AJP - Cell Physiology, Vol 260, Issue 2 C242-C248, Copyright © 1991 by American Physiological Society
Quasi-simultaneous measurement of ionized calcium and alpha-granule release in individual platelets
A. Oda, J. F. Daley, J. Kang, M. Smith, J. A. Ware and E. W. Salzman
Department of Surgery, Beth Israel Hospital, Boston, Massachusetts 02215.
The effect of alpha-thrombin and ADP on calcium mobilization and
alpha-granule release in individual platelets was investigated by flow
cytometry. alpha-Thrombin (4.5 nM) caused a uniform rise of free cytosolic
calcium ([Ca2+]i) among indo-1-loaded human platelets. Despite the
uniformity of this effect, approximately 20% of the cells failed to secrete
alpha-granule content, as shown by binding of fluorescein isothiocyanate
(FITC)-conjugated S12 monoclonal antibody. ADP (10 microM) caused a similar
brisk and uniform rise of calcium but did not increase S12 binding to any
platelets. On the other hand, with alpha-thrombin (0.5 nM), calcium
mobilization was heterogeneous and paralleled granule release. [Ca2+]i
increased rapidly in some platelets, while only slowly in others. When an
electronic gate was set according to FITC-S12 fluorescence, cells with a
greater secretory response proved to be those with a higher calcium level.
With both alpha-thrombin and ADP, chelation of external calcium by EGTA (2
mM) reduced calcium response of individual cells. NiCl2 (1 mM) also
inhibited calcium rise of individual platelets to the same extent as EGTA
(2 mM) in spite of the presence of 1 mM CaCl2 in the extracellular media.
The effects of EGTA and NiCl2 were not limited to a particular
subpopulation of cells. These data suggest that the putative
Ni2(+)-inhibitable divalent cation channel(s) may be responsible for the
increased influx of calcium that occurs during platelet activation by
alpha-thrombin and ADP. It appears that these calcium channels contribute
to the elevation of [Ca2+]i among virtually all the platelets.
