Immunological characterization of eristostatin and echistatin binding sites on α_IIb β₃ and α_Vβ₃ integrins

Two disintegrins with a high degree of amino acid sequence similarity, echistatin and eristostatin, showed a low level of interaction with Chinese hamster ovary (CHO) cells, but they bound to CHO cells transfected with α_IIbβ₃ genes (A5 cells) and to CHO cells transfected with α_vβ₃ genes (VNRC3 cells) in a reversible and saturable manner. Scatchard analysis revealed that eristostatin bound to 816000 sites per A5 cell (K_d 28 nM) and to 200000 sites (K_d 14 nM) per VNRC3 cell respectively. However, VNRC3 cells did not bind to immobilized eristostatin. Echistatin bound to 495000 sites (K_d 53 nM) per A5 cell and to 443000 sites (K_d 20 nM) per VNRC3 cell. As determined by flow cytometry, radiobinding assay and adhesion studies, binding of both disintegrins to A5 cells and resting platelets and binding of echistatin to VNRC3 cells resulted in the expression of ligand-induced binding sites (LIBS) on the β₃ subunit. Eristostatin inhibited, more strongly than echistatin, the binding of three monoclonal antibodies: OPG2 (RGD motif dependent), A2A9 (α_IIbβ₃ complex dependent) and 7E3 (α_IIbβ₃ and α_vβ₃ complex dependent) to A5 cells, to resting and to activated platelets and to purified α_IIbβ₃. Experiments in which echistatin and eristostatin were used alone or in combination to inhibit the binding of 7E3 and OPG2 antibodies to resting platelets suggested that these two disintegrins bind to different but overlapping sites on α_IIbβ₃ integrin. Monoclonal antibody LM 609 and echistatin seemed to bind to different sites on α_vβ₃ integrin. However, echistatin inhibited binding of 7E3 antibody to VNRC3 cells and to purified α_vβ₃, suggesting that α_vβ₃ and α_IIbβ₃ might share the same epitope to which both echistatin and 7E3 bind. Eristostatin had no effect in these systems, providing further evidence that it binds to a different epitope on α_vβ₃.