Simultaneous detection of changes in cytoplasmic Ca2+, aminophospholipid exposure and microvesiculation in activated platelets

We have used flow cytometry to compare the temporal relationship between cytoplasmic Ca(2+)-fluxes and micro-vesiculation during platelet activation. Changes in fluorescence of the Ca(2+)-dye, fluo-3, and in forward light scatter as a measure of the decrease in platelet size that accompanies micro-vesiculation, were assessed simultaneously. In other experiments, changes in Ca(2+) levels and aminophospholipid exposure were assessed using fura-red, which is a long wavelength range indicator, and FITC-annexin V. Results obtained using the ionophore A23 187 and the ATPase inhibitor, thapsigargin, showed that micro-vesiculation is a relatively late event compared with intracellular Ca(2+) elevation. The relatively slow binding kinetics of annexin V prevented the establishment of a temporal relationship between increases in intracellular Ca(2+) and aminophospholipid exposure. Nevertheless, the combined use of fura-red and annexin V highlighted the heterogeneous response seen on some occasions with thapsigargin and always with a thrombin plus collagen mixture, and confirmed that individual platelets that bound annexin V were also those with elevated intracellular Ca(2+) levels.