Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

Rises in cytosolic Ca2+ concentration ([Ca2+](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 [Ca2+](cyt), but these only monitor the net effect of manipulations on the processes controlling [Ca2+](cyt) (Ca2+) buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca2+ or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca2+ signalling, we here monitor Ca2+ flux around the platelet by measuring net Ca2+ fluxes to or from the extracellular space and the intracellular Ca2+ stores, which act as the major sources and sinks for Ca2+ influx into and efflux from the cytosol, as well as monitoring the cytosolic Na+ concentration ([Na+](cyt)), which influences platelet Ca2+ fluxes via Na+/Ca2+ exchange. The intracellular store Ca2+ concentration ([Ca2+](st)) was monitored using Fluo-5N, the extracellular Ca2+ concentration ([Ca2+](ext)) was monitored using Fluo-4 whilst [Ca2+](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 [Ca2+](cyt) in the absence of extracellular Ca2+. PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca2+ release and Ca2+ removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) 1,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca2+](cyt) but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na+](cyt) which would be expected to reduce Ca2+ removal via the Na+/Ca2+ 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 Mn2+ quench of Fura-2 fluorescence. PKC inhibition was without effect on throinbin-evoked rises in [Ca2+](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 [Ca2+](cyt) by acceleration of SERCA activity, whilst rises in [Ca2+](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 Ca2+ signalling. (C) 2015 Elsevier Ltd. All rights reserved.