Identification of small peptide analogues having agonist and antagonist activity at the platelet thrombin receptor

Two tripeptide analogues ( N-[3-methyl-1- S[[2- S-[(methyl-amino)carbonyl]-1-pyrrolidinyl]carbonyl]butyl- d-analine) (SC40476) and N-[3-methyl- S-(1-pyrrolidinylcarbonyl)butyl]- d-alanine, ethyl ester, hydrochloride (SC42619)), inhibit aggregation of, and secretion from, human platelets induced by thrombin but cause no significant inhibition of esterolysis or fibrin formation catalysed by this enzyme. Inhibition by SC40476 of the aggregatory response induced by thrombin is incomplete. Neither peptide analogue inhibits aggregation induced by ADP, collagen, vasopressin or 11,9-epoxymethanoprostaglandin H 2 (U-46619). Enhancement of the response is observed when non-saturating concentrations of these agonists are employed. SC42619 causes a parallel shift to the right in the concentration-response curve describing aggregation induced by thrombin. The Schild plot of these data has a slope of 1.05 and the pA 2 is 2.9 ± 0.1. Both SC40476 and SC42619 induce a small but significant decrease in the single platelet content of platelet suspensions. Neither peptide analogue increases platelet cytosolic [Ca 2+] measured using quin 2 or Fura 2. Both analogues cause inhibition of the increase in cytosolic [Ca 2+] induced by thrombin. Inhibition by SC42619 is competitive with respect to thrombin when the extracellular [Ca 2+] is reduced to < 0.1 μM but is non-competitive in the presence of 1 mM Ca 2+. SC42619 also inhibits the increase in cytosolic [Ca 2+] induced by ADP in the presence of 1 mM Ca 2+ but not the smaller increase caused by this agonist when the medium contains < 0.1 μM Ca 2+. SC42619 inhibits Mn 2+ influx induced by thrombin and ADP. SC40476 and SC42619 inhibit the enhanced incorporation of [ 32P] into phosphatidic acid observed on stimulation by thrombin of platelets pre-labelled with [ 32P]-phosphate. Addition of the peptide analogues alone fails to increase significantly the 32P content of phosphatidate, phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine. SC40476 causes no detectable hydrolysis of glycoprotein V as detected by release of the proteolytic product (glycoprotein V fr). The results indicate that SC40476 and SC42619 interact selectively with the platelet thrombin receptor. Both peptide analogues act as effective antagonists for this receptor but also possess weak agonist activity which may also result from interaction with the thrombin receptor. The molecular basis for this latter activity has not been defined. SC42619 non-sel