title: Roles of phospholipase C and Ca(2+)-ATPase in calcium responses of single, fibrinogen-bound platelets.
abstract: Fura-2-loaded platelets were immobilized on fibrinogen, and cytosolic free calcium concentration ([Ca2+]i) was measured by video imaging of the fluorescence signal. In the immobilized, single platelets, ADP and low doses of thrombin evoked repetitive spikes of [Ca2+]i, whereas higher thrombin concentrations gave elevated plateaus in [Ca2+]i. Stimulation of the cells with thrombin after the addition of ADP changed the frequency of spiking, but not the maximal levels of [Ca2+]i reached. In suspensions of platelets, ADP and low doses of thrombin evoked transient elevation of myo-inositol phosphates, suggesting that the initiation of spiking was due to stimulation of phospholipase C. In platelet suspensions, the Ca(2+)-ATPase inhibitor thapsigargin (TG) evoked a gradual rise in [Ca2+]i, which was potentiated by preactivation of the platelets and inhibited by prostaglandin E1 and nitroprusside. In single platelets, TG induced a sudden increase in [Ca2+]i after a long but variable delay, followed by a phase of slow oscillations. The effects of preactivation with ADP were 2-fold: the delay time before the response to TG was shortened and the maximal level of [Ca2+]i reached with TG was higher than the level of the preceding Ca2+ spikes. Apparently, Ca2+ responses induced by the inhibition of Ca(2+)-ATPases are potentiated by prior elevation of [Ca2+]i and reduced by substances that inhibit agonist-evoked increases in [Ca2+]i. The data point to a mechanism of Ca(2+)-induced Ca2+ release and the presence of TG-sensitive and TG-insensitive Ca2+ stores in platelets. Rapid spiking may involve both pools, whereas the latter alone may account for the slow TG-evoked oscillations.
authors.affiliation: Department of Biochemistry, University of Limburg, Maastricht, The Netherlands.
authors.forename: J W
authors.initials: JW
authors.lastname: Heemskerk
authors.forename: P
authors.initials: P
authors.lastname: Vis
authors.forename: M A
authors.initials: MA
authors.lastname: Feijge
authors.forename: J
authors.initials: J
authors.lastname: Hoyland
authors.forename: W T
authors.initials: WT
authors.lastname: Mason
authors.forename: S O
authors.initials: SO
authors.lastname: Sage
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D001081:Apyrase / Q000494:pharmacology; D001241:Aspirin / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000097:blood*; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D005340:Fibrinogen / Q000494:pharmacology*; D016257:Fura-2; D006801:Humans; D007295:Inositol Phosphates / Q000097:blood*; D007700:Kinetics; D015539:Platelet Activation; D013050:Spectrometry, Fluorescence; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D013917:Thrombin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000097:blood*; D014741:Video Recording
chemical_list: D007295:Inositol Phosphates; D013729:Terpenes; D004533:Egtazic Acid; D000244:Adenosine Diphosphate; D019284:Thapsigargin; D005340:Fibrinogen; D010738:Type C Phospholipases; D013917:Thrombin; D001081:Apyrase; D000252:Calcium-Transporting ATPases; D001241:Aspirin; D002118:Calcium; D016257:Fura-2