Variability of the thrombin- and ADP-induced Ca2+ response among human platelets measured using fluo-3 and fluorescent videomicroscopy

The intracellular free Ca2+ concentration ([Ca2+](cyt)) of individual human platelets localized between siliconized glass cover slips was determined at rest and after stimulation with thrombin and ADP using the Ca2+ indicator fluo-3 (0.97 +/- 0.30 mmol/l cell volume) with fluorescence video microscopy. Resting [Ca2+](cyt) in the presence of 2 mM external Ca2+ showed only small inter-platelet variability ([Ca2+](cyt) = 86 +/- 30 (S.D.) nM). Resting [Ca2+](cyt) of individual fluo-3-loaded platelets measured as a function of time had a S.D. of 10 nM or 12% (S.D./mean). Individual platelets showed no affinity for the siliconized support and their [Ca2+](cyt) showed no tendency to oscillate in either the resting or in the activated state. When 0.2 U/ml thrombin or 20 mu M ADP were added, all platelets showed a characteristic Ca2+ transient whereby [Ca2+](cyt) increased to peak values within 8-12 sec and then declined. The Ca2+ transients measured with fluo-3 were in approximate synchrony but peak [Ca2+](cyt) values showed large inter-platelet variability. The ensemble average peak [Ca2+](cyt) for thrombin and ADP were 672 +/- 619 (S.D.) nM and 640 +/- 642 (S.D.) nM, respectively. Thus inter-platelet variations (S.D./mean) were 92% or 100% as large as the average measured values. Mathematically-constructed averages of the single platelet experiments agreed reasonably well with platelet-averaged values obtained in parallel experiments with stirred platelet suspensions in a plastic cuvette, measured with a conventional spectrofluorometer. Peak [Ca2+](cyt) values reflecting dense tubular Ca2+ release alone (external Ca2+ removed) also showed large interplatelet variation (171 +/- 105 (S.D.) nM with thrombin and 183 +/- 134 (S.D.) nM with ADP). Dense tubular Ca2+ release induced by cyclopiazonic acid (a dense tubular Ca2+-ATPase inhibitor) gave peak [Ca2+](cyt) of 289 +/- 170 nM. Thus the size of the dense tubular Ca2+ pool has an inter-platelet variation of 59% (S.D./mean). Variability of the dense tubular pool size accounts for some, but not all, of the large interplatelet variation in peak [Ca2+](cyt) seen with thrombin and ADP activation.