Thrombin-induced calcium movements in platelet activation.
Thrombin-induced calcium movements in platelet activation.
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血小板活化中凝血酶诱导的钙运动。
DOI：
10.1016/0167-4889(87)90244-8
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Haynes,DH
中科院分区：
文献类型：
--
作者：
Jy,W;Haynes,DH
以细胞内钙离子浓度([Ca~(2+)]Cyt)和氯四环素(CTC)为指标，研究凝血酶诱导的人血小板钙离子通量及其与血小板聚集的偶联作用。证明CTC荧光变化与致密管腔内游离钙离子浓度成正比。细胞内Quin2浓度为1 mM，分析表明它不干扰本文报道的过程。从钙内流、钙释放、钙封存和钙排出等方面分析静息状态和凝血酶激活过程中细胞内钙的变化。通过在凝血酶激活前1分钟清除细胞外钙离子来区分内流和内流。当细胞外钙浓度为2 mM时，凝血酶诱导的细胞内钙内流是细胞[Ca~(2+)]升高的主要原因(80%以上)。凝血酶诱导的钙离子内流和释放的EC50值略有不同(0.17U/ml比0.35U/ml)。异搏定、苯普地尔和Cd~(2+)可抑制内流的贡献(IC50值分别为19μM、2μM和50μM)。根据凝血酶激活的通道对内流结果进行分析。吲哚美辛预处理实验表明，花生四烯酸途径的激活约占。50%的内流相关[Ca~(2+)]升高。维拉帕米或Cd~(2+)不抑制细胞内[Ca~(2+)]浓度升高，但可被高IC_(50)(25μM)的苯普地尔抑制。它只被消炎痛抑制15-20%，因此不依赖于血栓素A2的形成。释放反应不需要钙离子内流。凝血酶激活的血小板聚集率约与[Ca~(2+)]细胞的4次方有关，明显的Kmof为0.4μM。比较部分凝血酶激活和完全凝血酶激活、部分维拉帕米抑制的条件下的聚集率表明，这种对[Ca~(2+)]细胞的依赖是聚集行为的主要决定因素。分析表明，当生理浓度的外源性钙离子存在时，钙内流是凝血酶升高细胞内钙离子的主要途径。
The thrombin-induced Ca2+fluxes and their coupling to platelet aggregation of the human platelet were studied using quin2 as a measure of the cytoplasmic Ca2+concentration ([Ca2+]cyt) and chlorotetracycline (CTC) as a measure of internally sequestered Ca2+. Evidence is given that the CTC fluorescence change is proportional to the free internal Ca2+concentration in the dense tubular lumen. The intracellular quin2 concentration was 1 mM and analysis showed that it did not perturb the processes reported herein. The value of [Ca2+]cytat rest and during thrombin activation was analyzed in terms of Ca2+influx, Ca2+release, Ca2+sequestration, and Ca2+extrusion. Influx was distinguished from internal release by removing extracellular Ca2+1 min before thrombin activation. In the presence of 2 mM external Ca2+, the thrombin-induced Ca2+influx accounts for most of the increase in [Ca2+]cyt(over 80%). Thrombin-induced Ca2+influx and release have somewhat different EC50values (0.17 U/ml vs. 0.35 U/ml). The contribution of influx can be inhibited by verapamil, bepridil and Cd2+(IC50values of 19 μM, 2 μM and 50 μM). The influx results were analyzed in terms of a thrombin-activated channel. Indomethacin pretreatment experiments suggest that activation of the arachidonic pathway accounts for approx. 50% of the influx-related [Ca2+]cytelevation. Elevation of [Ca2+]cytby intracellular release is not inhibited by verapamil or Cd2+but is inhibited by bepridil with a high IC50(25 μM). It is only 15–20% inhibited by indomethacin and is thus not dependent on thromboxane A2formation. The release reaction does not require Ca2+influx. The rate of thrombin-activated platelet aggregation is shown to have an approximately fourth-power dependence on [Ca2+]cytwith an apparentKmof 0.4 μM. Comparisons of aggregation rates of the partially thrombin-activated vs. fully thrombin-activated, partially verapamil-inhibited conditions suggest that this dependence on [Ca2+]cytis the major determinant of the aggregation behavior. Analysis shows that calcium influx is the major pathway for elevating [Ca2+]cytby thrombin when physiological concentrations of external Ca2+are present.