TRAP-6 amide

The CNS expresses many components of an extracellular protease signalling system, including the protease-activated receptor-1 (PAR-1) whose tethered ligand is generated by thrombin. Activation of PAR-1 potentiates NMDA receptor activity in hippocampal neurons. Because NMDA activity mediates hyperalgesia, we tested the hypothesis that PAR-1 receptors also regulate pain processing. In contrast to the potentiating effect of thrombin in the hippocampus, NMDA-induced behaviours and the transient mechanical hyperalgesia (von Frey fibres) induced by intrathecally injected NMDA in mice were inhibited by thrombin in a dose-related fashion. This anti-hyperalgesic effect was mimicked by SFLLRN, the natural ligand at PAR-1 binding sites, but not SLIGRL-amide, a PAR-2 agonist. The effects of SFLLRN were less potent and shorter in duration than that of thrombin, consistent with its more transient effect on PAR-1 sites. Both thrombin and SFLLRN inhibited acetic acid-induced abdominal stretch (writhing) behaviours, which were also sensitive to NMDA antagonism, but not hot plate or tail flick latencies, which were insensitive to NMDA antagonists. TFLLR-amide, a selective ligand for PAR-1 sites, mimicked the effects of thrombin while RLLFT-amide, an inactive, reverse peptide sequence, did not. In addition, the effect of TFLLR-amide was prevented by RWJ-56110, a PAR-1 antagonist. Thrombin and TFLLR-amide produced no oedema (Evans Blue extravasation) in the spinal cord that would account for these effects. Based on the reported ability of thrombin to mobilize endothelin-1 from astrocytes, we tested the role of this compound in thrombin's activity. BQ123, an endothelin A receptor antagonist, prevented thrombin's inhibition of writhing and NMDA-induced behaviours while BQ788, an endothelin B receptor antagonist, did not. Thus, activation of PAR-1 sites by thrombin in the CNS appears to inhibit NMDA-mediated nociception by a pathway involving endothelin type A receptors.

Platelet reactivity testing is important for the diagnosis of bleeding disorders, and increasingly to optimise anti-platelet therapy. Traditional light transmission aggregometry is considered the gold standard, whilst 96-well plate aggregometry, founded on similar principles, provides a higher throughput screening method. Despite the widespread use of both, methodologies and outputs vary widely between laboratories. We report a methodological approach towards providing a standardised optical detection of platelet aggregation (optimul method) based upon 96-well plate aggregometry. Individual wells of half-area 96-well plates were coated with gelatine and one of seven concentrations of arachidonic acid (AA), adenosine diphosphate (ADP), collagen, epinephrine (EPI), ristocetin, TRAP-6 amide or U46619, before being lyophilised, vacuum-sealed, foil-packed and stored at room temperature for up to 24 weeks. For platelet testing, 40 µl of platelet-rich plasma was added to each well. Platelet aggregation was determined by changes in light absorbance, release of ATP/ADP by luminescence and release of thromboxane (TX) A(2) by ELISA. Some experiments were conducted in the presence of aspirin (30 µM) or prasugrel active metabolite (PAM; 3 µM). Optimul plates stored for up to 12 weeks permitted reliable detection of concentration-dependent platelet aggregation, ATP/ADP release and TXA₂ production. PAM caused reductions in platelet responses to AA, ADP, collagen, EPI, TRAP-6 and U46619, whilst aspirin inhibited responses to AA, collagen and EPI. We conclude that the optimul method offers a viable, standardised approach, allowing platelet reactivity testing and could provide a broad platelet function analysis without the need for dedicated equipment.

A series of Phe-Gly dipeptide-derived piperazinones containing an aromatic urea moiety and a basic amino acid has been synthesized and evaluated as inhibitors of human platelet aggregation induced by the PAR1 agonist SFLLRN and as cytotoxic agents in human cancer cells. The synthetic strategy involves coupling of a protected basic amino acid benzyl amide to 1,2- and 1,2,4-substituted-piperazinone derivatives, through a carbonylmethyl group at the N1-position, followed by formation of an aromatic urea at the exocyclic moiety linked at the C2 position of the piperazine ring and removal of protecting groups. None of the compounds showed activity in the biological evaluation.