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

Abstract

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.

Zinc regulates reactive oxygen species generation in platelets

“…[10,11] Recent work has demonstrated that intracellular Zn 2+ ([Zn 2+ ]i) increases in the platelet cytosol as a result of agonist stimulation, an a manner consistent with a secondary messenger. [12] In this work it was shown that agonistevoked increases in [Zn 2+ ]i were sensitive to the platelet redox state, suggestive of a role for thiol groups on Zn 2+ binding proteins.…”

“…Zn 2+ is present in platelet α granules and also in the platelet cytosol where it is likely to associate with cation-binding proteins [12]. Platelet activation results in increases in cytosolic Zn 2+ concentration, consistent with release from intracellular stores.…”

“…[2,34] Activated platelets release ROS, potentially affecting platelets and other cells in a paracrine manner. [35] Whilst exogenous Zn 2+ has been found to act as a platelet agonist [11,12,36], the effect of ESR was employed to analyse superoxide anion production, as this is the most direct and reliable technique to identify chemical species that present one or more unpaired electrons. [14] Platelets were loaded with the cell-permeable superoxidespecific spin probe CMH (200μM) prior to ESR analysis.…”

“…Platelet activation results in increases in cytosolic Zn 2+ concentration, consistent with release from intracellular stores. [12] To assess the influence of intracellular Zn 2+ on ROS generation, platelet suspensions were preincubated with the intracellular Zn 2+ chelator TPEN (50μM) prior to stimulation with thrombin (1U/mL) or CRP-XL (1μg/mL). In TPEN pretreated platelets, agonist-stimulated DHE fluorescence was reduced to basal levels.…”

“…These data are consistent with a role for [Zn 2+ ]i in regulation of agonist-dependent ROS production. Whether this effect is attributable to agonist-evoked increases in [Zn 2+ ]i, [12] or to the perturbation of obligate Zn 2+ -binding proteins is unclear.…”