Elevation of pHi is not an essential step in calcium mobilisation in fura-2-loaded human platelets.

Human platelets were co-loaded with the fluorescent indicators BCECF and fura-2 to measure pHi and [Ca2+]i and incubated with aspirin to block cyclooxygenase. Either pHi and shape change and aggregation or pHi and [Ca2+]i were measured simultaneously in the same stirred cuvette, at 37 degrees C. In Hepes-buffered saline containing 1 mM Ca2+, mean resting pHi was 6.98 +/- 0.01 (SE, n = 59). Changes of pHi up to +/- 0.35 units, imposed by additions of NH4Cl, CO2 or nigericin, produced no shape change or aggregation and only insignificant changes in [Ca2+]i. Sufficient thrombin to raise [Ca2+]i over 1 microM and cause rapid shape change and aggregation increased pHi by no more than 0.05 units, and the increase in pHi lagged behind the elevation of [Ca2+]i. We conclude that changes in pHi do not form a necessary or sufficient component of the pathways leading to receptor-mediated Ca2+ mobilisation or the stimulation of shape change or aggregation.