Aminopeptidase modulation of the pharmacological responses to synthetic thrombin receptor agonists

Abstract

Thrombin is a contractile stimulus of isolated rabbit aortic rings and apparently produces its effects through the recently characterized cleavable receptor. A synthetic hexapeptide, NAT 6-NH 2 (new amino terminus), was found to be the minimal active structure for full activation of this receptor. The N-terminal Ser residue of NAT 6-NH 2 is crucial for biological activity. In this study we examined the metabolism of NAT 6-NH 2 in rabbit plasma, where it was rapidly degraded by aminopeptidase M. In the presence of the aminopeptidase inhibitor amastatin, no metabolism was observed. On this basis a metabolically resistant analogue, [Sar 1]NAT 6-NH 2, was designed. We compared the biological activity of thrombin, NAT 6-NH 2 and [Sar 1]NAT 6-NH 2 in the rabbit aorta and found that [Sar 1]NAT 6-NH 2 was more potent than NAT 6-NH 2; however, in the presence of amastatin the concentration-effect curve for NAT 6-NH 2 was shifted to the left of that for [Sar 1]NAT 6-NH 2. The effects of [Sar 1]NAT 6-NH 2 and of thrombin were not modified by the presence of the aminopeptidase inhibitor. We also studied the effect of amastatin on the in vivo hypotensive response to NAT 6-NH 2 and found that it was also influenced by aminopeptidase M inhibition. Our results show that aminopeptidase protection is important when evaluating responses to synthetic agonists of the thrombin cleavable receptor and that an in vivo model, the anesthesized and heparinized rabbit, may be useful for the development of agonists and antagonists of this receptor.