THROMBIN-INDUCED CYTOSOLIC ALKALINIZATION IN HUMAN PLATELETS OCCURS WITHOUT AN APPARENT INVOLVEMENT OF HCO-3/CL- EXCHANGE

We have estimated the changes in cytosolic pH (pHi) that occur when human platelets are stimulated by thrombin. Changes in pHi 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 pHi, from 7.05±0.04 to 7.45±0.05. Both H+ efflux and pHi 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 pHi. After blockade of Na+/H+ exchange by 100 μM ethylisopropylamiloride (EIPA), thrombin induced a decrease in pHi 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. © 1990 Springer-Verlag.