Analysis of anti-PDE3 activity of 2-morpholinochromone derivatives reveals multiple mechanisms of anti-platelet activity.

A series of synthetic 2-morpholinochromones have been evaluated as inhibitors of platelet phosphodiesterase, PDE3A. While previous assertions about the anti-PDE3 activity of this class have been confirmed, in some cases the reported anti-platelet activities clearly derive from a non-PDE3 regulated mechanism. The potential utility of 2-morpholinochromones either as PDE3 inhibitors and/or anti-thrombotic agents thus remains only poorly examined.

Proteinase-activated receptor 2: differential activation of the receptor by tethered ligand and soluble peptide analogs.

Activation of rat proteinase-activated receptor 2 (PAR2) by trypsin involves the unmasking of the tethered sequence S(37)LIGRL(42) that either tethered or on its own as a free peptide, activates PAR2. We aimed to determine whether different peptide sequences acting either as trypsin-revealed tethered ligands or as soluble peptides had the same relative activities for triggering the receptor. A comparison was also made between the different soluble and tethered receptor activating sequences in receptor constructs with extracellular loop 2 (ECL2) residues E(232)E(233) (PAR2SR/EE) mutated to R(232)R(233) (PAR2SR/RR). Using site-directed mutagenesis, we prepared PAR2 constructs with trypsin-revealed tethered ligand sequences corresponding to the synthetic receptor-activating peptides (PAR2APs): SLIGRL-NH(2) (SR-NH(2)), SLIGAL-NH(2) (SA-NH(2)), and SLIGEL-NH(2) (SE-NH(2)). Kirsten virus-transformed rat kidney cells stably expressing 1) wild-type PAR2 with site-mutated tethered ligands (PAR2SA/EE and PAR2SE/EE); 2) wild-type PAR2 with ECL2 mutated to R(232)R(233) (PAR2SR/RR); and 3) PAR2 constructs with both the RR mutation in ECL2 and a mutation in the tethered ligand (PAR2SA/RR and PAR2SE/RR) were assessed for receptor-mediated calcium signaling and cell growth inhibition, upon activation either by trypsin or the above-mentioned PAR2APs. Trypsin exerted equivalent and full agonist activity on the PAR2 constructs, causing a maximum response between 20 to 80 nM. In contrast, the PAR2APs as free peptide agonists showed marked potency differences in all wild-type receptors with mutated tethered ligands (SR-NH(2) >> SA-NH(2) >> SE-NH(2)) and in all ECL2 RR mutated constructs (SE-NH(2) > SR-NH(2) >> SA-NH(2)). We conclude that for receptor activation, the trypsin-revealed PAR2 tethered ligand sequence interacts differently for receptor activation than does the same peptide sequence as a free peptide.

Functional expression of a human thrombin receptor in Sf9 insect cells: evidence for an active tethered ligand.

Desensitization of recombinant human thrombin receptors expressed in Sf9 insect cells was compared with native thrombin receptors in megakaryoblast erythroleukaemia (HEL) cells. Addition of thrombin (2 units/ml) or agonist peptide SFLLRN (10 microM) to HEL cells, or to Sf9 cells infected with recombinant baculovirus containing the thrombin receptor cDNA, produced an increase in the free cytosolic Ca2+ concentration ([Ca2+]i) as measured by fura-2. The response in HEL cells was transient, reflecting a rapid homologous desensitization. In contrast, [Ca2+]i in Sf9 cells expressing the thrombin receptor increased rapidly to a peak value that slowly declined, but remained elevated for at least 12 min following stimulation by thrombin. The sustained [Ca2+]i response to thrombin was not reversed by washout of thrombin or by any subsequent addition of hirudin. Pretreatment of Sf9 cells with either thrombin (2 units/ml) or SFLLRN (10 or 50 microM) for 5 min produced a shift in the ED50 for SFLLRN (added 10 min after washout) from 0.4 microM to 20 and 7 microM, respectively. Thus, desensitization of thrombin receptors expressed in Sf9 cells occurs slowly and reflects a decrease in receptor affinity. The sustained [Ca2+]i response in Sf9 cells stimulated by thrombin may reflect continuous activation by the tethered ligand. To test this hypothesis, the effect of protease treatment during the sustained phase of the response was examined. Addition of either aminopeptidase M or thermolysin reversed the sustained response to SFLLRN, but only thermolysin reversed the sustained response to thrombin. Thermolysin had no effect on the change in [Ca2+]i observed following carbachol stimulation of Sf9 cells expressing the M5 muscarinic receptor. Furthermore, following thermolysin treatment, the cells remained responsive to a subsequent application of SFLLRN. These results demonstrate that the tethered ligand remains active for extended periods of time after thrombin stimulation and suggests that further hydrolysis by extracellular proteases may represent an important mechanism of rapid receptor deactivation.

Mechanisms of thrombin receptor agonist specificity. Chimeric receptors and complementary mutations identify an agonist recognition site.

Identification of the docking interactions by which peptide agonists activate their receptors is critical for understanding signal transduction at the molecular level. The human and Xenopus thrombin receptors respond selectively to their respective hexapeptide agonists, SFLLRN and TFRIFD. A systematic analysis of human/Xenopus thrombin receptor chimeras revealed that just two human-for-Xenopus amino acid substitutions, Phe for Asn87 in the Xenopus receptor's amino-terminal exodomain and Glu for Leu260 in the second extracellular loop, conferred human receptor-like specificity to the Xenopus receptor. This observation prompted complementation studies to test the possibility that Arg5a in the human agonist peptide might normally interact with Glu260 in the human receptor. The mutant agonist peptide SFLLEN was a poor agonist at the wild type human receptor but an effective agonist at a mutant human receptor in which Glu260 was converted to Arg. An "arginine scan" of the receptor's extracellular surface revealed additional complementary mutations in the vicinity of position 260 and weak complementation at position 87 but not elsewhere in the receptor. Strikingly, a double alanine substitution that removed negative charge from the Glu260 region of the human receptor also effectively complemented the SFLLEN agonist. The functional complementation achieved with single Arg substitutions was thus due at least in part to neutralization of a negatively charged surface on the receptor and not necessarily to introduction of a new salt bridge. By contrast, charge neutralization did not account for the gain of responsiveness to SFLLRN seen in the human/Xenopus receptor chimeras. Thus two independent approaches, chimeric receptors and arginine scanning for complementary mutations, identified the Glu260 region and to a lesser degree Phe87 as important determinants of agonist specificity. These extracellular sites promote receptor responsiveness to the "correct" agonist and inhibit responsiveness to an "incorrect" agonist. They may participate directly in agonist binding or regulate agonist access to a nearby docking site.