A truncated form of the Agouti-related protein (AgRP), a member of the cystine-knot family, has shown promise as a scaffold for engineering novel peptides with new molecular recognition properties. In this study, we replaced a constrained six amino acid loop in AgRP with a nine amino acid loop containing an Arg–Gly–Asp integrin recognition motif, and randomized the neighboring residues to create a library of ∼20 million AgRP variants. We displayed the AgRP mutants as fusions on the surface of yeast and used high-throughput fluorescence-activated cell sorting (FACS) to isolate peptides that bound specifically to the platelet integrin αIIbβ3, a clinically important target for the prevention and treatment of thrombosis. These AgRP peptides had equilibrium dissociation (KD) constants for αIIbβ3 integrin ranging from 60 to 90 nM, and did not bind to αvβ3, αvβ5, or α5β1 integrins. Using an alternate library screening strategy, we identified AgRP peptides that bound to both αIIbβ3 and αvβ3 integrins with KD values ranging from 40 to 70 nM and 20 to 30 nM, respectively, and did not bind to αvβ5 or α5β1 integrins. Unique consensus sequences were identified within both series of AgRP peptides suggesting alternative molecular recognition events that dictate different integrin binding specificities. In addition, the engineered AgRP peptides prevented platelet aggregation as well as or slightly better than the FDA-approved cyclic peptide eptifibatide. Collectively, these data demonstrate that cystine-knot peptides can be generated with high affinity and specificity to closely-related integrins, and provide insights into molecular interactions between small, structured peptide ligands and their receptors. Copyright © 2010 John Wiley & Sons, Ltd.