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 (EC 3.4.11.2) 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. In addition, aminopeptidase M in plasma and on endothelial cells can inactivate the peptides by cleavage of the N-terminal serine. This observation needs to be considered in designing and interpreting experiments using these peptides. Plasma aminopeptidase M does not, however, appear to inactivate the tethered ligand produced by thrombin's cleavage of the receptor on platelets ex vivo, but this does not exclude a potential effect on platelets or endothelial cells in vivo at low thrombin concentrations.