Inhibition of fibrinogen binding to immobilized αIIbβ3 (A) and vitronectin binding to immobilized αvβ3 (B) by echistatin and its analogues. Three hundred nanograms of integrin was immobilized on a 96-well enzyme-linked immunosorbent assay (ELISA) plate under conditions described in the Materials and Methods. After blocking the plate, the ligand (fibrinogen or vitronectin, 1 μg per sample) was added to the wells together with different concentrations of echistatin (○), D27W echistatin (•), echistatin 1-41 (▿), or R24A echistatin (▾) and incubated at 37°C for 30 minutes. The amount of bound ligand was detected using antifibrinogen or antivitronectin polyclonal antibodies as described in the Materials and Methods. Error bars represent the standard deviation for three independent experiments.