Paul W. Manley

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 H2 (U-46619). Enhancement of the response is observed when nonsaturating 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 pA2 is 2.9 +/- 0.1. Both SC40476 and SC42619 induced a small but significant decrease in the single platelet content of platelet suspensions. Neither peptide analogue increases platelet cytosolic [Ca2+] measured using quin 2 or Fura 2. Both analogues cause inhibition of the increase in cytosolic [Ca2+] induced by thrombin. Inhibition by SC42619 is competitive with respect to thrombin when the extracellular [Ca2+] is reduced to less than 0.1 microM but is non-competitive in the presence of 1 mM Ca2+. SC42619 also inhibits the increase in cytosolic [Ca2+]induced by ADP in the presence of 1 mM Ca2+ but not the smaller increase caused by this agonist when the medium contains less than 0.1 microM Ca2+. SC42619 inhibits Mn2+ 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 VFR). 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-selectively inhibits Ca2+ influx induced by several agonists but this effect does not appear to contribute to the observed inhibition of the aggregatory and secretory responses.

Thrombin receptor antagonists: Structure-activity relationships for the platelet thrombin receptor and effects on prostacyclin synthesis by human umbilical vein endothelial cells

Structure-activity studies on a series of analogues of N-(3-methyl-S-(1-pyrrolidinyl carbonyl) butyl)-D-alanine ethyl ester hydrochloride (SC42619) have defined the features of this dipeptide analogue required for observation of thrombin receptor antagonist activity on the human platelet. The affinity for SC42619, and for its structural analogue SC43583 is enhanced by pretreatment of the platelets with chymotrypsin. Endothelial cell prostacyclin (PGI2) synthesis induced by thrombin and trypsin is selectively inhibited by SC42619 provided that prolonged exposure to this antagonist is avoided. However inhibition of PGI2 synthesis by SC42619 is not overcome by increasing the thrombin concentration. The data provide further support for identification of SC42619 and certain of its analogues as selective antagonists at the platelet thrombin receptor but suggest that these compounds may have more complex, and possibly non-selective effects on the endothelial cell.

5-[6-1 -(Cyclohexyl-1 H-tetrazol-5-YL)hexyl]-1,8-naphthyridin-2-(1H)-one, SC-44368, a Potent Anti-aggregatory Agent which Selectively Inhibits Platelet Cyclic AMP Phosphodiesterase

SC-44368 (5-[6-(1-cyclohexyl-1H-tetrazol-5-y)hexyl]-1,8-naphthyridin-2(1H)-one) is a potent and selective competitive inhibitor of platelet cyclic AMP-dependent phosphodiesterase (cAMP-PDE) (Ki: 1.65 μM). For the phosphodiesterase isoenzyms from human platelets SC-44368 shows a 26-fold selectivity (IC50 ratio) for the inhibition of the cAMP-PDE over the cyclic GMP-dependent phosphodiesterase (cGMP-PDE). By comparison, 3-isobutyl-1-methyl-xanthine (IBMX) inhibited the cAMP-PDE and cGMP-PDE from human platelets with approximately equal efficacy. Broad inhibitory activity was evident against human platelet aggregatory responses in vitro. IC50 values of 18.1 ± 5.3 μM (25 nM platelet activating factor, PAF), 17.3 ± 3.0 μM (1.0 μg/ml collagen) and 24.2 ± 10.3 μM (1μM ADP) were obtained against maximum increases in platelet-rich plasma (PRP) light transmission achieved by each agonist. SC-44368 potentiated the prostacyclin-induced increase of intra-platelet cAMP levels but did not potentiate the sodium nitroprusside-induced increase of intraplatelet cGMP levels. In an ex vivo model of platelet aggregation SC-44368 (3 mg/kg, i.v.) produced a potent inhibition of collagen-induced platelet aggregation. SC-44368 produced only weak hypotensive activity in the rat. Thus, SC-44368 is a novel cAMP-PDE inhibitor which possesses potent, broad spectrum anti-aggregatory properties.