Tao J, Rose B, Haynes D H
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, FL, USA.
Biochim Biophys Acta. 1996 May 28;1311(3):164-74. 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/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 microM 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.
使用钙离子指示剂Fluo-3（细胞内体积为0.97±0.30 mmol/l），通过荧光视频显微镜测定了置于硅化玻璃盖玻片之间的单个血小板的细胞内游离钙离子浓度（[Ca2+]cyt），分别在静息状态以及用凝血酶和ADP刺激后进行测定。在存在2 mM细胞外钙离子的情况下，静息[Ca2+]cyt仅显示出较小的血小板间变异性（[Ca2+]cyt = 86±30（标准差）nM）。作为时间函数测量的单个加载Fluo-3的血小板的静息[Ca2+]cyt的标准差为10 nM或12%（标准差/平均值）。单个血小板对硅化支持物无亲和力，其[Ca2+]cyt在静息或激活状态下均无振荡趋势。当加入0.2 U/ml凝血酶或20 μM ADP时，所有血小板均显示出特征性的钙离子瞬变，即[Ca2+]cyt在8 - 12秒内升至峰值，然后下降。用Fluo-3测量的钙离子瞬变大致同步，但[Ca2+]cyt峰值显示出较大的血小板间变异性。凝血酶和ADP的总体平均峰值[Ca2+]cyt分别为672±619（标准差）nM和640±642（标准差）nM。因此，血小板间变异（标准差/平均值）分别是平均测量值的92%或100%。单个血小板实验的数学构建平均值与在塑料比色皿中搅拌血小板悬浮液的平行实验中获得的血小板平均值相当吻合，后者是用传统荧光分光光度计测量的。仅反映致密管状钙离子释放（去除细胞外钙离子）的[Ca2+]cyt峰值也显示出较大的血小板间变异（凝血酶刺激时为171±105（标准差）nM，ADP刺激时为183±134（标准差）nM）。由环匹阿尼酸（一种致密管状钙离子 - ATP酶抑制剂）诱导的致密管状钙离子释放产生的[Ca2+]cyt峰值为289±170 nM。因此，致密管状钙离子池的大小在血小板间变异为59%（标准差/平均值）。致密管状池大小的变异性解释了凝血酶和ADP激活时[Ca2+]cyt峰值中部分但不是全部的大血小板间变异。
