Abstract

We have used a guinea pig gastric longitudinal (LM) smooth muscle bioassay system to evaluate the contractile activities of a previously described thrombin receptor-derived polypeptide, S42FLLRNPNDKYEPF55 (one-letter amino acid code) (TRP42-55) and of a series of peptides derived from this sequence. The contractile activities of the polypeptides were compared with the actions of thrombin. Shortened peptides of the sequences S42FLLRNPND50, S42FLLRN47, and S42FLLR46 (TRP42-46) all exhibited contractile activities that were equivalent to or greater than those of the parent polypeptide, TRP42-55. Both TRP42-55 and TRP42-46 mimicked the action of thrombin, in terms of two different signal transduction pathways that were activated either in the LM preparation or in the related but distinct gastric circular muscle assay. In the LM preparation, the peptide FSLLR also exhibited appreciable, but much reduced, activity. Minimal activity was exhibited in the LM by the sequence SFLLA, but the lysine-containing analogue S42FLLK46 was about one fifth as potent as TRP42-46. In contrast, the receptor-derived sequences S42FLL45, S42FL44-NH2, F43LLR46, and S42ALLR46, as well as arginine-containing polypeptides beginning with the SF motif, SFRG and SFRGHITR, were inactive in the LM bioassay system, at concentrations of greater than or equal to 200 microM, as either agonists or antagonists against TRP42-55. In addition to its actions in the LM and circular muscle preparations, the active pentapeptide, TRP42-46, also exhibited thrombin-mimetic intrinsic activity in a rat aortic arterial ring relaxation bioassay, whereas the pentapeptide S42FLLA46 and the tetrapeptide S42FLL45 were inactive. We conclude that the intrinsic biological activity of the thrombin receptor-derived peptide resides in the pentapeptide TRP42-46 and that the phenylalanine and arginine residues at positions 43 and 46 play key roles in the activity of this pentapeptide in smooth muscle systems.