Thrombin receptor activating peptides: importance of the N-terminal serine and its ionization state as judged by pH dependence, nuclear magnetic resonance spectroscopy, and cleavage by aminopeptidase M

Peptides derived from the recently identified thrombin receptor were tested for their ability to induce platelet aggregation in platelet-rich plasma. The 14 amino acid peptide identified as the new N-terminus after thrombin cleavage (T-14) and an 11 amino acid peptide (T-11) lacking the 3 C-terminal amino acids of T-14 were studied. Both induced platelet aggregation at micromolar concentrations, with T-11 about twice as potent as T-14. Induction of platelet aggregation by these two peptides showed an unusual pH dependence, being more potent at pH 7.2 than at pH 8.1; thrombin-induced aggregation showed a reverse pH dependence. Proton NMR studies of T-11 demonstrated that the chemical shift of the C-alpha proton of the N-terminal serine had a pH dependence that mirrored the aggregation potency. Acetylating the N-terminus of T-11 resulted in loss of aggregating activity, and this peptide did not show the pH-dependence change in chemical shift. The T-14 and T-11 peptides lost aggregating activity when incubated in plasma due to cleavage of the N-terminal serine by an enzyme identified as aminopeptidase M based on its pattern of inhibition and the ability of purified aminopeptidase M (EC3.4.11.2) to cleave the T-11 peptide. Endothelial cell aminopeptidase M was also able to cleave T-11. Inhibiting aminopeptidase M with amastatin enhanced aggregation induced by T-11 but not thrombin. These studies suggest that ionization of the N-terminus of the T-11 and T-14 peptides may be important in initiating platelet aggregation.