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Abstract

We have estimated the changes in cytosolic pH (pH_i) that occur when human platelets are stimulated by thrombin. Changes in pH_i were estimated (i) from the H⁺ efflux across the plasma membrane using an extracellular pH electrode and (ii) using an intracellular pH-sensitive fluorescent dye (BCECF). Stimulation of platelets with thrombin (0.5 unit/ml) resulted in an H⁺ efflux that averaged 7.7±1.6 μmol/1011 platelets (means±SD) leading to an increase in pH_i, from 7.05±0.04 to 7.45±0.05. Both H⁺ efflux and pH_i changes were unaffected by 0.1 mM 4,4-diisothiocyanostilbene-2,2 disulphonate (DIDS), 0.1 mM 4′-acetamido 4′-isothiostilbene-2,2′-disulphonic acid (SITS), or 0.5 mM bumetanide, suggesting no involvement of anion transport systems, e.g. an HCO₃^-/Cl^- exchange. Removal of HCO₃^-or Cl^- from the suspending buffer had no effect on the extent of the rise in pH_i. After blockade of Na⁺/H⁺ exchange by 100 μM ethylisopropylamiloride (EIPA), thrombin induced a decrease in pH_i the rate of which averaged 0.39 unit/min in HCO₃^--containing medium, and 0.57 unit/min in HCO₃^--free medium. The cytosolic buffer capacity for H⁺ was determined by the nigericin/ NH₄Cl technique in BCECF-loaded platelets and averaged 25.3 mmol/(1xpH) in buffer containing 8 mM HCO₃^-, but only 17.2 mmol/(1xpH) in HCO₃^--free buffer. The total amount of H⁺ transferred by Na⁺/H⁺ exchange can be estimated from our measurements at 10 mmol/l platelet cytosol in the absence of HCO₃^-and to 14 mmol/l platelet cytosol in the presence of HCO₃^-, and is in good agreement with the estimated amount of Na⁺ uptake by ADP-stimulated platelets. We conclude that net extrusion of H⁺ from stimulated platelets is predominantly mediated by Na⁺/H⁺ exchange without an apparent contribution of HCO₃^-/Cl^- exchange.