Fig. 7.

Increasing platelet [Zn ²⁺ ] _i using Zn ²⁺ ionophores increases platelet activation markers. ( A ) Washed platelet suspensions were stimulated by thrombin (Thr, 1 U/mL), clioquinol (Cq) (300 µM), pyrithione (Py) (300 µM) or A23187 (100 µM) and changes of PAC-1 (white), CD62P (grey) and CD63 (black) binding were obtained after 60 minutes. ( B ) Washed platelet suspensions were stimulated by CRP-XL (1 µg/mL), U46619 (10 µM) or thrombin (1 U/mL), following pre-treatment with TPEN (50 µM), and changes of PAC-1 (white), CD62P (grey) and CD63 (black) binding were obtained after 60 minutes. ( C ) Washed platelet suspensions were treated with Ca ²⁺ or Zn ²⁺ ionophores, or conventional platelet agonists, prior to analysis of annexin-V binding by flow cytometry. □ Clioquinol (300 µM), ▵ pyrithione (300 µM), ○ A23187 (300 µM), • CRP (1 µg/mL), ▪ thrombin, (1 U/mL), ▪ vehicle (DMSO). ( D ) Platelet suspensions were pre-treated with the caspase inhibitor Z-VAD (▵, 1 µM), the Zn ²⁺ chelator, TPEN (▪, 25 µm) or vehicle (□) prior to stimulation with clioquinol (300 µM). ○ Unstimulated platelets. Changes in the percentage of platelets binding to annexin-V were recorded. Washed platelets suspensions were pre-treated with Z-VAD (1 µM), or TPEN (50 µM) prior to stimulation with conventional agonists, CRP-XL (1 µg/mL, E ), U46619 (10 µM, F ) or thrombin (1 U/mL, G ). Changes in annexin-V binding were monitored using flow cytometry. ○ Vehicle, □ Z-VAD (1 µM), ▵ TPEN (50 µM), ▿ DMSO (no agonist). Data are means ± standard error of the mean (SEM) of at least 3 independent experiments. Significance is denoted as *** p  < 0.001, ** p  < 0.01 or * p  < 0.05.