The term "disintegrin" was first used in 1990 to describe a group of viper venom-derived, nonenzymatic small proteins that shared numerous structural and functional properties. These proteins, which have been found in a great number of viper species studied since that time possess both a remarkable sequence homology and an equally notable variability in potency and selectivity in their interactions with integrin receptors. The discovery that small disintegrins may actually have been derived from much larger mosaic proteins possessing catalytic activity, and that species other than snakes (both plant and animal) produce proteins containing disintegrin-like domains, has led to much research related to both the proteins themselves and the receptors to which they bind. The purpose of this review is to discuss the literature and the authors' own data on the structure and function of disintegrins and their relevance to the studies on proteins containing disintegrin-like domains, such as hemorrhagins and ADAMs.

There are key differences between the amino acid residues of the RGD loops and the C termini of echistatin, a potent antagonist of ␣ IIb ␤ 3 , ␣ v ␤ 3 and ␣ 5 ␤ 1 , and eristostatin, a similar disintegrin selectively inhibiting ␣ IIb ␤ 3 . In order to identify echistatin motifs required for selective recognition of ␣ v ␤ 3 and ␣ 5 ␤ 1 integrins, we expressed recombinant echistatin, eristostatin, and 15 hybrid molecules. We tested them for their ability to inhibit adhesion of different cell lines to fibronectin and von Willebrand factor and to express ligand-induced binding site epitope. The integrins are a family of cell surface glycoproteins that act as receptors for extracellular matrix (ECM) 1 proteins, or for membrane-bound counter-receptors on other cells. Each integrin is a heterodimer that contains an ␣ and a ␤ subunit, the pairing of which specifies the ligand binding abilities of integrins (1). Integrins can bind adhesive ligands and upon this binding undergo conformational changes leading to the exposure of neoepitopes recognized by specific monoclonal antibodies called anti-ligand-induced binding site (LIBS) antibodies (2).The Arg-Gly-Asp (RGD) sequence is the cell attachment site of a large number of adhesive ECM, blood, and cell surface proteins, and nearly half of the over 20 known integrins recognize this sequence in their adhesion protein ligands (3). In addition to the RGD motif, many adhesive receptors recognize other integrin-binding domains, such as the KQAGDV sequence (4) of fibrinogen ␥-chain, ILDV sequence of the CS1 region of fibronectin (5, 6), and RRETAWA sequence identified from a phage display library (7). It is known that the Arg and Asp residues are necessary but not sufficient to ensure binding activity. The RGD sequence is generally found to be associated with a series of probable ␤-bends, which result in a highly ordered structure. This highly ordered conformation might form the basis of the specific binding of many proteins containing this cell surface recognition sequence. Additional determinants of integrin specificity and the high affinity of RGDcontaining adhesive proteins for integrins may be the result of the specific conformation of the RGD sequence or by the amino acids immediately adjacent to the RGD site, creating an extended RGD locus.Disintegrins are snake venom proteins capable of binding to integrins and interfering with integrin function (8 -10). Disintegrins typically have an RGD sequence as their active site, except for barbourin containing a KGD sequence (11), and a new class of heterodimeric disintegrins such as EC3 and EMF10, containing MLD, VGD, and other recognition motifs (12). It appears that low molecular weight disintegrins, binding to integrins with an affinity approaching that of monoclonal antibodies, may represent a useful probe to identify functionally important motifs in the cell adhesion receptors. Scarborough et al. (13) postulated that the amino acid residue Cterminal to the RGD sequence determines selectivity of disintegrin for an integrin re...

The existence of disintegrins, non-enzymatic, small molecular weight proteins from viper venom, has been known for 2 decades, and their impact on cellular research has been substantial and far-reaching. Disintegrins have been the molecular scaffold used in the design of therapeutics for the prevention of thrombosis and cancer. Their sequencing has provided insights into the evolution of proteins over millennia. Production of recombinant disintegrin mutants and fusion proteins has allowed investigations into molecular mechanisms at work in cell-extracellular matrix interactions. Structural homologies with non-snake proteins have shown disintegrin-like molecules in species ranging from slime mold to humans. Intracellular signaling events have been elucidated through the use of venom disintegrins, including events related to programmed cell death, motility, cell proliferation and viral pathogenesis. Disintegrin sequences (protein or genes) have been placed in microbubbles and liposomes and been found to target neovascular endothelium and metastatic tumors in two mouse models. The purpose of this review is to highlight the members of this disintegrin family discovered since 1998 as well as the increased understanding of their usefulness in therapeutics and technical assays.

Echistatin is a viper venom disintegrin containing RGD loop maintained by disulfide bridges. It binds with a high affinity to αvβ3 and αIIbβ3 and it induces extensive conformational changes in these integrins resulting in expression of ligand-induced binding site (LIBS) epitopes. We investigated the activities of echistatin and its three analogues (R24A, D27W, echistatin 1-41). R24A echistatin did not react with αIIbβ3 and αvβ3 integrins and did not cause LIBS effect. D27W echistatin showed increased binding to αIIbβ3 and decreased binding to αvβ3. This substitution impaired the ability of echistatin to induce LIBS in αvβ3 integrin. Deletion of nine C-terminal amino acids of echistatin decreased its ability to bind αIIbβ3 and inhibit platelet aggregation. Truncated echistatin failed to induce LIBS epitopes on cells transfected with αIIbβ3 and αvβ3 genes. The ability of echistatin 1-41 to compete with binding of vitronectin to immobilized αvβ3 and monoclonal antibody 7E3 to platelets and to VNRC3 cells was decreased, although this analogue, after immobilization, retained its ability to bind purified αvβ3. We propose a hypothesis in which echistatin's RGD loop determines selective recognition of αIIbβ3 and αvβ3 integrin, whereas the C-terminal domain supports its binding to resting integrin and significantly contributes to the expression of LIBS epitope and to conformational changes of the receptor, leading to a further increase of the binding affinity of echistatin and of the inhibitory effect.

Viper venom disintegrins contain the RGD/KGD motif. They inhibit platelet aggregation and cell adhesion, but show structural and functional heterogeneity. We investigated the interaction of four prototypic disintegrins with alpha IIb beta 3 expressed on the surface of resting and activated platelets. The binding affinity (Kd) of 125I-albolabrin, 125I-echistatin, 125I-bitistatin and 125I-eristostatin toward resting platelets was 294, 153, 48 and 18 nM respectively. The Kd value for albolabrin decreased 3-fold and 6-fold after ADP- or thrombin-induced activation. The Kd values for bitistatin and echistatin also decreased with ADP, but there was no further decrease with thrombin. In contrast, eristostatin bound with the same high affinity to resting and activated platelets. The pattern of fluorescein isothiocyanate (FITC)-eristostatin and FITC-albolabrin binding to resting and activated platelets was consistent with observations using radiolabelled material. Eristostatin showed faster and more irreversible binding to platelets, and greater potency compared with albolabrin in inducing conformational neo-epitopes in beta 3. The anti-alpha IIb beta 3 monoclonal antibody OP-G2 that is RGD-dependent inhibited disintegrin binding to activated platelets more strongly than binding to resting platelets and it inhibited the binding to platelets of albolabrin more strongly than eristostatin. The specificity of disintegrin interaction with alpha IIb beta 3 was confirmed by demonstrating cross-linking of these peptides to alpha IIb beta 3 on normal platelets, but not to thrombasthenic platelets deficient in alpha IIb beta 3.