Dataset Information

Thrombin-induced phosphoinositide hydrolysis in platelets. Receptor occupancy and desensitization.

ABSTRACT: The relationship between occupancy of thrombin receptors on platelets and enhanced phosphoinositide hydrolysis was analysed by examination of the dose-response relationship, the effects of thrombin inhibitors and the contribution of secondary effects. Washed human platelets were labelled with [3H]inositol, and agonist-induced accumulation of labelled inositol phosphates was measured. The dose-response curves and the time courses for alpha-thrombin- or gamma-thrombin-induced accumulation of inositol phosphates were similar to those for dense-granule secretion. Addition of the thrombin inhibitor hirudin to thrombin-activated platelets revealed that the continuous presence of active thrombin was required to maintain the accumulation of labelled inositol phosphates; the total production of inositol phosphates increased with longer periods of exposure to thrombin, reaching a maximum between 5 and 10 min. After activation with thrombin, the ability of a second, greater, addition of thrombin to induce additional phosphoinositide hydrolysis decreased with time; it was absent within 10 min after the first addition. The failure to sustain accumulation of labelled inositol phosphates or to respond to a second addition of thrombin beyond 10 min was not due to depletion of the pool of labelled precursors, because the platelets retained their ability to respond to collagen. Addition of ADP-consuming enzymes decreased sensitivity to thrombin, but inhibition of cyclo-oxygenase with indomethacin did not impair the thrombin-induced hydrolysis of phosphoinositides. It was concluded that thrombin-induced hydrolysis of phosphoinositides has characteristics consistent with mediation by a receptor that is similar to that that triggers dense-granule secretion, requires continuous presence of active thrombin to be maintained, is mediated by a receptor that displays thrombin-induced desensitization, and is only partially enhanced by secondary agents.

SUBMITTER: Huang EM

PROVIDER: S-EPMC1147657 | biostudies-other | 1987 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

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.

Project description:Fura-2-loaded human platelets were used to study Ca2+ release from intracellular compartments, as well as Ca2+ influx from the extracellular space. We investigated the response towards the endoperoxide/thromboxane-receptor agonist. U46619, and the inhibitor of the endoplasmic-reticulum Ca(2+)-ATPase, thapsigargin. U46619 dose-dependently depleted intracellular Ca2+ stores, followed by active sequestration of released Ca2+. Ca2+ influx induced by U46619 largely relies on receptor occupancy. Removing the thromboxane analogue from its receptor by using the endoperoxide/thromboxane-receptor antagonist BM 13177 largely blunted U46619-mediated Ca2+ influx. The Ca(2+)-ATPase inhibitor thapsigargin evoked a gradual rise in intracellular Ca2+, which was potentiated by a preceding activation of platelets with the receptor agonist U46619. This agonist-sensitizing effect also depends on receptor occupancy. Removing U46619 from its receptor by addition of the endoperoxide/thromboxane-receptor antagonist BM13177 suppressed the sensitizing effect completely. Furthermore, interrupting downstream receptor signalling events by raising intracellular levels of cyclic nucleotides (cyclic AMP, cyclic GMP) again suppressed the U46619-sensitizing effect on thapsigargin-induced Ca2+ release. This study indicates that the process of Ca2+ release followed by resequestration in response to a platelet agonist by its own is not sufficient to produce the sensitizing effect. Rather, a continuously occupied receptor triggering sustained downstream signalling events seems to be required for sensitization. The presence of a receptor agonist may induce an increased cycling of Ca2+ between the agonist-responsive and the thapsigargin-dischargeable compartment, leading to faster and more intense accumulation of Ca2+ in the cytosolic compartment after inhibition of the Ca(2+) ATPase. Suggestively, receptor occupancy increases the Ca(2+)-releasing potency of thapsigargin by coupling the thapsigargin-sensitive Ca(2+)-storing compartments with an agonist-responsive compartment that exhibits a high leakage rate in stimulated platelets.

Dataset Information

Thrombin-induced phosphoinositide hydrolysis in platelets. Receptor occupancy and desensitization.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets