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Abstract

One of the earliest events following stimulation of human platelets with thrombin is a rise in the cytosolic pH, pH_i, mediated by exchange, and an increase in the cytosolic free Ca²⁺ concentration, [Ca²⁺]_i. In the present study we investigated whether an increase in pH_i alone, induced by the ionophore monensin, is sufficient for platelet activation. Although monensin (20 μM) raised pH_i from 7.10 ± 0.05 (n=21) to 7.72 ± 0.17 (n=13), neither Ca²⁺ influx nor mobilization were detectable upon this treatment in fura2-loaded platelets. In contrast, thrombin (0.05 U/ml) raised pHi to 7.31 ± 0.10 (n=10) and increased [Ca²⁺]_i by more than 250 nM both in the presence and absence of extracellular Ca²⁺. Thrombin also caused the formation of phosphatidic acid and phosphorylation of the 20 kDa and 47 kDa proteins in platelets labeled with ³²P. Monensin, however, induced none of these responses. It is concluded that an increase in pH_i alone is not a sufficient trigger for platelet activation but enhances intracellular signal transduction in platelets stimulated by natural agonists.