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O.V. Kim et al.
increased fluorescence observed after treatment of platelets with A23187, used as a positive control. In accor- dance with the flow cytometry data, the time-lapse fluo- rescent confocal microscopy of PRP clots pre-incubated for 90 min with a calpain substrate also revealed activation of platelet calpains in response to thrombin. Specifically, fluorescence of the calpain cleavage product detected in platelets 90 min after thrombin treatment was 6.5-fold higher than that detected in untreated platelets (Figure 8B).
Remarkably, the rate of enzymatic reaction calculated as the first derivative of the dynamic fluorescence signal reached a maximum (the highest calpain activity) after 35 min of thrombin-induced platelet activation, coinciding with a decrease in the mitochondrial membrane potential (Figure 8C) and the beginning of platelet fragmentation (Figure 8D).
Accordingly, pre-treatment of platelets with a calpain inhibitor ALLN prior to platelet activation resulted in a 3-
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Figure 7. The lack of caspase 3/7 activity in thrombin-stimulated platelets. (A, B) Representative confocal micrographs of activated platelets pre-incubated with a fluorogenic substrate of caspases 3 and 7. Platelets were visualized after activation with thrombin (A) or the calcium ionophore A23187 (B, positive control) in platelet-rich plasma (PRP) clots in the presence of Ca2+. Scale bars = 4 μm. (C) Quantitative estimation of the dynamic caspase-3/7 activity in platelets based on confocal microscopy (mean ± standard deviation, n=3). P<0.01 for the difference between thrombin-treated platelets and the positive control (two-tailed Mann- Whitney U test). (D-F) Representative flow cytometry dot plots of isolated platelets analyzed for caspase-3/7 activity with a fluorogenic substrate after incubation with thrombin (D), the calcium ionophore A23187 (E) and without treatment (F). The inset boxed numbers (mean ± standard deviation) indicate the average caspase-3/7- positive fractions of platelets stimulated with thrombin (D) and A23187 (E) and untreated platelets (F) (n=3, P<0.0001). (G) Western blot analysis of caspase-3 cleav- age in platelet lysates obtained from control untreated platelets (Ctrl), platelets treated with thrombin (Thr) and platelets treated with the calcium ionophore A23187 used as a positive control. No caspase-3/7 activation was revealed in platelets treated with 1 U/mL thrombin at 37oC either in PRP (A-C) or Tyrode buffer (D-G) in the presence of Ca2+.
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haematologica | 2019; 104(9)