SIMULTANEOUS DETECTION OF CHANGES IN CYTOPLASMIC CA2+, AMINOPHOSPHOLIPID EXPOSURE AND MICROVESICULATION IN ACTIVATED PLATELETS

We have used now cytometry to compare the temporal relationship betwee n cytoplasmic Ca2+-fluxes and microvesiculation during platelet activa tion, Changes in fluorescence of the Ca2+-dye, fluo-3, and in forward light scatter as a measure of the decrease in platelet size that accom panies microvesiculation, were assessed simultaneously, In other exper iments, changes in Ca2+ levels and aminophospholipid exposure were ass essed 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 microvesiculation is a rel atively late event compared with intracellular Ca2+ elevation, The rel atively slow binding kinetics of annexin V prevented the establishment of a temporal relationship between increases in intracellular Ca2+ an d aminophospholipid exposure, Nevertheless, the combined use of fura-r ed and annexin V highlighted the heterogeneous response seen on some o ccasions with thapsigargin and always with a thrombin plus collagen mi xture, and confirmed that individual platelets that bound annexin V we re also those with elevated intracellular Ca2+ levels.