A synthetic cell-permeable peptide corresponding to the highly conserved α-integrin signature motif, Palmityl-K989VGFFKR995 (Pal-FF), induces integrin activation and aggregation in human platelets. Systematic replacement of the F992-F993 with amino acids of greater or lesser hydrophobicity to create Pal-KVGxxKR peptides demonstrate that hydrophobic amino acids (isoleucine, phenylalanine, tyrosine, tryptophan) are essential for agonist potency. In marked contrast, substitution with small and/or hydrophilic amino acids (glycine, alanine, serine) causes a switch in the biological activity resulting in inhibition of platelet aggregation, adhesion, ADP secretion, and thromboxane synthesis. These substituted, hydrophilic peptides are not true pharmacological antagonists, as they actively induce a phosphotyrosine signaling cascade in platelets. Singly substituted peptides (Pal-AF and Pal-FA) cause preferential retention of pro- or anti-thrombotic properties, respectively. Because the α-integrin signature motif is an established docking site for a number of diverse cytoplasmic proteins, we conclude that eliminating critical protein-protein interactions mediated through the hydrophobic amino acids, especially F993, favors an anti-thrombotic pathway in platelets. Agents derived from the inhibitory peptides described in this study may represent a new therapeutic strategy for anti-platelet or anti-integrin drug development. © 2009 American Chemical Society.