The effect of a panel of proline mutants of dendroaspin, an inhibitor of platelet aggregation and cell adhesion, including A42-dendroaspin, A47-dendroaspin, A49-dendroaspin, A42,47-dendroaspin and A47,49-dendroaspin, was investigated using platelet-aggregation and cell-adhesion assays. Here we show that a single alanine-for-proline substitution did not affect potency when measured as the ability either to inhibit platelet aggregation induced by ADP (IC50 ≈ 170nM) or to block transfected A375-SM cell adhesion to fibrinogen in the presence of Mn2+ as compared with wild-type dendroaspin. By comparison, double proline substitution with alanines significantly reduced the potency in both assays by approx. 5–8-fold. These observations, therefore, suggest that proline residues flanking the RGD motif in dendroaspin and other RGD-containing venom proteins, e.g. disintegrins, may contribute to maintaining a favourable conformation for the solvent-exposed RGD site for its recognition by integrin receptors.
Present address: The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, U.S.A.