CC BY 4.0 · Thromb Haemost 2019; 119(01): 128-139
DOI: 10.1055/s-0038-1676589
Cellular Haemostasis and Platelets
Georg Thieme Verlag KG Stuttgart · New York

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

Niaz S. Ahmed
1  School of Life Sciences, Anglia Ruskin University, Cambridge, United Kingdom
,
Maria E. Lopes Pires
1  School of Life Sciences, Anglia Ruskin University, Cambridge, United Kingdom
,
Kirk A. Taylor
2  Cardio-Respiratory Interface Section, National Heart and Lung Institute, Imperial College London, London, United Kingdom
,
Nicholas Pugh
1  School of Life Sciences, Anglia Ruskin University, Cambridge, United Kingdom
› Author Affiliations
Funding This work was supported by British Heart Foundation project grants (PG/14/47/30912 and PG/18/64/33922).
Further Information

Publication History

26 June 2018

31 October 2018

Publication Date:
31 December 2018 (online)

Abstract

Background 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.

Objectives 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.

Methods 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.

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 [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 αIIbβ3 activation and release of dense, but not α, granules. Furthermore, Zn2+-ionophores induced externalization of phosphatidylserine.

Conclusion 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, αIIbβ3 up-regulation and dense granule release, in a redox-sensitive manner.

Authors' Contributions

N.S.A., M.L.P. and N.P. designed and conducted experiments, and wrote the manuscript. N.S.A., M.L.P., K.T. and N.P. designed experiments and wrote the manuscript.


Supplementary Material