors have counter-receptors on endothelial cells and also specific ligands that are extracellular matrix (ECM) proteins [3].Integrins are comprised of noncovalently linked a and b chains that can associate in various combinations and thus determine the ligand-binding specificities of the intact heterodimer [4,5]. On the other hand, binding of integrins to the ECM is mediated by an integrin-recognition RGD motif found on some ECM components such as fibronectin, vitronectin and fibrinogen [6]. Integrin-ligand binding and receptor clustering initiate a signaling cascade that involves receptor activation, increase in tyrosine kinase activity and protein phosphorylation, and reorganization of the actin cytoskeleton [5,7]. Focal adhesion kin...

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The alpha2ß1 integrin is a major collagen receptor that plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Alternagin-C (ALT-C), a disintegrin-like protein purified from the venom of the Brazilian snake Bothrops alternatus, competitively interacts with the alpha2ß1 integrin, thereby inhibiting collagen binding. When immobilized in plate wells, ALT-C supports the adhesion of fibroblasts as well as of human vein endothelial cells (HUVEC) and does not detach cells previously bound to collagen I. ALT-C is a strong inducer of HUVEC proliferation in vitro. Gene expression analysis was done using an Affimetrix HU-95A probe array with probe sets of ~10,000 human genes. In human fibroblasts growing on collagen-coated plates, ALT-C up-regulates the expression of several growth factors including vascular endothelial growth factor, as well as some cell cycle control genes. Up-regulation of the vascular endothelial growth factor gene and other growth factors could explain the positive effect on HUVEC proliferation. ALT-C also strongly activates protein kinase B phosphorylation, a signaling event involved in endothelial cell survival and angiogenesis. In human neutrophils, ALT-C has a potent chemotactic effect modulated by the intracellular signaling cascade characteristic of integrin-activated pathways. Thus, ALT-C acts as a survival factor, promoting adhesion, migration and endothelial cell proliferation after binding to alpha2ß1 integrin on the cell surface. The biological activities of ALT-C may be helpful as a therapeutic strategy in tissue regeneration as well as in the design of new therapeutic agents targeting alpha2ß1 integrin

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