Filamin A regulates caspase-3 cleavage in platelets in a protein kinase C (PKC)-dependent manner

Apoptosis is a critical process for the maintenance of cell populations, and involves mito-chondrial depolarization, the sequential cleavage of caspase-9 and-3, followed by the externalization of phosphatidylserine (PS) on the plasma membrane. The actin cytoskel-eton and its accessory proteins are known regulators of apoptotic signaling in nucleated cells but their roles in platelet apoptosis are undefined. Filamin A (FLNA) is a ubiquitously expressed actin-crosslinking protein that also serves as an intracellular signaling scaffold. Here we used platelets from mice with a platelet-specific FLNA deficiency (Flnafl/Y, Pf4-cre/+, termed platelet-specific knockout) to test the role of FLNA in platelet apoptosis. Treatment with the BH3-mimetic drug ABT-737 induced caspase-3 cleavage and PS exposure in platelets from floxed mice (Flnafl/Y, termed control) but these effects were essentially abrogated in FLNA-null platelets (platelet-specific knockout). Protein kinase C (PKC), a known FLNA ligand, was also activated by ABT-737, and PKC's phosphorylation of its downstream substrates was attenuated in FLNA-null platelets. The PKC inhibitor bisindolylmaleimide (BIM) also reduced caspase-3 cleavage, thus essentially phenocopy-ing the FLNA-null platelets. Notably, the caspase-3 cleavage defect in FLNA-null platelets was rescued by the PKC-activating phorbol ester PMA, suggesting that FLNA and PKC share a common pathway in regulating platelet apoptosis. Mitochondrial depolarization and caspase-9 cleavage were unaffected by BIM treatment, suggesting that PKC specif-ically controls the downstream caspase-3 point of the pro-apoptotic signaling pathway. These data point to a novel role for FLNA in the regulation of platelet apoptosis, thus pro-viding an improved understanding of how circulating platelet counts are maintained.