The influence of platelet activating factor on the effects of platelet agonists and antiplatelet agents in vitro.

We assessed the effect of the intercellular mediator of inflammation, platelet activating factor (PAF), on platelet function. The interaction between PAF and the platelet agonists ADP, thrombin and convulxin was analyzed in vitro in whole blood with the use of flow cytometry and was further characterized with the use of receptor antagonists to PAF (ABT-491), P2Y1 (MRS-2179), and P2Y12 (cangrelor) as well as a monoclonal anti-PSGL-1 antibody (anti-CD162). Low concentrations of PAF (0.1 nM) synergistically augmented platelet activation induced by other agonists (P < 0.01). Augmentation by PAF was receptor mediated and did not require platelet-leukocyte interaction. With >99% inhibition of P2Y receptor-mediated platelet activation, greater than additive activation was still observed with the combination of ADP plus PAF. Accordingly, PAF synergistically augments platelet activation in response to ADP and thrombin, and the extent of inhibition exerted by P2Y receptor antagonists is decreased in the presence of PAF.

Antiplatelet therapy.

Within the last few years antiplatelet therapy has developed exponentially, with new agents being tested in an increasing number of clinical scenarios. The mechanism of action of these newer agents and evidence of benefit is prevented in this review.

Involvement of basic amino acid residues in transmembrane regions 6 and 7 in agonist and antagonist recognition of the human platelet P2Y(12)-receptor.

The P2Y(12)-receptor plays a prominent role in ADP-induced platelet aggregation. In the present study, we searched for amino acid residues involved in ligand recognition of the human P2Y(12)-receptor. Wild-type or mutated receptors were expressed in 1321N1 astrocytoma cells and Chinese hamster ovary (CHO) cells. There were no major differences in cellular expression of the constructs. Cellular cAMP production and cAMP response element (CRE)-dependent luciferase expression was increased by isoproterenol (astrocytoma cells) or forskolin (CHO cells). In cells expressing wild-type receptors, R256K or S101A mutant constructs, 2-methylthio-ADP inhibited the induced cAMP production with IC(50) concentrations of about 0.3nM. In cells expressing R256A constructs, the IC(50) concentration amounted to 25nM. In cells expressing H253A/R256A, Y259D and K280A constructs, 2-methylthio-ADP failed to affect the cellular cAMP production. Moreover, in cells expressing Y259D and K280A constructs, 2-methylthio-ADP did also not change the forskolin-induced CRE-dependent luciferase expression and caused only small increases in the serum response element-dependent luciferase expression. The antagonist cangrelor had similar potencies at wild-type receptors and R256A constructs (apparent pK(B)-value at wild-type receptors: 9.2). In contrast, reactive blue-2 had a lower potency at the R256A construct (apparent pK(B)-value at wild-type receptors: 7.6). In summary, the data indicate the involvement of Arg256, Tyr259 and, possibly, H253 (transmembrane region TM6) as well as Lys280 (TM7) in the function of the human P2Y(12)-receptor. Arg256 appears to play a role in the recognition of nucleotide agonists and the non-nucleotide antagonist reactive blue-2, but no role in the recognition of the nucleotide antagonist cangrelor.

A novel pyridazinone derivative as a nonprostanoid PGI2 agonist.

A novel optically pure pyridazinone derivative was synthesized and identified as a nonprostanoid PGI2 agonist. It inhibited ADP-induced aggregation of human platelets with an IC50 value of 0.081 microM and has high oral bioavailability (56%) with a long half-life (4.3 h) in rats.