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

Abstract

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.(ABSTRACT TRUNCATED AT 250 WORDS)

The proteinase activated receptor-2 (PAR-2) mediates mitogenic responses in human vascular endothelial cells.

“…Thus, these observations establish the specificity of the PAR-2 ligand for its receptor and exclude the possibility that endothelial cell responses are mediated by trans-activation of the TR. Indeed, these observations are consistent with previous TR structure-function studies demonstrating the importance of the phenylalanine in position 2 of the Tr-activating peptide (TR [42][43][44][45][46][47] ) to affect receptor activation (6)(7)(8). Furthermore, the inability of TR antisense oligonucleotides to affect PAR [39][40][41][42][43][44] -mediated responses excludes the possibility that the cell-surface coexpression of both receptors are physically linked or dually required for optimal activation-dependent responses.…”

“…This irreversible cleavage reveals a new amino terminus that functions as a "tethered ligand," presumably binding intramolecularly to other receptor domain(s) to effect downstream receptor coupling events (1,4,5). Synthetic peptidomimetics, based on the new amino terminus created after cleavage, function as full agonists for receptor activation, bypassing the need for proteolytic receptor activation (1,(6)(7)(8). Elucidation of receptor domains involved in ligandmediated receptor activation have been identified in this and other laboratories (9,10).…”

How the protease thrombin talks to cells

“…This proteolytic switch removes amino-terminal sequence that sterically hinders ligand function and generates a new protonated amino group at the amino terminus created by receptor cleavage. In the SFLLRN peptide, the cognate protonated amino group is critical for agonist activity (7,8). Parallels with zymogen activation in serine proteases are apparent (2, 9).…”

Long‐term effects of thrombin require sustained activation of the functional thrombin receptor

“…Recently, Coller et al have demonstrated that aminopeptidase M in plasma and on endothelial cells cleaves and thereby inactivates TRAP [19]. Accordingly, inhibition of aminopeptidase M enhances platelet aggregation induced by TRAP pointing to the possibility of rapid degradation of TRAP in in vivo and in vitro systems.…”