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

Area of Science:

• * Biochemistry and Molecular Biology
• * Cellular Physiology

Background:

• * Platelets play a crucial role in hemostasis and thrombosis.
• * Intracellular ion concentrations (Ca2+, Na+) and membrane potential are critical for platelet function.
• * Thapsigargin is a known inhibitor of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA).

Purpose of the Study:

• * To investigate the effects of thapsigargin on intracellular Ca2+, pH, Na+, and membrane potential in human platelets.
• * To elucidate the mechanisms underlying thapsigargin-induced changes in platelet ion homeostasis.
• * To explore the role of Na+-dependent processes and Ca2+ channels in thapsigargin's action.

Main Methods:

• * Monitoring of intracellular Ca2+ using fura-2.
• * Measurement of intracellular pH using 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein.
• * Assessment of intracellular Na+ using sodium-binding benzofuran isophthalate.
• * Determination of membrane potential using 3,3'-dipropylthiadicarbocyanine iodide.
• * Utilizing Mn2+ as a Ca2+ surrogate to study channel activation.

Main Results:

• * Thapsigargin increased cytosolic Ca2+ and caused cytosolic alkalinization, indicating stimulation of Na+-H+ exchange.
• * Thapsigargin activated Ca2+ channels at low cytosolic Ca2+ levels and enhanced Ca2+ influx in a Na+-dependent manner.
• * Thapsigargin elevated cytosolic Na+ and induced platelet hyperpolarization via stimulation of Ca2+-sensitive K+ channels.

Conclusions:

• * Thapsigargin exerts multifactorial effects on human platelets, influencing multiple ionic parameters.
• * These findings provide insights into the regulation of platelet ionic homeostasis.
• * Thapsigargin serves as a valuable tool for studying platelet ion channel function and regulation.