Human platelets contain a pool of free zinc in dense granules

Walter H A Kahr; Sara J Henderson; Fred G Pluthero; Harry F G Heijnen; Nima Vaezzadeh; Alan R Stafford; James C Fredenburgh; Jeffrey I Weitz

doi:10.1016/j.rpth.2024.102352

Human platelets contain a pool of free zinc in dense granules

Res Pract Thromb Haemost. 2024 Feb 15;8(2):102352. doi: 10.1016/j.rpth.2024.102352. eCollection 2024 Feb.

Authors

Walter H A Kahr^{1

2

3}, Sara J Henderson^{4

5}, Fred G Pluthero³, Harry F G Heijnen⁶, Nima Vaezzadeh^{4

7}, Alan R Stafford⁸, James C Fredenburgh^{4

8}, Jeffrey I Weitz^{4

5

7

8}

Affiliations

¹ Division of Haematology/Oncology, Department of Paediatrics, University of Toronto and the Hospital for Sick Children, Toronto, Ontario, Canada.
² Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
³ Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
⁴ Thrombosis and Atherosclerosis Research Institute, McMaster University, Hamilton, Ontario, Canada.
⁵ Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.
⁶ Departments of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht, The Netherlands.
⁷ Department of Medical Sciences, McMaster University, Hamilton, Ontario, Canada.
⁸ Department of Medicine, McMaster University, Hamilton, Ontario, Canada.

Abstract

Background: Activated platelets release procoagulant factors that include Ca²⁺ and Zn²⁺. Releasable Ca²⁺ stores have been identified in platelet dense granules and the dense tubular system, but similar stores of free Zn²⁺ have not been identified.

Objectives: Guided by studies of platelet Ca²⁺, we employed minimally disruptive methods to identify and localize concentrated free Zn²⁺ in human platelets.

Methods: Resting platelets from normal donors (NDs), patients with gray platelet syndrome (GPS) lacking α-granules, and patients with Hermansky-Pudlak syndrome (HPS) deficient in dense granules were loaded with cell-permeant fluorescent probes specific to free Ca²⁺ or Zn²⁺. Ion concentrations were detected in fixed cells as bright puncta via high-resolution confocal microscopy. Ions were also directly detected via transmission electron microscopy and energy dispersive X-ray analysis. Levels of total platelet Ca, Zn, and Mg were measured via inductively coupled plasma optical emission spectroscopy.

Results: Fluorescent Zn²⁺ puncta counts were similar in ND and GPS platelets and markedly lower in HPS platelets, pointing to dense granules as likely reservoirs of free Zn²⁺. This localization was supported by direct detection of Ca²⁺, Zn²⁺, and Na⁺ in platelet dense granules via transmission electron microscopy and energy dispersive X-ray analysis. Measurements of total platelet Ca, Zn, and Mg via inductively coupled plasma optical emission spectroscopy indicated that free Zn²⁺ represents a small proportion of total platelet zinc, consistent with the strong affinity of Zn²⁺ for binding proteins, including several abundant in platelet α-granules.

Conclusion: We conclude that normal human platelets contain a pool of free Zn²⁺ concentrated in dense granules that is available for secretion upon platelet activation and potentially contributes to hemostasis.

Keywords: dense granules; hemostasis; platelets; secretion; zinc.