Aminopeptidase modulation of the pharmacological responses to synthetic thrombin receptor agonists.

Thrombin is a contractile stimulus of isolated rabbit aortic rings and apparently produces its effects through the recently characterized cleavable receptor. A synthetic hexapeptide, NAT6-NH2 (new amino terminus), was found to be the minimal active structure for full activation of this receptor. The N-terminal Ser residue of NAT6-NH2 is crucial for biological activity. In this study we examined the metabolism of NAT6-NH2 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, [Sar1]NAT6-NH2, was designed. We compared the biological activity of thrombin, NAT6-NH2 and [Sar1]NAT6-NH2 in the rabbit aorta and found that [Sar1]NAT6-NH2 was more potent than NAT6-NH2; however, in the presence of amastatin the concentration-effect curve for NAT6-NH2 was shifted to the left of that for [Sar1]NAT6-NH2. The effects of [Sar1]NAT6-NH2 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 NAT6-NH2 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.