Positional importance of Pro(53) adjacent to the Arg(49)-Gly(50)-Asp(51) sequence of rhodostomin in binding to integrin alpha IIb beta 3

Rhodostomin (RHO), a disintegrin isolated from snake venom, has been demons trated to inhibit platelet aggregation through interaction with integrin al pha IIb beta3, but there is a lack of direct evidence for RHO-integrin alph a IIb beta3 binding. In addition, no study on the length of Arg(49)-Gly(50) -Asp(51) (RGD) loop of RHO influencing on its binding to integrin alpha IIb beta3 has been reported. In the present study we have developed a highly s ensitive dot-blot and glutathione S-transferase-RHO pull-down assays; the l atter was coupled with a biotin-avidin-horseradish peroxidase enhanced-chem iluminescence detection system. These were able to demonstrate the direct b inding of RHO to integrin alpha IIb beta3. The pull-down assay further show ed that four alanine-insertion mutants upstream of the RGD motif and three insertions downstream of the RGD were able to decrease integrin alpha IIb b eta3 binding activity to only a limited extent. By contrast, two insertions immediately next to RGD and one insertion in front of the Cys(57) caused a lmost complete loss of binding activity to alpha IIb beta3. The results of the platelet-aggregation-inhibition assay and platelet-adhesion assay for t he insertion mutants were consistent with results ofthe pull-down assay. It is thus concluded that, although an insertion of a single alanine residue in many positions of the RGD loop has only minor effects on RHO binding to integrin alpha IIb beta3 the specific position of Pro(53) residue adjacent to the RGD sequence is important for RHO binding to platelet integrin alpha IIb beta3.