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.(ABSTRACT TRUNCATED AT 250 WORDS)
Coller
B S
BS
Department of Medicine, State University of New York, Stony Brook 11794-8151.
Ward
P
P
Ceruso
M
M
Scudder
L E
LE
Springer
K
K
Kutok
J
J
Prestwich
G D
GD
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000378:metabolism*; D018826:CD13 Antigens; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects*; D011956:Receptors, Cell Surface / Q000737:chemistry*; D018179:Receptors, Thrombin; D012694:Serine / Q000737:chemistry*; D013329:Structure-Activity Relationship
D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D012694:Serine; D000626:Aminopeptidases; D018826:CD13 Antigens