Agonist-Evoked Increases in Intra-Platelet Zinc Couple to Functional Responses

Background Zinc (Zn (2+) ) is an essential trace element that regulates intracellular processes in multiple cell types. While the role of Zn (2+) as a platelet agonist is known, its secondary messenger activity in platelets has not been demonstrated. Objectives This article determines whether cytosolic Zn (2+) concentrations ([Zn (2+) ] (i) ) change in platelets in response to agonist stimulation, in a manner consistent with a secondary messenger, and correlates the effects of [Zn (2+) ] (i) changes on activation markers. Methods Changes in [Zn (2+) ] (i) were quantified in Fluozin-3 (Fz-3)-loaded washed, human platelets using fluorometry. Increases in [Zn (2+) ] (i) were modelled using Zn (2+) -specific chelators and ionophores. The influence of [Zn (2+) ] (i) on platelet function was assessed using platelet aggregometry, flow cytometry and Western blotting. Results Increases of intra-platelet Fluozin-3 (Fz-3) fluorescence occurred in response to stimulation by cross-linked collagen-related peptide (CRP-XL) or U46619, consistent with a rise of [Zn (2+) ] (i) . Fluoresence increases were blocked by Zn (2+) chelators and modulators of the platelet redox state, and were distinct from agonist-evoked [Ca (2+) ] (i) signals. Stimulation of platelets with the Zn (2+) ionophores clioquinol (Cq) or pyrithione (Py) caused sustained increases of [Zn (2+) ] (i) , resulting in myosin light chain phosphorylation, and cytoskeletal re-arrangements which were sensitive to cytochalasin-D treatment. Cq stimulation resulted in integrin α (IIb) β (3) activation and release of dense, but not α, granules. Furthermore, Zn (2+) -ionophores induced externalization of phosphatidylserine. Conclusion These data suggest that agonist-evoked fluctuations in intra-platelet Zn (2+) couple to functional responses, in a manner that is consistent with a role as a secondary messenger. Increased intra-platelet Zn (2+) regulates signalling processes, including shape change, α (IIb) β (3) up-regulation and dense granule release, in a redox-sensitive manner.