Influence of cytoplasmic pH on the aggregation and Ca2+ mobilization in rabbit platelets

The aggregability of rabbit platelets was studied under various cytoplasmic pHs (pHi). Nigericin, a K+/H+ ionophore, which can induce a decrease in pHi, at 2-10 microM in 2 min incubation reduced both platelet aggregation and an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) stimulated with thrombin or U46619. The reduced aggregability recovered 10 min after incubation with nigericin in parallel with an increase in pHi. In contrast, when pHi was increased by simultaneous addition of NH4Cl, methylamine or monensin, aggregation in response to a low concentration of thrombin, U46619, arachidonic acid or A23187 was enhanced significantly. The enhancing effect of NH4Cl was lowered by prolonged incubation with NH4Cl, by which the increased pHi was improved concomitantly. Indomethacin, an inhibitor of cyclooxygenase, failed to inhibit the enhancement of aggregation by NH4Cl under stimulation with U46619. In addition, treatment with NH4Cl enhanced an increase in [Ca2+]i in response to U46619 in a concentration-dependent manner, although the treatment by NH4Cl alone did not affect [Ca2+]i. When pHi was artificially altered during the ranges of 6.6-7.4 by treatment with nigericin in K+-rich medium, aggregation by low concentrations of thrombin was dependent on the pHi. These data indicate that pHi is an important factor for platelet activation including intracellular Ca2+ mobilization and aggregation.