Adenosine diphosphate (ADP) is an important platelet agonist and ADP released from platelet dense granules amplifies responses to other agonists. There are three known subtypes of ADP receptor on platelets: P2X(1), P2Y(1) and P(2T) receptors. Sustained ADP-induced aggregation requires co-activation of P2Y(1) and P(2T) receptors. AR-C69931MX, a selective P(2T) receptor antagonist and novel antithrombotic agent, was studied to characterize further the function of the P(2T) receptor. The roles of the P2Y(1) receptor and thromboxane A(2) were assessed using the selective P2Y(1) antagonist A2P5P and aspirin respectively. Aggregation was measured by whole blood single-platelet counting and platelet-rich plasma turbidimetry, using hirudin anticoagulation. Dense granule release was estimated using ([14)C]-5-hydroxytryptamine (HT)-labelled platelets. Ca(2+) mobilization, P-selectin expression, Annexin V binding and microparticle formation were determined by flow cytometry. P(2T) receptor activation amplified ADP-induced aggregation initiated by the P2Y(1) receptor, as well as amplifying aggregation, secretion and procoagulant responses induced by other agonists, including U46619, thrombin receptor-activating peptide (TRAP) and collagen, independent of thromboxane A(2) synthesis, which played a more peripheral role. P(2T) receptor activation sustained elevated cytosolic Ca(2+) induced by other pathways. These studies indicate that the P(2T) receptor plays a central role in amplifying platelet responses and demonstrate the clinical potential of P(2T) receptor antagonists.

The central role of the P(2T) receptor in amplification of human platelet activation, aggregation, secretion and procoagulant activity.

Differential inhibition by low-dose aspirin of human venous prostacyclin synthesis and platelet thromboxane synthesis.

Prostacyclin and thromboxanes. Implications for their role in tumor cell metastasis.

The consequences of inhibiting the metabolism of prostaglandin G2 to thromboxane A2 in man were studied by using an inhibitor of thromboxane synthase, 4-[2-(IH-imidazol-1-yl)ethoxy] benzoic acid hydrochloride (dazoxiben). Single doses of 25, 50, 100, and 200 mg of dazoxiben were administered to healthy volunteers at 2-wk intervals in a randomized, placebo-controlled, double-blind manner. Serum thromboxane B2 and aggregation studies in whole blood and platelet-rich plasma were measured before dosing and at 1, 4, 6, 8, and 24 h after dosing. Both serum thromboxane B2 and the platelet aggregation response to arachidonic acid (1.33 mM) were reversibly inhibited in a dose-dependent manner. Aggregation induced by 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (0.4 and 4.0 microM) in platelet-rich plasma as well as both aggregation and nucleotide release induced by collagen (95 micrograms/ml) in platelet-rich plasma and whole blood were unaltered by dazoxiben. Additional evidence for a platelet-inhibitory effect of the compound was a significant prolongation of the bleeding time at 1 h after administration of the highest dose (200 mg) of dazoxiben. Endogenous prostacyclin biosynthesis was assessed by measurement of the major urinary metabolite of prostacyclin, 2,3-dinor-6-keto-PGF1 alpha (PGI-M). PGI-M excretion was increased by dazoxiben; it rose a mean 2.4-fold from predosing control values at 0-6 h after administration of the highest dose studied (200 mg).

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M.J. Parry

Papers

Effects of a selective inhibitor of thromboxane synthetase on human blood platelet behaviour