The Disintegrin Echistatin Stabilizes Integrin αIIbβ3ʹs Open Conformation and Promotes Its Oligomerization

We have employed echistatin, a 5.4 kDa snake venom disintegrin, as a model protein to investigate the paradox that small ligand-mimetics can bind to the resting αIIbβ3 integrin while adhesive macromolecules cannot. We characterized the interactions between purified human αIIbβ3 and two recombinant echistatin variants: rEch (1-49) M28L, chosen for its selectivity toward β3-integrins, and rEch (1-40) M28L, a carboxy-terminal truncation mutant. While both contain an RGD integrin targeting sequence, only rEch (1-49) M28L was an effective inhibitor of αIIbβ3 function. Electron microscopy of rotary shadowed specimens yielded a variety of αIIbβ3 conformers ranging from compact, spherical particles (maximum dimension 22 nm) to the classical “head with two tails” forms (32 nm). The population of larger particles (42–56 nm) increased from 17% to 28% in the presence of rEch (1-49) M28L, indicative of ligand-induced oligomerization. Sedimentation velocity measurements demonstrated that both full length and truncated echistatin perturbed αIIbβ3ʹs solution structure, yielding slower-sedimenting open conformers. Dynamic light scattering showed that rEch (1-49) M28L protected αIIbβ3 from thermal aggregation, raising its transition mid-point from 46 °C to 69 °C; a smaller shift resulted with rEch (1-40) M28L. Sedimentation equilibrium demonstrated that both echistatin ligands induced substantial αIIbβ3 dimerization. vanʹt Hoff analysis revealed a pattern of entropy/enthalpy compensation similar to tirofiban, a small RGD ligand-mimetic that binds tightly to αIIbβ3, but yields smaller conformational perturbations than echistatin. We propose that echistatin may serve as a paradigm for understanding multidomain adhesive macromolecules because its ability to modulate αIIbβ3ʹs structure resides on an RGD loop, while full disintegrin activity requires an auxiliary site that includes the carboxy-terminal nine amino acid residues.