SUBSTITUTING ISOSERINE FOR SERINE IN THE THROMBIN RECEPTOR ACTIVATIONPEPTIDE 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 pe ptides are readily cleaved and inactivated by plasma, serum, and endot helial cell-associated aminopeptidase M. The rapid degradation and ina ctivation of the peptides makes it difficult to assess dose-response r elationships precisely or to conduct long term incubations with cells in the presence of plasma or serum. To overcome these problems, we fir st substituted D-serine for the NH2-terminal L-serine in an active pep tide ligand (SFLLRNPNDKY). The D-serine derivative resisted degradatio n 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 s erine in a related peptide ligand (SFLLRN) produced an active peptide ((iso-S)FLLRN) that is approximately 15-20% as potent as SFLLRN as jud ged by platelet aggregation. To assess the stability of the peptides i n plasma, SFLLRN (1 mM) was incubated with 50% plasma for various peri ods of time; after 15 min, 65% of the peptide remained intact, and aft er 2 h only 4% remained intact. Loss of aggregating activity parallele d the loss of intact peptide. In contrast, even after 2 h of incubatio n with plasma, 83% of the (iso-S)FLLRN remained intact and the aggrega ting activity was essentially unchanged. Qualitative differences in th e 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 wa s less evident with (iso-S)FLLRN, and inhibition of aminopeptidase M b y amastatin enhanced aggregation in platelet-rich plasma induced by SF LLRN but not (iso-S)FLLRN. The (iso-S)FLLRN peptide should permit impr oved analysis of the effects of constant levels of peptide-induced thr ombin receptor stimulation in the presence of plasma or serum, or when testing the effects of the peptide on cells that contain surface-asso ciated aminopeptidase M.