Delineating Zinc Influx Mechanisms during Platelet Activation

Zinc (Zn²⁺) is released by platelets during a hemostatic response to injury. Extracellular zinc ([Zn²⁺]_o) initiates platelet activation following influx into the platelet cytosol. However, the mechanisms that permit Zn²⁺ influx are unknown. Fluctuations in intracellular zinc ([Zn²⁺]_i) were measured in fluozin-3-loaded platelets using fluorometry and flow cytometry. Platelet activation was assessed using light transmission aggregometry. The detection of phosphoproteins was performed by Western blotting. [Zn²⁺]_o influx and subsequent platelet activation were abrogated by blocking the sodium/calcium exchanged, TRP channels, and ZIP7. Cation store depletion regulated Zn²⁺ influx. [Zn²⁺]_o stimulation resulted in the phosphorylation of PKC substates, MLC, and β3 Integrin. Platelet activation via GPVI or Zn²⁺ resulted in ZIP7 phosphorylation in a Casein Kinase 2-dependent manner and initiated elevations of [Zn²⁺]_i that were sensitive to the inhibition of Orai1, ZIP7, or IP₃R-mediated pathways. These data indicate that platelets detect and respond to changes in [Zn²⁺]_o via influx into the cytosol through TRP channels and the NCX exchanger. Platelet activation results in the externalization of ZIP7, which further regulates Zn²⁺ influx. Increases in [Zn²⁺]_i contribute to the activation of cation-dependent Enzymes. Sensitivity of Zn²⁺ influx to thapsigargin indicates a store-operated pathway that we term store-operated Zn²⁺ entry (SOZE). These mechanisms may affect platelet behavior during thrombosis and hemostasis.

NCX; TRP channels; ZIP7; aggregation; cation signaling; platelets; store-operated calcium entry; store-operated zinc entry; zinc; zinc entry; zinc-induced platelet activation.