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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.

SUBMITTER: Pollock WK

PROVIDER: S-EPMC1153551 | biostudies-other | 1984 May

REPOSITORIES: biostudies-other

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Project description:Cytosolic Ca2+ levels and arachidonate liberation were investigated in platelets loaded with the fluorescent Ca2+ indicator dye fura-2, and labelled with [3H]arachidonate. Fura-2 was used in preference to quin2 because the latter interfered with [3H]arachidonate labelling of phospholipids. From a resting free Ca2+ level of around 100 nM, ionomycin (10-200 nM) evoked an instantaneous, concentration-dependent increase in cytosolic Ca2+ that only resulted in [3H]arachidonate liberation (up to 4-fold over control) at Ca2+ levels greater than 1 microM. Addition of collagen (10 micrograms/ml) evoked an elevation in Ca2+ up to 461 +/- 133 nM. These changes in Ca2+ were accompanied by a 2-4-fold elevation in [3H]arachidonate with depletion of [3H]phosphatidylcholine by 17 +/- 4% and [3H]phosphatidylinositol by 41 +/- 7%. Indomethacin (10 microM) reduced the elevation in Ca2+ by collagen to 115 +/- 18 nM but did not significantly inhibit the 2-4-fold increase in [3H]arachidonate. [3H]Phosphatidylcholine and [3H]phosphatidylinositol were decreased by 9 +/- 7% and 10 +/- 6%, respectively, with collagen in the presence of indomethacin. Stimulation of phosphoinositide turnover by collagen in the presence and absence of indomethacin was indicated by [32P]phosphatidate formation in cells prelabelled with [32P]Pi. This phosphatidate formation was decreased (75%) by the presence of indomethacin. In the presence of indomethacin, phorbol myristate acetate (20 nM) alone or in combination with ionomycin (30 nM) failed to stimulate arachidonate liberation despite a marked stimulation of aggregation. These results indicate that, whereas ionomycin requires Ca2+ in the microM range for arachidonate liberation, collagen, notably in the presence of indomethacin, does so at basal Ca2+ levels. The mechanisms underlying the regulation of arachidonate release by collagen are not clear, but do not appear to involve activation of protein kinase C, or an elevation of cytosolic free Ca2+.

Project description:Human platelets incubated with [32P]Pi and [3H]arachidonate were transferred to a Pi-free Tyrode's solution by gel filtration. The labile phosphoryl groups of ATP and ADP as well as Pi in the metabolic pool of these platelets had equal specific radioactivity which was identical to that of[32P]phosphatidate formed during treatment of the cells with thrombin for 5 min. Therefore, the 32P radioactivity of phosphatidate was a true, relative measure for its mass. The thrombin-induced formation of[32P]-phosphatidate had the same time course and dose-response relationships as the concurrent secretion of acid hydrolases. 125I-alpha-Thrombin bound maximally to the platelets within 13s and was rapidly dissociated from the cells by hirudin; readdition of excess 125I-alpha-thrombin caused rapid rebinding of radioligand. This binding-dissociation-rebinding sequence was paralleled by a concerted start-stop-restart of phosphatidate formation and acid hydrolase secretion. [3H]Phosphatidylinositol disappearance was initiated upon binding but little affected by thrombin dissociation and rebinding. ATP deprivation caused similar changes in the time courses for [32P]-phosphatidate formation and acid hydrolase secretion which were different from those of [3H]phosphatidylinositol disappearance. The metabolic stress did not alter the magnitude (15%) of the initial decrease in phosphatidylinositol-4,5-bis[32P]phosphate, but did abolish the subsequent increase of phosphatidylinositol-4,5-bis[32P]-phosphate in the thrombin-treated platelets. It is concluded that in thrombin-treated platelets (1) phosphatidate synthesis, but not phosphatidylinositol disappearance, is tightly coupled to receptor occupancy and acid hydrolase secretion in platelets, (2) successive phosphorylations to phosphatidylinositol-4,5-bisphosphate is unlikely to be the main mechanism for phosphatidylinositol disappearance, and (3) only a small fraction (15%) of phosphatidylinositol-4,5-bisphosphate is susceptible to hydrolysis.

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

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