title: 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.
authors.affiliation: Department of Biochemistry, Hunterian Institute, Royal College of Surgeons of England, London, U.K.
authors.forename: K S
authors.initials: KS
authors.lastname: Authi
authors.forename: G H
authors.initials: GH
authors.lastname: Rao
authors.forename: B J
authors.initials: BJ
authors.lastname: Evenden
authors.forename: N
authors.initials: N
authors.lastname: Crawford
mesh_terms: D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003402:Creatine Kinase / Q000494:pharmacology; D006150:Guanine Nucleotides / Q000031:analogs & derivatives*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008345:Manganese / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000097:blood; D010974:Platelet Aggregation / Q000187:drug effects; D012503:Saponins / Q000494:pharmacology; D012701:Serotonin / Q000097:blood; D013268:Stimulation, Chemical; D013873:Thionucleotides / Q000494:pharmacology*; D013917:Thrombin / Q000494:pharmacology*
chemical_list: D006150:Guanine Nucleotides; D010712:Phosphatidic Acids; D012503:Saponins; D013873:Thionucleotides; D006153:Guanosine Diphosphate; C032829:6-thioguanosine 5'-diphosphate; D012701:Serotonin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008345:Manganese; D006160:Guanosine Triphosphate; D003402:Creatine Kinase; D013917:Thrombin; D002118:Calcium