Thrombin-induced platelet aggregation is mediated in part by the intracellularly activated calpain expressed onto the external side of the membrane. We have previously shown that P1, Phe-Gln-Val-Val-Cys(Npys)-Gly-NH2 [Npys = 3-nitro-2-pyridinesulfenyl], an affinity analog corresponding to the highly conserved sequence Gln-Val-Val-Ala-Gly-NH2, present in domains 2 and 3 of human kininogens, was an irreversible inhibitor of platelet calpain (second-order rate constant = 5.85 mM-1 s-1). P1 also selectively blocked thrombin-induced platelet aggregation. We have now synthesized twenty-three other peptides, analogous to P1, and evaluated them to define the specificity of the amino acid sequence in P1 to selectively block thrombin-induced platelet aggregation. We find that replacement by Leu of Val and by Tyr of Phe adjacent to Gln is minimally tolerated and the resulting peptides are partially effective in selectively blocking thrombin-induced platelet aggregation. The presence of valine adjacent to cysteine in P1 is essential for the inhibitor to selectively block thrombin-induced platelet aggregation. The presence of valine adjacent to cysteine in P1 is essential for the inhibitor to selectively block thrombin-induced platelet aggregation. Extensions of the N-terminal sequence in P1 did not improve its selectivity. Ac-Ala-Gln-Val-Val-Ala-Gly-NH2 (Ac, acetyl), a peptide containing the conserved sequence but lacking the Npys function, neither inhibited platelet calpain nor platelet aggregation induced by thrombin. Presence of the peptide sequence and Npys function are both required in P1 for its selective action in inhibiting platelet aggregation induced by thrombin.