Substituting isoserine for serine in the thrombin receptor activation peptide SFLLRN confers resistance to aminopeptidase M-induced cleavage and inactivation

Peptides containing sequences derived from the new NH2 terminus of the seven-transmembrane domain thrombin receptor after thrombin cleavage can activate platelets directly. We recently demonstrated that such peptides are readily cleaved and inactivated by plasma, serum, and endothelial cell-associated aminopeptidase M. The rapid degradation and inactivation of the peptides makes it difficult to assess dose-response relationships precisely or to conduct long term incubations with cells in the presence of plasma or serum. To overcome these problems, we first substituted D-serine for the NH2-terminal L-serine in an active peptide ligand (SFLLRNPNDKY). The D-serine derivative resisted degradation in plasma, but it is less than 1% as active as the L-serine peptide. Substituting a racemic mixture of the beta-amino acid isoserine for serine in a related peptide ligand (SFLLRN) produced an active peptide ((iso-S)FLLRN) that is approximately 15-20% as potent as SFLLRN as judged by platelet aggregation. To assess the stability of the peptides in plasma, SFLLRN (1 mM) was incubated with 50% plasma for various periods of time; after 15 min, 65% of the peptide remained intact, and after 2 h only 4% remained intact. Loss of aggregating activity paralleled the loss of intact peptide. In contrast, even after 2 h of incubation with plasma, 83% of the (iso-S)FLLRN remained intact and the aggregating activity was essentially unchanged. Qualitative differences in the patterns of platelet aggregation produced by the peptides were also observed. Thus, the distinct pattern of aggregation followed by rapid disaggregation observed with submaximal aggregating doses of SFLLRN was less evident with (iso-S)FLLRN, and inhibition of aminopeptidase M by amastatin enhanced aggregation in platelet-rich plasma induced by SFLLRN but not (iso-S)FLLRN. The (iso-S)FLLRN peptide should permit improved analysis of the effects of constant levels of peptide-induced thrombin receptor stimulation in the presence of plasma or serum, or when testing the effects of the peptide on cells that contain surface-associated aminopeptidase M.