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

Area of Science:

• Cellular Biology
• Biochemistry
• Physiology

Background:

• Cytosolic calcium concentration ([Ca2+]cyt) rises are critical for platelet activation, but underlying mechanisms remain unclear.
• Previous studies often measure net Ca2+ changes, limiting insights into specific Ca2+ sources, sinks, and transporters.
• Protein kinase C (PKC) is implicated in platelet signaling, but its precise role in regulating Ca2+ fluxes is not fully understood.

Purpose of the Study:

• To investigate the specific effects of protein kinase C (PKC) on platelet calcium (Ca2+) signaling dynamics.
• To differentiate the roles of PKC in ADP- and thrombin-evoked Ca2+ responses by monitoring Ca2+ fluxes and cytosolic Na+ concentration ([Na+]cyt).
• To elucidate the mechanisms by which PKC modulates Ca2+ handling, including SERCA and Na+/K+-ATPase activities.

Main Methods:

• Monitored Ca2+ fluxes to/from extracellular space and intracellular stores, alongside cytosolic Ca2+ ([Ca2+]cyt) and Na+ ([Na+]cyt) concentrations.
• Utilized fluorescent indicators Fluo-5N, Fluo-4, Fura-2, and SFBI for measuring [Ca2+]st, [Ca2+]ext, [Ca2+]cyt, and [Na+]cyt, respectively.
• Employed selective PKC inhibitors (Ro-31-8220, bisindolylmaleimide I), SERCA inhibitors (thapsigargin, 2,5-di(tert-butyl) l,4-benzohydroquinone), and manipulated extracellular ion concentrations.

Main Results:

• PKC inhibition potentiated ADP- and thrombin-evoked [Ca2+]cyt rises, particularly in the absence of extracellular Ca2+.
• PKC inhibition differentially affected ADP- and thrombin-evoked responses: potentiating ADP-evoked Ca2+ release but reducing thrombin-evoked release and extracellular Ca2+ removal.
• PKC appears to limit ADP-evoked [Ca2+]cyt by enhancing SERCA activity and thrombin-evoked [Ca2+]cyt by enhancing both SERCA and Na+/K+-ATPase activity, influencing Na+/Ca2+ exchange.

Conclusions:

• PKC plays distinct roles in modulating Ca2+ signaling in response to different platelet agonists.
• PKC limits ADP-evoked Ca2+ transients primarily through accelerating SERCA activity.
• PKC limits thrombin-evoked Ca2+ transients by accelerating both SERCA and Na+/K+-ATPase, thereby regulating Na+/Ca2+ exchanger activity and cytosolic Na+ levels.