Dataset Information

Action of guanosine 5'-[beta-thio]diphosphate on thrombin-induced activation and Ca2+ mobilization in saponin-permeabilized and intact human platelets.

ABSTRACT: The non-hydrolysable guanine analogues guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta-thio]diphosphate (GDP[S]) have been used extensively (as promoters and inhibitors respectively) to probe the importance of G-protein function. We report on the use of GDP[S] in permeabilized and intact platelets. The stimulatory analogue GTP[S] (9-60 microM) induces shape change, aggregation and 5-hydroxy[14C]-tryptamine secretion when added to saponin (12-14 micrograms/ml)-permeabilized platelets, but not to intact platelets. In line with the activation responses in permeabilized cells, GTP[S] induces an increase in [32P]-phosphatidic acid, which is indicative of phospholipase C activity. GDP[S] (greater than 400 microM) totally inhibits GTP[S] (90 microM)-stimulated phospholipase C activity and functional responses in saponized platelets. GDP[S] (1 mM) was also effective at inhibiting low-dose thrombin (0.1 unit/ml)-induced aggregation and secretion responses (without affecting shape change) in permeabilized platelets with inhibition of [32P]-phosphatidic acid formation. At higher doses of thrombin (greater than 0.5 unit/ml), both functional responses and [32P]phosphatidic acid formation are restored in the presence of GDP[S]. Studies on intact cells revealed that GDP[S] was as effective at inhibiting low-dose thrombin-induced functional responses as in the permeabilized cells, but there was no inhibition of [32P]phosphatidic acid formation, indicating that the agent is nonmembrane-penetrating. This reflected the fact that GDP[S] has additional inhibitory sites on the surface of platelets. In Fura-2-loaded cells GDP[S] inhibited thrombin-induced Ca2+ mobilization, as measured by Fura-2 fluorescence, in a dose-dependent manner. In studies with and without Ca2+ present on the outside, the effect of GDP[S] was to block Ca2+ influx. These studies indicate that, although GDP[S] is a valuable tool in studying G-protein function in permeabilized cells, it also has inhibitory activities on the surface of platelets, and one of these has been identified as an effect on the Ca2+-influx channel after agonist stimulation.

SUBMITTER: Authi KS

PROVIDER: S-EPMC1135324 | biostudies-other | 1988 Nov

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:In an earlier study we reported the effect of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in releasing Ca2+ from highly purified human platelet intracellular membrane vesicles. [Authi & Crawford (1985) Biochem. J. 230, 247-253]. We have now investigated the metabolic and functional consequences of introducing Ins(1,4,5)P3 into saponin-permeabilized platelets. Washed human platelets when resuspended in a suitable medium were permeabilized with saponin (10-14 micrograms/ml) to allow entry of low-Mr water-soluble molecules without significant release of the cytoplasmic marker enzyme protein lactate dehydrogenase. Saponin-permeabilized platelets show identical platelet responses (shape change, aggregation and release of 5-hydroxy[14C]tryptamine) to both collagen (5 micrograms/ml) and thrombin (0.1 unit/ml) as obtained with intact cells, indicating that there is minimal disturbance to the surface membrane receptor topography for these two agonists. Ins(1,4,5)P3 (1-10 microM) added to saponin-treated platelets (but not to intact platelets) induced dose-related shape change, aggregation and release of 5-hydroxy[14C]tryptamine which at maximal doses was comparable with responses obtained with thrombin or collagen. The cyclo-oxygenase inhibitors indomethacin and aspirin, if added prior to saponization and Ins(1,4,5)P3 addition, completely inhibited both aggregation and release of 5-hydroxy[14C]tryptamine (EC50 for indomethacin, 50 nM; for aspirin, 30 microM). We believe that Ins(1,4,5)P3 induces the release of Ca2+ from intracellular storages sites which stimulates the Ca2+-dependent phospholipase A2 releasing arachidonic acid from membrane phospholipids. Arachidonic acid is then converted to the aggregatory prostanoids (prostaglandin H2 and thromboxane A2) resulting in the observed responses. This concept is supported by the use of the thromboxane receptor antagonists EPO 45 and EPO 92, both of which also completely inhibit Ins(1,4,5)P3-induced responses in saponin-permeabilized platelets. Electron microscopy of the platelet preparations revealed that thrombin- and collagen-induced platelet aggregates of intact and saponized cells were identical, showing extensive pseudopod formation and dense granule release. The Ins(1,4,5)P3-induced aggregates also showed similar dense granule release but an almost total absence of pseudopod formation. These results are discussed in the light of the second messenger role of Ins(1,4,5)P3 in stimulus-response coupling in platelets.

Project description:With enzyme preparations from Phaseolus aureus seedlings, the initial rate of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]glucose is not increased by additions of GDP-alpha-d-mannose. However, final incorporation is increased by addition of GDP-alpha-d-mannose, since the total reaction-time is extended. In contrast, the initial rate of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]mannose is increased by all concentrations of GDP-alpha-d-glucose that are less than that of the GDP-alpha-d-[(14)C]mannose. Maximum stimulation of the initial rate occurs at a GDP-alpha-d-[(14)C]mannose/GDP-alpha-d-glucose concentration ratio of about 4:1. However, eventual incorporation from GDP-alpha-d-[(14)C]mannose is decreased by the addition of GDP-alpha-d-glucose, since the reaction rate falls off sharply after about 2min. Reciprocal plots of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]mannose result in biphasic graphs. The two straight-line portions of the plot are joined by a curved line in the concentration range between 2-3 and 50mum. Extrapolated K(m) values for the two linear components are 0.4-1.0 and 700-1500mum. The effect of GDP-alpha-d-glucose on the kinetics of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]mannose is complex, and depends on relative concentrations of the two sugar nucleotides. (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]glucose also results in biphasic reciprocal plots. One component appears to have K(m) about 2-3mum, the other about 200-400mum. In this reaction, GDP-alpha-d-mannose appears to be a competitive inhibitor with K(i) 20-30mum. With particulate preparations of P. aureus, GDP-alpha-d-[(14)C]glucose appears to be a precursor for the synthesis of one polysaccharide, a glucomannan, the mannose moieties of which are derived from an intermediate existing in the particulate preparation. From the rate results, GDP-alpha-d-[(14)C]mannose appears to be a precursor for at least two polysaccharides, one of which is a glucomannan.

Dataset Information

Action of guanosine 5'-[beta-thio]diphosphate on thrombin-induced activation and Ca2+ mobilization in saponin-permeabilized and intact human platelets.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets