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.
Highlights
Zinc (Zn2þ) is an essential trace element, serving as a cofactor for 10 to 15% of proteins encoded within the human genome.[1]
Stimulation of platelets with the Zn2þ ionophores clioquinol (Cq) or pyrithione (Py) caused sustained increases of [Zn2þ]i, resulting in myosin light chain phosphorylation, and cytoskeletal rearrangements which were sensitive to cytochalasin-D treatment
Zn2þionophores induced externalization of phosphatidylserine. These data suggest that agonist-evoked fluctuations in intra-platelet Zn2þ couple to functional responses, in a manner that is consistent with a role as a secondary messenger
Summary
Zinc (Zn2þ) is an essential trace element, serving as a cofactor for 10 to 15% of proteins encoded within the human genome.[1] It is acknowledged as an extracellular signalling molecule in glycinergic and GABAergic neurones, and is released into the synaptic cleft following excitation.[2,3] Zn2þ is concentrated in atherosclerotic plaques and released from damaged epithelial cells, and is released from platelets along with their α-granule cargo following collagen stimulation.[4] increased concentrations of unbound or labile (free) Zn2þ are likely to be present at areas of haemostasis, and may be much higher in the microenvironment of a growing thrombus. Zn2þ plays a role in haemostasis by contributing to wound healing,[5] and regulating coagulation, for example, as a co-factor for factor XII.[6] Labile Zn2þ acts as a received June 26, 2018 accepted after revision October 31, 2018. While the role of Zn2þ as a platelet agonist is known, its secondary messenger activity in platelets has not been demonstrated
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