Thromboxane-induced phosphatidate formation in human platelets. Relationship to receptor occupancy and to changes in cytosolic free calcium

• Published in: Biochemical journal 1984-05, Vol.219 (3), p.833-842
• Main Authors: Pollock, W K, Armstrong, R A, Brydon, L J, Jones, R L, MacIntyre, D E
• Format: Article
• Language: English
• Subjects: Blood Platelets - drug effects; Blood Platelets - metabolism; calcium; Calcium - blood; cytosol; Cytosol - metabolism; Dose-Response Relationship, Drug; Humans; In Vitro Techniques; man; Phosphatidic Acids - blood; phospholipids; Phospholipids - biosynthesis; Platelet Aggregation - drug effects; platelets; Prostaglandin Endoperoxides, Synthetic - pharmacology; Prostaglandin H2; Prostaglandins H - pharmacology; Prostaglandins, Synthetic - pharmacology; receptors; Receptors, Prostaglandin - blood; Receptors, Thromboxane; thromboxane A2; Thromboxane A2 - pharmacology; Thromboxanes - pharmacology
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2190833

The inter-relationships between receptor occupancy, inositol phospholipid metabolism and elevation of cytosolic free Ca2+ in thromboxane A2-induced human platelet activation were investigated by using the stable thromboxane A2 mimetic, 9,11-epoxymethanoprostaglandin H2, and the thromboxane A2 receptor antagonist, EPO45. 9,11-Epoxymethanoprostaglandin H2 stimulated platelet phosphatidylinositol metabolism as indicated by the rapid accumulation of [32P]phosphatidate and later accumulation of [32P]phosphatidylinositol in platelets pre-labelled with [32P]Pi. These effects of 9,11-epoxymethanoprostaglandin H2 were concentration-dependent and half-maximal [32P]phosphatidate formation occurred at an agonist concentration of 54 +/- 8 nM. With platelets labelled with the fluorescent Ca2+ indicator quin 2, resting cytosolic free Ca2+ was 86 +/- 12 nM. 9,11-Epoxymethanoprostaglandin H2 induced a rapid, concentration-dependent elevation of cytosolic free Ca2+ to a maximum of 300-700 nM. Half-maximal stimulation was observed at an agonist concentration of 80 +/- 23 nM. The thromboxane A2 receptor antagonist EPO45 selectively inhibited 9,11-epoxymethanoprostaglandin H2-induced [32P]phosphatidate formation and elevation of cytosolic free Ca2+, indicating that both events are sequelae of receptor occupancy. Human platelets contain a single class of stereospecific, saturable, high affinity (KD = 70 +/- 13 nM) binding sites for 9,11-epoxymethano[3H]prostaglandin H2. The concentration-response curve for receptor occupancy (9,11-epoxymethano-[3H]prostaglandin H2 binding) is similar to that for 9,11-epoxymethanoprostaglandin H2-induced [32P]phosphatidate formation and for elevation of cytosolic free Ca2+. These observations indicate that human platelet thromboxane A2 receptor occupation is closely linked to inositol phospholipid metabolism and to elevation of cytosolic free Ca2+. Both such events may be necessary for thromboxane A2-induced human platelet activation.