Effect of low concentration of epinephrine on human platelet aggregation analyzed by particle counting method and confocal microscopy

Abstract

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 μm) without any change in light transmission. The redistribution of filamentous actin (F-actin) and the expression of activated glycoprotein llb-Illa complex (GPllb-llla), 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 Ca 2+ ((Ca 2+) i) in individual platelets involved in the aggregate formation by CLSM with fluo-3, a Ca 2+-indicating dye. Ep caused a weak (Ca 2+) i increase in some individual platelets involved in the formation of small aggregates. This (Ca 2+) i increase was associated with platelet aggregation, because no (Ca 2+) i rise was detected in single platelets. Furthermore, platelets stimulated by Ep in the presence of RGDS or Ro 44-9883, a GPllb-llla antagonist, did not form small aggregates or trigger a (Ca 2+) i rise. Prior incubation with low concentrations of Ep (20, 100 nmol/L) enhanced the initial formation of small (diameter 7 to 30 μm), medium (diameter 30 to 50 μm), or large (diameter 50 to 70 μm) aggregates induced by a subthreshold concentration of adenosine diphosphate (0.25 μmol/L) as determined by the particle counting method. However, no apparent synergistic (Ca 2+) 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 GPllb-llla complex. (2) An increase in (Ca 2+) 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 (Ca 2+) i.