The effect of the single substitution of arginine within the RGD tripeptide motif of a modified neurotoxin dendroaspin on its activity of platelet aggregation and cell adhesion.

The Arg-Gly-Asp (RGD) tripeptide unit is a cell-cell and cell-extracellular matrix recognition sequence of some integrins that is found within several extracellular matrix glycoproteins and dendroaspin, a disintegrin-like venom protein isolated from the snake venom of the Dendroaspis jamsonii. In the present study, the RGD motif in dendroaspin was substituted by Lys-Gly-Asp (KGD), His-Gly-Asp (HGD), Gln-Gly-Asp (QGD) and Ala-Gly-Asp (AGD) denoted as KGD-den, HGD-den, QGD-den and AGD-den, respectively. Each of the mutants exhibited activity as inhibitor of ADP-induced platelet aggregation with IC50 values of 0.26, 2.5, 6, and 17 microM for KGD-den, HGD-den, QGD-den, and AGD-den, respectively, as compared with RGD-den (IC50 = 0.18 microM). Interestingly, HGD-den was approx. two-fold more potent and a more selective inhibitor than either the KGD-den or QGD-den counterpart at blocking A375-SM human melanoma cell adhesion to fibrinogen (beta3-mediated). KGD-den, HGD-den, and QGD-den were preferentially antagonists of A375-SM human melanoma cell adhesion to fibrinogen rather than to fibronectin (alpha5beta1-, beta3-mediated). Both HGD-den and KGD-den were equipotent as inhibitors of human erythroleukaemia (HEL) cell adhesion to fibrinogen (IC50 = 0.15 microM) and also preferential inhibitors of HEL cell adhesion to fibrinogen (beta3 and beta1-mediated) rather than to fibronectin. These findings show that the presence of the arginine within the RGD motif of dendroaspin is not obligatory and substitution of this residue can modulate inhibitory potency and integrin binding selectivity.