Thapsigargin‐evoked changes in human platelet Ca2+, Na+, pH and membrane potential.

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Abstract
1. In this work we explored the effect of thapsigarin on the intracellular Ca2+, pH, Na+ and membrane potential in human platelets. These parameters were monitored using the fluorescent probes fura‐2, 2',7'‐bis‐(2‐carboxyethyl)‐5,6‐carboxyfluorescein, sodium‐binding benzofuran isophthalate, and 3,3'‐dipropylthiadicarbocyanine iodide. 2. Thapsigargin caused an increase in the cytosolic Ca2+, coupled with cytosolic alkalinization. Thapsigargin‐induced alkalinization was Na(+)‐dependent, indicating that thapsigargin stimulated the Na(+)‐H+ exchange. 3. Using Mn2+ as a Ca2+ surrogate, we showed that thapsigargin activated Ca2+ channels at relatively low levels of cytosolic Ca2+, suggesting that a rise in cytosolic free Ca2+ is not the signal for the activation of these channels. 4. Thapsigargin‐induced increase in the cytosolic free Ca2+ was greater in Na(+)‐containing medium than in Na(+)‐free medium, suggesting that Na(+)‐dependent mechanisms participate in the regulation of platelet cytosolic Ca2+. 5. Thapsigargin not only increased the cytosolic Ca2+, but also elevated the cytosolic free Na+. The latter effect was more pronounced in Ca(2+)‐free medium, a finding that may indicate that some of the Na+ enters through Ca2+ entry pathways. 6. Finally, thapsigargin evoked sustained platelet hyperpolarization which was attenuated by charybdotoxin, indicating thapsigargin‐induced stimulation of Ca(2+)‐sensitive K+ channels. 7. Together these observations demonstrate a multifactorial effect of thapsigargin on platelets that can be utilized to further understand platelet ionic homeostasis.

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