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

• Published in: Biochimica et biophysica acta 1996-05, Vol.1311 (3), p.164-174
• Main Authors: Tao, Jianguo, Rose, Birgit, Haynes, Duncan H
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate - pharmacology; ADP; Aniline Compounds; Blood Platelets - drug effects; Blood Platelets - metabolism; Calcium; Calcium - blood; Calcium - pharmacology; Calcium imaging; Cytoplasm - metabolism; Cytoplasmic; Fluo-3; Fluorescent Dyes; Human; Humans; Indoles - pharmacology; Kinetics; Microscopy, Video - methods; Platelet; Platelet Activation; Spectrometry, Fluorescence; Thrombin; Thrombin - pharmacology; Xanthenes
• ISSN: 0167-4889; 0006-3002; 1879-2596
• DOI: 10.1016/0167-4889(96)00003-1

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/1 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 μM 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.