In order to demonstrate that the amino acid residues flanking the RGD sequence were important for inhibiting the ADP-induced platelet aggregation, we analyzed the role of the amino acid residues in the domain preceding the RGD loop on the activity of disintegrins. Our approach was to develop hybrids between the disintegrins kistrin and elegantin targeting residues in this domain and within the RGD loop. The basic sequence within elegantin KKKR( 44 ) T ( 45 ) I ( 46 ) /A (50)RGDN(54)P(55) was changed by mutagensis to SKAG ( 44 ) T ( 45 ) I ( 46 )/P(50)RGDM(54)P(55) and to SKAG(44)I(46)/P(50)RGDM(54)P(55), thereby resembling the corresponding S(39)RAGT(43)/P(50)RGDM(52)P(53) sequence in kistrin. This changed KKKR(44)T(45)I(46)/A(50)RGDN(54)P(55)right curved arrow SKAG(44)T(45)I(46)/P(50)RGDM(54)P(55) dramatically reduced the activity of elegantin as an inhibitor of platelet aggregation. In contrast, deletion of T(45) (KKKR(44)T(45)I(46)/A(50)RGDN(54)P(55)right curved arrow SKAG(44)T(45)I(46)/P(50)RGDM(54)P(55))increased activity of elegantin as an inhibitor platelet aggregation. It was further shown that their electrophoresis properties were very different. These data highlight the importance of the domain encompassing residues 39--45 and the amimo acid residues flanking the RGD sequence on disintegrin structure-function.