Oda A, Daley J F, Kang J, Smith M, Ware J A, Salzman E W
Department of Surgery, Beth Israel Hospital, Boston, Massachusetts 02215.
Am J Physiol. 1991 Feb;260(2 Pt 1):C242-8. doi: 10.1152/ajpcell.1991.260.2.C242.
The effect of alpha-thrombin and ADP on calcium mobilization and alpha-granule release in individual platelets was investigated by flow cytometry. alpha-Thrombin (4.5 nM) caused a uniform rise of free cytosolic calcium ([Ca2+]i) among indo-1-loaded human platelets. Despite the uniformity of this effect, approximately 20% of the cells failed to secrete alpha-granule content, as shown by binding of fluorescein isothiocyanate (FITC)-conjugated S12 monoclonal antibody. ADP (10 microM) caused a similar brisk and uniform rise of calcium but did not increase S12 binding to any platelets. On the other hand, with alpha-thrombin (0.5 nM), calcium mobilization was heterogeneous and paralleled granule release. [Ca2+]i increased rapidly in some platelets, while only slowly in others. When an electronic gate was set according to FITC-S12 fluorescence, cells with a greater secretory response proved to be those with a higher calcium level. With both alpha-thrombin and ADP, chelation of external calcium by EGTA (2 mM) reduced calcium response of individual cells. NiCl2 (1 mM) also inhibited calcium rise of individual platelets to the same extent as EGTA (2 mM) in spite of the presence of 1 mM CaCl2 in the extracellular media. The effects of EGTA and NiCl2 were not limited to a particular subpopulation of cells. These data suggest that the putative Ni2(+)-inhibitable divalent cation channel(s) may be responsible for the increased influx of calcium that occurs during platelet activation by alpha-thrombin and ADP. It appears that these calcium channels contribute to the elevation of [Ca2+]i among virtually all the platelets.
通过流式细胞术研究了α-凝血酶和ADP对单个血小板中钙动员和α-颗粒释放的影响。α-凝血酶（4.5 nM）使indo-1标记的人血小板中游离胞质钙（[Ca2+]i）均匀升高。尽管这种效应具有一致性，但约20%的细胞未能分泌α-颗粒内容物，异硫氰酸荧光素（FITC）偶联的S12单克隆抗体结合实验表明了这一点。ADP（10 μM）引起类似的快速且均匀的钙升高，但并未增加S12与任何血小板的结合。另一方面，对于α-凝血酶（0.5 nM），钙动员是异质性的，且与颗粒释放平行。一些血小板中的[Ca2+]i迅速增加，而另一些则增加缓慢。当根据FITC-S12荧光设置电子门时，分泌反应较强的细胞被证明是钙水平较高的细胞。对于α-凝血酶和ADP，EGTA（2 mM）螯合细胞外钙均降低了单个细胞的钙反应。尽管细胞外培养基中存在1 mM CaCl2，但NiCl2（1 mM）也将单个血小板的钙升高抑制到与EGTA（2 mM）相同的程度。EGTA和NiCl2的作用并不局限于特定的细胞亚群。这些数据表明，推测的Ni2(+)-可抑制二价阳离子通道可能是α-凝血酶和ADP激活血小板过程中钙内流增加的原因。似乎这些钙通道几乎对所有血小板中[Ca2+]i的升高都有作用。
