Zinc is an intracellular secondary messenger in platelets

Zinc (Zn2+) is an essential trace element which regulates intracellular processes in multiple cell types. Whilst Zn2+ has been shown to be released from intracellular compartments of a variety of cell types in response to external stimuli, its role in platelets has yet to be confirmed. This work aimed to determine whether agonist-evoked activation of platelets induced changes in cytosolic zinc concentrations ([Zn2+]i) contributing to modulation of activatory processes in platelets, in a manner consistent with a secondary messenger. Fluorometry was used to assess intracellular Zn2+ [Zn2+]i changes via zinc-specific fluorophores. Fluctuations in [Zn2+]i were modulated using Zn2+-specific chelators and ionophores. Light transmission aggregometry, flow cytometry and western blotting were employed to assess the influence of [Zn2+]i on platelet function. Stimulation by CRP-XL or U46619 evoked increases in intraplatelet Fluozin-3 (FZ-3) fluorescence consistent with [Zn2+]i elevation. Increases in FZ-3 fluorescence were abrogated by Zn2+ chelators and were distinct from agonist-evoked [Ca2+]i signals. Fluctuations of [Zn2+]i induced by CRP-XL or U46619 were sensitive to changes in platelet redox states. Sustained increases of [Zn2+]i were induced by the Zn2+ ionophores, Clioquinol or Pyrithione, which resulted in significant platelet shape change, integrin αIIbβ3 upregulation, dense granule but not α granule secretion and phosphatidylserine exposure. Chelation of [Zn2+]i also resulted in the inhibition of agonist-induced platelet activity. Furthermore, elevation of [Zn2+]i was also found to occur in a similar manner to [Ca2+]i elevation from stores within platelets in addition to redox state modulation. In conclusion, this is the first description of agonist-dependent increases in platelet [Zn2+]i, indicative of a role as a secondary messenger. The Zn2+ release is sensitive to mediators of the platelet redox state, and so may be attributable to release from oxidised thiol groups on Zn2+ -binding proteins. Modulation of [Zn2+]i indicated a role for Zn2+ in platelet activatory processes. The rise in [Zn2+]i in response to CRP-XL and U46619 demonstrates the physiological importance of Zn2+, implying the importance to Zn2+ in platelet activation during thrombus formation.