Calcium mobilization in human platelets using indo-1 and flow cytometry
LK Jennings, ME Dockter, CD Wall, CF Fox and DM Kennedy
Department of Medicine, University of Tennessee, Memphis.
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.
Volume 74,
Issue 8,
pp. 2674-2680,
12/01/1989
Copyright © 1989 by The American Society of Hematology
