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 & beta;3 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.

Automated summary

Zinc ions are released by platelets and can activate platelets. The mechanisms of zinc influx and platelet activation were investigated using fluorescence and flow cytometry. The results showed that blocking the sodium/calcium exchange, TRP channels, and ZIP7 can inhibit zinc influx and platelet activation. These mechanisms may affect thrombosis and hemostasis.