Figure 1. Effect of GPN on agonist-dependent Ca²⁺ release from intracellular stores.

Resting human platelets (A) or platelets incubated for 10 min at 37 °C in the presence of 50 μM GPN (B) were loaded with Lysosensor Green DND-189 to stain the acidic organelles and fluorescence was detected using a confocal microscope as described in the Materials and methods section. Images shown are representative of six separate experiments. (C) Human platelets were loaded either with Lysosensor Green DND-189 (thick line) or with fura 2 (thin line) and then treated with 50 μM GPN as indicated in a Ca²⁺-free medium. Fluorescence was detected using a fluorescence spectrophotometer as described in the Materials and methods section. Results shown are representative of four to six experiments, which, in the case of loading with Lysosensor Green, were also used to obtain the images shown in (A) and (B). (D–G) fura 2-loaded human platelets were incubated for 10 min at 37 °C in the presence of 50 μM GPN or the vehicle (DMSO as control). At the time of experiment 100 μM EGTA was added. Cells were then stimulated with TBHQ (20 μM; D), thrombin (0.1 unit/ml; E), ADP (10 μM; F), or AVP (0.1 μM; G). Changes in [Ca²⁺]_c were monitored as described in the Materials and methods section and traces are representative of four to six independent experiments.