Rhodostomin (RHO), a disintegrin isolated from snake venom, has been demonstrated to inhibit platelet aggregation through interaction with integrin αIIbβ3, but there is a lack of direct evidence for RHO–integrin αIIbβ3 binding. In addition, no study on the length of Arg49-Gly50-Asp51 (RGD) loop of RHO influencing on its binding to integrin αIIbβ3 has been reported. In the present study we have developed a highly sensitive dot-blot and glutathione S-transferase–RHO pull-down assays; the latter was coupled with a biotin–avidin–horseradish peroxidase enhanced-chemiluminescence detection system. These were able to demonstrate the direct binding of RHO to integrin αIIbβ3. The pull-down assay further showed that four alanine-insertion mutants upstream of the RGD motif and three insertions downstream of the RGD were able to decrease integrin αIIbβ3 binding activity to only a limited extent. By contrast, two insertions immediately next to RGD and one insertion in front of the Cys57 caused almost complete loss of binding activity to αIIbβ3. The results of the platelet-aggregation-inhibition assay and platelet-adhesion assay for the insertion mutants were consistent with results of the 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 αIIbβ3, the specific position of Pro53 residue adjacent to the RGD sequence is important for RHO binding to platelet integrin αIIbβ3.
- insertion mutagenesis
- pull-down assay
↵1 Present address: Graduate Institute of Molecular and Cellular Biology, Tzu-Chi University, Hualien, Taiwan.
Abbreviations used: RHO, rhodostomin; GST, glutathione S-transferase; HRP, horseradish peroxidase; ECL® (Amersham), enhanced chemiluminescence; RGD, the motif Arg-Gly-Asp in one-letter amino acid notation; vWF, von Willebrand factor; PKA, protein kinase A; NIH, National Institutes of Health.
- The Biochemical Society, London ©2001