Delineating Zinc Influx Mechanisms during Platelet Activation

Zinc (Zn(2+)) is released by platelets during a hemostatic response to injury. Extracellular zinc ([Zn(2+)](o)) initiates platelet activation following influx into the platelet cytosol. However, the mechanisms that permit Zn(2+) influx are unknown. Fluctuations in intracellular zinc ([Zn(2+)](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. [Zn(2+)](o) influx and subsequent platelet activation were abrogated by blocking the sodium/calcium exchanged, TRP channels, and ZIP7. Cation store depletion regulated Zn(2+) influx. [Zn(2+)](o) stimulation resulted in the phosphorylation of PKC substates, MLC, and β3 integrin. Platelet activation via GPVI or Zn(2+) resulted in ZIP7 phosphorylation in a casein kinase 2-dependent manner and initiated elevations of [Zn(2+)](i) that were sensitive to the inhibition of Orai1, ZIP7, or IP(3)R-mediated pathways. These data indicate that platelets detect and respond to changes in [Zn(2+)](o) via influx into the cytosol through TRP channels and the NCX exchanger. Platelet activation results in the externalization of ZIP7, which further regulates Zn(2+) influx. Increases in [Zn(2+)](i) contribute to the activation of cation-dependent enzymes. Sensitivity of Zn(2+) influx to thapsigargin indicates a store-operated pathway that we term store-operated Zn(2+) entry (SOZE). These mechanisms may affect platelet behavior during thrombosis and hemostasis.