BIOCHEMICAL-CHARACTERIZATION OF THE BINDING OF ECHISTATIN TO INTEGRINALPHA(V)BETA(3) RECEPTOR

Echistatin is a 49-amino-acid peptide belonging to the family of disin tegrins that are derived from snake venoms and are potent inhibitors o f platelet aggregation and cell adhesion. Integrin alpha(v) beta(3) re ceptor plays a critical role in several physiological processes such a s tumor-induced angiogenesis, tumor cell metastasis, osteoporosis and wound repair. In this study, we have characterized the binding of echi statin to purified integrin alpha(v) beta(3) receptor and the form exp ressed on human embryonic kidney 293 cells. We show that both purified and membrane-bound integrin alpha(v) beta(3) binds to echistatin with a high affinity, which can be competed efficiently by linear and cycl ic peptides containing the RGD sequence. Previous studies have shown t hat alpha(v) beta(3) binds to vitronectin in a nondissociable manner, whereas an RGD-containing peptide derived from vitronectin binds in a dissociable manner with a K-d of 9.4 x 10(-7) M. Our studies indicate that radiolabeled echistatin binds to alpha(v) beta(3) in a nondissoci able manner, similar to native echistatin. However, echistatin does no t support the adhesion of 293 cells expressing alpha(v) beta(3) recept or because of poor binding to plastic dishes and is a potent antagonis t of the adhesion of these cells to vitronectin. These studies demonst rate that echistatin binding to alpha(v) beta(3) is of high affinity a nd irreversible similar to vitronectin and provides an alternate ligan d for high-throughput screening for alpha(v) beta(3) antagonists.