Human platelets contain a pool of free zinc in dense granules

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.