Bcl-x_L–inhibitory BH3 mimetic ABT-737 depletes platelet calcium stores

To the editor:

Recently, several studies have been published in Blood that have examined the effect of Bcl-x_L–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.

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-x_L, 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.