Role of the amino- and carboxyl-terminal domains of thrombin receptor-derived polypeptides in biological activity in vascular endothelium and gastric smooth muscle: evidence for receptor subtypes.

Using guinea pig gastric longitudinal muscle (GLM) and rat gastric longitudinal muscle (RLM) contractile assays and a rat aortic ring (RA) endothelium-dependent relaxation assay, we have examined the biological activities of a number of human and rat thrombin receptor-derived polypeptides (TRPs) modified at amino-terminal and carboxyl-terminal residues. Our study focused primarily on the human pentapeptide [S42FLLR46 (P5)], previously shown to retain full thrombin-like activity. Whereas N-acetylation (N-acetyl-P5) abolished biological activity in the GLM and RA assays, amidation or esterification of the carboxyl-terminal carboxyl group [P5-NH2, P5-OCH3, or S42FLLRNP48-NH2 (P7-NH2)] enhanced peptide potency by about 10-fold in both the GLM and RA assays, compared with the unmodified TRPs (P5 and P7). Removal from P5 of either the amino-terminal hydroxyl group of serine (to yield A42FLLR46) or both the amino-terminal hydroxyl group and the primary amino group of P5 [to yield propionyl-F43LLR46 (Pr-P4)] produced peptides that were active in both the GLM and RA assays. Substitution of the carboxyl-terminal guanidinium group of P5 with a less basic primary amino acid residue (S42FLLK46) resulted in a peptide with a lower potency than that of P5 in the GLM and RA assays, whereas substitution of D-arginine for L-arginine at the carboxyl terminus abolished biological activity. Substitution of norleucine for arginine (S42FLLNorleuN) resulted in a peptide active in the GLM but not in the RA assay. For selected agonists (Pr-P4, P5, P7, and P7-NH2), the potencies in the GLM and RA assays, relative to that of P5, differed; for the GLM the order was P7-NH2 > P7 > P5 congruent to Pr-P4, whereas for the RA the order was P7-NH2 > or = Pr-P4 >> P5 > or = P7. Data comparable to those obtained with the GLM assay were also obtained with a RLM assay, wherein the potency series was P7-NH2 > P7 > P5. The relative potencies of pentapeptides based on the rat receptor sequence [SFFLR (Ra-P5), SFFLR-NH2 (Ra-P5-NH2), and SFFLRNP (Ra-P7)] differed in the RLM and RA assays. In the RLM the order was Ra-P5-NH2 > P7-NH2 > P5-NH2 > Ra-P7 = P7 > P5 = Ra-P5, whereas in the RA the relative potency series was P7-NH2 > P5-NH2 > Ra-P5-NH2 > Ra-P7 > P5 > or = P7 > Ra-P5.(ABSTRACT TRUNCATED AT 400 WORDS)