Influence of the calcium-sensitive fluorophore, Quin 2, on platelet function
Influence of the calcium-sensitive fluorophore, Quin 2, on platelet function
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DOI：
10.1182/blood.v67.2.354.bloodjournal672354
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发表时间：
1986-02
期刊：
影响因子：
20.3
通讯作者：
G. Rao;J. Peller;C. Semba;J. White
中科院分区：
文献类型：
--
作者：
G. Rao;J. Peller;C. Semba;J. White
最近使用Quin 2（一种用于监测胞质游离钙变化的荧光团）的研究表明，强激动剂可引起血小板荧光的显著剂量依赖性增加。然而，弱激动剂刺激很少或没有增加的光发射的Quin 2加载的血小板，这表明钙通量不参与这些代理的激活。本研究已寻求一种替代的解释弱刺激失败，导致含Quin 2的血小板胞浆游离钙上升。检查用于制备、洗涤、加载、凝胶过滤和评价荧光团填充细胞的条件与保留对弱激动剂激活的敏感性的相容性。用Quin 2测量细胞溶质钙变化的技术需要多次洗涤、未搅拌的血小板。在这些条件下，血小板不聚集或分泌响应弱激动剂。Quin 2在浓度大于40 μ mol/L时，抑制血小板对强激动剂的反应，并完全阻断它们对弱激动剂的反应。Quin 2对血小板功能的抑制似乎与游离钙的高缓冲能力有关，尽管不能排除其他机制。这一建议得到了以下观察结果的支持：Quin 2诱导的阻滞可以通过膜调节来克服，这是一个钙依赖性过程。然而，由于这两种激动剂都很弱，在用于监测钙的实验条件下，不能证明细胞溶质钙与功能恢复同时显著升高。因此，用于制备用于Quin 2评价的血小板的条件和Quin 2本身似乎是弱激动剂未能引起荧光团负载细胞中钙移位证据的原因。
: 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.