title: Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca²⁺ signalling in human platelets.
abstract: Rises in cytosolic Ca(2+) concentration ([Ca(2+)]cyt) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca(2+)]cyt, but these only monitor the net effect of manipulations on the processes controlling [Ca(2+)]cyt (Ca(2+) buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca(2+) or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca(2+) signalling, we here monitor Ca(2+) flux around the platelet by measuring net Ca(2+) fluxes to or from the extracellular space and the intracellular Ca(2+) stores, which act as the major sources and sinks for Ca(2+) influx into and efflux from the cytosol, as well as monitoring the cytosolic Na(+) concentration ([Na(+)]cyt), which influences platelet Ca(2+) fluxes via Na(+)/Ca(2+) exchange. The intracellular store Ca(2+) concentration ([Ca(2+)]st) was monitored using Fluo-5N, the extracellular Ca(2+) concentration ([Ca(2+)]ext) was monitored using Fluo-4 whilst [Ca(2+)]cyt and [Na(+)]cyt were monitored using Fura-2 and SFBI, respectively. PKC inhibition using Ro-31-8220 or bisindolylmaleimide I potentiated ADP- and thrombin-evoked rises in [Ca(2+)]cyt in the absence of extracellular Ca(2+). PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca(2+) release and Ca(2+) removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca(2+)]cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na(+)]cyt which would be expected to reduce Ca(2+) removal via the Na(+)/Ca(2+) exchanger (NCX). Thrombin-evoked rises in [Na(+)]cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non-selective cation permeability of the plasma membrane as assessed by Mn(2+) quench of Fura-2 fluorescence. PKC inhibition was without effect on thrombin-evoked rises in [Ca(2+)]cyt following SERCA inhibition and either removal of extracellular Na(+) or inhibition of Na(+)/K(+)-ATPase activity by removal of extracellular K(+) or treatment with digoxin. These data suggest that PKC limits ADP-evoked rises in [Ca(2+)]cyt by acceleration of SERCA activity, whilst rises in [Ca(2+)]cyt evoked by the stronger platelet activator thrombin are limited by PKC through acceleration of both SERCA and Na(+)/K(+)-ATPase activity, with the latter limiting the effect of thrombin on rises in [Na(+)]cyt and so forward mode NCX activity. The use of selective PKC inhibitors indicated that conventional and not novel PKC isoforms are responsible for the inhibition of agonist-evoked Ca(2+) signalling.
authors.affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Lever
authors.affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom.
authors.forename: Azhar
authors.initials: A
authors.lastname: Hussain
authors.affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom.
authors.forename: Benjamin B
authors.initials: BB
authors.lastname: Sun
authors.affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom.
authors.forename: Stewart O
authors.initials: SO
authors.lastname: Sage
authors.affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom; Institute for Science and Technology in Medicine, Keele University, Guy Hilton Research Centre, Thornburrow Drive, Hartshill, Stoke-on-Trent ST4 7QB, United Kingdom. Electronic address: a.g.s.harper@keele.ac.uk.
authors.forename: Alan G S
authors.initials: AG
authors.lastname: Harper
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D015539:Platelet Activation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D020033:Protein Isoforms / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism
chemical_list: D015220:Calcium Channels; D007211:Indoles; D007527:Isoenzymes; D010975:Platelet Aggregation Inhibitors; D020033:Protein Isoforms; D000244:Adenosine Diphosphate; D011493:Protein Kinase C; D013917:Thrombin; D002118:Calcium; C064758:Ro 31-8220
keywords: Calcium; Na(+)/K(+)-ATPase; Platelet; Protein kinase C; Sarco/endoplasmic reticulum Ca(2+)-ATPase