To the editor:

Recently, several studies have been published in Blood that have examined the effect of Bcl-xL–inhibitory BH3 mimetics on platelet function and lifespan.1-3  These reports agree that 2 related BH3 mimetics, ABT-737 and ABT-263, induce platelet apoptosis. This is characterized by caspase-3 activation and caspase-dependent phosphatidylserine externalization, and may underlie the thrombocytopenia observed during administration of these drugs.2,4  However, in addition, the study by Vogler et al made 2 specific observations regarding the effect of BH3 mimetics on platelet calcium signaling: first, that they induce a transient calcium signal; and second, that prolonged treatment with BH3 mimetics depletes intracellular calcium stores.1  In contrast, Schoenwaelder and Jackson, in correspondence to the editor, dispute these observations.5 

We have also investigated the effect of ABT-737 on platelet calcium signaling. In agreement with Vogler et al, we find that ABT-737 (10μM) can induce a calcium signal (Figure 1A). However, the signal that we detect is small relative to that induced by thrombin. The maximum increase in cytosolic calcium concentration above basal was estimated as ∼ 50nM, and was variable between platelets from different donors. For clarity, we haveexpanded this section of the trace to show the ABT-737–induced calcium signal. This small signal might not be sufficient to directly activate platelets, although it could reflect a larger but more local subcellular signal, or a subpopulation of platelets that respond differently compared with most platelets.

Figure 1

Effect of ABT-737 on platelet calcium signaling. (A) Washed platelets loaded with Fura-2 (1 × 108/mL) were treated with ABT-737 (10μM; gray trace) or DMSO (black trace) in the presence of extracellular CaCl2 (1mM), followed by stimulation with thrombin (0.1 U/mL). The initial section of the traces are expanded and separated below to show the small ABT-737–induced signal. Fluorescence was calibrated in terms of cytosolic calcium concentration to estimate the magnitude of the response.6  (B) Platelets in modified Tyrode buffer were treated with ABT-737 (10μM) or DMSO or for the indicated time then treated with EGTA (1.2mM) followed by ionomycin (Iono; 1μM) to artificially deplete calcium stores. Traces are representative of at least 3 independent experiments. All experiments were performed at 37°C under stirring conditions.

Figure 1

Effect of ABT-737 on platelet calcium signaling. (A) Washed platelets loaded with Fura-2 (1 × 108/mL) were treated with ABT-737 (10μM; gray trace) or DMSO (black trace) in the presence of extracellular CaCl2 (1mM), followed by stimulation with thrombin (0.1 U/mL). The initial section of the traces are expanded and separated below to show the small ABT-737–induced signal. Fluorescence was calibrated in terms of cytosolic calcium concentration to estimate the magnitude of the response.6  (B) Platelets in modified Tyrode buffer were treated with ABT-737 (10μM) or DMSO or for the indicated time then treated with EGTA (1.2mM) followed by ionomycin (Iono; 1μM) to artificially deplete calcium stores. Traces are representative of at least 3 independent experiments. All experiments were performed at 37°C under stirring conditions.

Close modal

We also found that incubation with ABT-737 induced calcium store depletion. However, this was not a rapid event, but rather required prolonged incubation with ABT-737. To assess Ca2+ store content, we treated platelets with 10μM ABT-737 for either a short time (5 minutes) or a long time (120 minutes). The extracellular Ca2+ was then chelated by EGTA (1.2mM) and ionomycin (1μM) added to artificially release the intracellular calcium stores (Figure 1B). Brief treatment with ABT-737 (5 minutes) did not substantially affect the ionomycin-induced Ca2+ signal, indicating that the filling state of the intracellular Ca2+ stores was unaffected. This is consistent with the observations reported by Schoenwaelder and Jackson.5  However, ionomycin-induced Ca2+ release was substantially reduced after longer treatment with ABT-737. This suggests that the ionomycin-sensitive intracellular Ca2+ stores are slowly depleted after inhibition of Bcl-xL, consistent with the data of Vogler et al.1 

Taken together, our data indicate that BH3 mimetics such as ABT-737 modify intracellular Ca2+ handling in platelets. Whether this contributes to platelet apoptosis and BH3 mimetic–induced thrombocytopenia requires further investigation.

Acknowledgments: This work was supported by The British Heart Foundation (RG/10/006/28299).

Contribution: M.T.H. designed and performed experiments, analyzed data, and wrote the paper; and A.W.P. designed research and wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Matthew T. Harper, University of Bristol School of Physiology and Pharmacology, Medical Sciences Building, University Walk, Bristol, BS8 1TD, United Kingdom; e-mail: [email protected].

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