Calcium mobilization in human platelets using indo-1 and flow cytometry

Abstract

Regulation of cytoplasmic free calcium concentration is believed to be important in the response of platelets to external stimuli. A relatively new fluorescent calcium indicator, indo-1, has properties by which alterations of cytoplasmic calcium can be evaluated in single platelets by flow cytometry. Activation of platelets at a temperature lower than 37 degrees C allows examination of the heterogeneity of intracellular free calcium levels and can distinguish variations among platelets in the initiation, duration, and magnitude of calcium fluxes. The clear advantage of flow cytometric analysis of platelet cytosolic calcium is that stimulus-response coupling can now be studied on a single cell basis. Platelets were activated by addition of human alpha-thrombin or ADP at 37 degrees C or at room temperature (22 degrees C). Activation at 37 degrees C approaches more closely an in vivo response and, as expected, increases in cytosolic calcium occurred within seconds of agonist addition. Transient increases in cytoplasmic calcium levels occurred when platelets were challenged with a low concentration of agonist. Heterogeneity in cytoplasmic calcium levels was also observed at 10(-5) mol/L ADP and 0.1 U/mL alpha-thrombin. Some of this heterogeneity was no longer observed at higher concentrations of agonist (10(-4) mol/L ADP and 0.5 U/mL thrombin), suggesting that a sufficient magnitude of signal is required to induce changes in platelet cytosolic calcium. Light-scatter properties of the activated platelets were also monitored simultaneously and showed changes in response to both agonists. The ability to measure changes in cytoplasmic free calcium by ratio flow cytofluorimetry provides a new approach to study of the role of alterations in intracellular calcium in response to agonists acting through different membrane receptors as well as providing a sensitive technique to detect functional subpopulations of platelets.