SIGNIFICANCE OF RGD LOOP AND C-TERMINAL DOMAIN OF ECHISTATIN FOR RECOGNITION OF ALPHA-IIB-BETA-3 AND ALPHA-V-BETA-3 INTEGRINS AND EXPRESSION OF LIGAND-INDUCED BINDING-SITE

Echistatin is a viper venom disintegrin containing RGD loop maintained by disulfide bridges. It binds with a high affinity to alpha v beta 3 and alpha IIb beta 3 and it induces extensive conformational changes in these integrins resulting in expression of ligand-induced binding s ite (LIES) epitopes, We investigated the activities of echistatin and its three analogues (R24A, D27W, echistatin 1-41). R24A echistatin did not react with alpha IIb beta 3 and alpha v beta 3 integrins and did not cause LIES effect, D27W echistatin showed increased binding to alp ha IIb beta 3 and decreased binding to alpha v beta 3. This substituti on impaired the ability of echistatin to induce LIES in alpha v beta 3 integrin. Deletion of nine C-terminal amino acids of echistatin decre ased its ability to bind alpha IIb beta 3 and inhibit platelet aggrega tion. Truncated echistatin failed to induce LIES epitopes on cells tra nsfected with alpha IIb beta 3 and alpha v beta 3 genes. The ability o f echistatin 1-41 to compete with binding of vitronectin to immobilize d alpha v beta 3 and monoclonal antibody 7E3 to platelets and to VNRC3 cells was decreased, although this analogue, after immobilization, re tained its ability to bind purified alpha v beta 3. We propose a hypot hesis in which echistatin's RGD loop determines selective recognition of alpha IIb beta 3 and alpha v beta 3 integrin, whereas the C-termina l domain supports its binding to resting integrin and significantly co ntributes to the expression of LIES epitope and to conformational chan ges of the receptor, leading to a further increase of the binding affi nity of echistatin and of the inhibitory effect. (C) 1997 by The Ameri can Society of Hematology.