title: Thromboxane-induced phosphatidate formation in human platelets. Relationship to receptor occupancy and to changes in cytosolic free calcium.
abstract: 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.
mesh_terms: D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D066298:In Vitro Techniques; D010712:Phosphatidic Acids / Q000097:blood*; D010743:Phospholipids / Q000096:biosynthesis; D010974:Platelet Aggregation / Q000187:drug effects; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology; D044262:Prostaglandin H2; D011463:Prostaglandins H / Q000494:pharmacology; D011465:Prostaglandins, Synthetic / Q000494:pharmacology; D011982:Receptors, Prostaglandin / Q000097:blood; D017482:Receptors, Thromboxane; D013928:Thromboxane A2 / Q000494:pharmacology*; D013931:Thromboxanes / Q000494:pharmacology*
chemical_list: D010712:Phosphatidic Acids; D010743:Phospholipids; D011450:Prostaglandin Endoperoxides, Synthetic; D011463:Prostaglandins H; D011465:Prostaglandins, Synthetic; D011982:Receptors, Prostaglandin; D017482:Receptors, Thromboxane; D013931:Thromboxanes; D044262:Prostaglandin H2; D013928:Thromboxane A2; C034549:EP 045; D002118:Calcium