Influence of the calcium-sensitive fluorophore, Quin 2, on platelet
function
GH Rao, JD Peller, CP Semba and JG White
Recent investigations using Quin 2, a fluorophore used to monitor cytosolic
free calcium shifts, have shown that strong agonists cause a dramatic
dose-dependent increase in platelet fluorescence. However, weak agonists
stimulated little or no increase in light emission of Quin 2-loaded
platelets, suggesting that calcium flux is not involved in activation by
these agents. The present study has sought an alternative explanation for
the failure of weak stimuli to cause a rise in cytosolic free calcium in
platelets containing Quin 2. Conditions used to prepare, wash, load,
gel-filter, and evaluate the fluorophore- filled cells were examined for
their compatibility with retention of sensitivity to activation by weak
agonists. The technique used to measure shifts in cytosolic calcium with
Quin 2 requires multiply washed, unstirred platelets. Under these
conditions, platelets do not aggregate or secrete in response to weak
agonists. Quin 2, at concentrations greater than 40 mumol/L, inhibits the
response of platelets to strong agonists, and completely blocks their
reaction to weak agonists. Quin 2 inhibition of platelet function appears
related to high buffering capacity for free calcium, although other
mechanisms cannot be ruled out. This suggestion is supported by the
observation that Quin 2-induced blockade can be overcome by membrane
modulation, which is a calcium-dependent process. However, since both
agonists are weak, significant elevation in cytosolic calcium concurrent
with functional restoration could not be demonstrated under the
experimental conditions used for monitoring calcium. Thus, the conditions
used to prepare platelets for Quin 2 evaluation and Quin 2 itself appear to
be responsible for the failure of weak agonists to cause evidence of a
calcium shift in fluorophore-loaded cells.
Volume 67,
Issue 2,
pp. 354-361,
02/01/1986
Copyright © 1986 by The American Society of Hematology
