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

Zinc (Zn²⁺) is an essential trace element that regulates intracellular processes in multiple cell types. While the role of Zn²⁺ as a platelet agonist is known, its secondary messenger activity in platelets has not been demonstrated.

This article determines whether cytosolic Zn²⁺ concentrations ([Zn²⁺]_i) change in platelets in response to agonist stimulation, in a manner consistent with a secondary messenger, and correlates the effects of [Zn²⁺]_i changes on activation markers.

Changes in [Zn²⁺]_i were quantified in Fluozin-3 (Fz-3)-loaded washed, human platelets using fluorometry. Increases in [Zn²⁺]_i were modelled using Zn²⁺-specific chelators and ionophores. The influence of [Zn²⁺]_i on platelet function was assessed using platelet aggregometry, flow cytometry and Western blotting.

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 [Zn²⁺]_i. Fluoresence increases were blocked by Zn²⁺ chelators and modulators of the platelet redox state, and were distinct from agonist-evoked [Ca²⁺]_i signals. Stimulation of platelets with the Zn²⁺ ionophores clioquinol (Cq) or pyrithione (Py) caused sustained increases of [Zn²⁺]_i, resulting in myosin light chain phosphorylation, and cytoskeletal re-arrangements which were sensitive to cytochalasin-D treatment. Cq stimulation resulted in integrin α_IIbβ₃ activation and release of dense, but not α, granules. Furthermore, Zn²⁺-ionophores induced externalization of phosphatidylserine.

These data suggest that agonist-evoked fluctuations in intra-platelet Zn²⁺ couple to functional responses, in a manner that is consistent with a role as a secondary messenger. Increased intra-platelet Zn²⁺ regulates signalling processes, including shape change, α_IIbβ₃ up-regulation and dense granule release, in a redox-sensitive manner.