Changes of intracellular pH and calcium concerned in platelet activation

Abstract

We have investigated the changes in intracellular pH (pHi) and cytosolic free Ca²⁺ concentration ([Ca²⁺]cyt) in platelet activation evoked by thrombin and thromboxane A₂ analogue, STA₂. pH was measured with the new fluorescent dye, 2′, 7′-bis (carboxyethyl) 5, 6-carboxyfluorescein (BCECF), and [Ca²⁺]cyt was determined by the fluorescent Ca²⁺ indicator, fura-2. Resting pHi of human platelets averaged 7.03±0.08 pH unit, which is similar to the values reported by others. There was an initial fall of 0.01-0.02 pH unit immediately after addition of thrombin. This was followed by an increase in pHi to between 0.07-0.18pH unit above the resting level. The resting [Ca²⁺]cyt level of around 100nM was increased abruptly to 1-3μM after stimulation with thrombin. Thrombin-induced platelet aggregation, increase in [Ca²⁺]cyt and intracellular alkalinization were inhibited by removal of Na⁺ or by treatment of platelet with Na⁺/H⁺ exchange inhibitor, amiloride.
STA₂ induced a transient increase in [Ca²⁺]cyt, however, it failed to cause a distinct alkalinization. Increase in [Ca²⁺]cyt and platelet aggregation evoked by STA₂ were observed even after removal of extracellular Na⁺. These results suggest that there may be another pathway in relation to platelet activation and Ca²⁺ mobilization independent of intracellular alkalinization.