Nakamura T, Ariyoshi H, Kambayashi J, Ikeda M, Kawasaki T, Sakon M, Monden M
Department of Surgery II, Osaka University Medical School, Japan.
J Lab Clin Med. 1997 Sep;130(3):262-70. doi: 10.1016/s0022-2143(97)90020-2.
The effect of a physiologic concentration of epinephrine (Ep) on platelet activation was studied by using a novel method that simultaneously measures platelet aggregation by changes in light transmission and counts particles of various sizes by using light scattering. Detailed morphologic changes associated with activation process were studied by using confocal laser scanning microscopy (CLSM). A low concentration of Ep (20 nmol/L) corresponding to a high physiologic concentration triggered the formation of small platelet aggregates (diameter 7 to 30 microm) without any change in light transmission. The redistribution of filamentous actin (F-actin) and the expression of activated glycoprotein IIb-IIIa complex (GPIIb-IIIa), detected by PAC-1 binding, were also observed in the platelets comprising the small aggregates. Attempts were then made to detect changes in cytoplasmic ionized Ca2+ ((Ca2+)i) in individual platelets involved in the aggregate formation by CLSM with fluo-3, a Ca2+-indicating dye. Ep caused a weak (Ca2+)i increase in some individual platelets involved in the formation of small aggregates. This (Ca2+)i increase was associated with platelet aggregation, because no (Ca2+)i rise was detected in single platelets. Furthermore, platelets stimulated by Ep in the presence of RGDS or Ro 44-9883, a GPIIb-IIIa antagonist, did not form small aggregates or trigger a (Ca2+)i rise. Prior incubation with low concentrations of Ep (20, 100 nmol/L) enhanced the initial formation of small (diameter 7 to 30 microm), medium (diameter 30 to 50 microm), or large (diameter 50 to 70 microm) aggregates induced by a subthreshold concentration of adenosine diphosphate (0.25 micromol/L) as determined by the particle counting method. However, no apparent synergistic (Ca2+)i increase was observed in platelets involved in aggregate formation. From these observations the following conclusions have been reached. (1) A high physiologic concentration of Ep (20 nmol/L) is capable of triggering the formation of small aggregates, resulting in the redistribution of F-actin and the expression of activated GPIIb-IIIa complex. (2) An increase in (Ca2+)i is observed in platelets comprising the small aggregates. This increase is not related to the binding of Ep to its receptor but most likely is triggered by platelet-platelet association. (3) The characteristic potentiating effect of Ep is not due to the synergistic increase in (Ca2+)i.
通过一种新方法研究了生理浓度肾上腺素（Ep）对血小板活化的影响，该方法可通过光透射变化同时测量血小板聚集，并利用光散射对各种大小的颗粒进行计数。利用共聚焦激光扫描显微镜（CLSM）研究了与活化过程相关的详细形态学变化。对应于高生理浓度的低浓度Ep（20 nmol/L）触发了小血小板聚集体（直径7至30微米）的形成，而光透射没有任何变化。在构成小聚集体的血小板中也观察到了丝状肌动蛋白（F-肌动蛋白）的重新分布以及通过PAC-1结合检测到的活化糖蛋白IIb-IIIa复合物（GPIIb-IIIa）的表达。然后尝试通过使用Ca2+指示染料fluo-3的CLSM检测参与聚集体形成的单个血小板中细胞质游离Ca2+（（Ca2+）i）的变化。Ep在一些参与小聚集体形成的单个血小板中引起了微弱的（Ca2+）i升高。这种（Ca2+）i升高与血小板聚集相关，因为在单个血小板中未检测到（Ca2+）i升高。此外，在存在RGDS或GPIIb-IIIa拮抗剂Ro 44-9883的情况下，受Ep刺激的血小板未形成小聚集体或触发（Ca2+）i升高。预先用低浓度的Ep（20、100 nmol/L）孵育可增强由亚阈值浓度的二磷酸腺苷（0.25 μmol/L）诱导的小（直径7至30微米）、中（直径30至50微米）或大（直径50至70微米）聚集体的初始形成，这是通过颗粒计数法确定的。然而，在参与聚集体形成的血小板中未观察到明显的协同（Ca2+）i升高。从这些观察结果得出以下结论。（1）高生理浓度的Ep（20 nmol/L）能够触发小聚集体的形成，导致F-肌动蛋白的重新分布和活化GPIIb-IIIa复合物的表达。（2）在构成小聚集体的血小板中观察到（Ca2+）i升高。这种升高与Ep与其受体的结合无关，但很可能是由血小板-血小板相互作用触发的。（3）Ep的特征性增强作用不是由于（Ca2+）i的协同升高。
