PB0415 - The role of filamin A in platelet thromboxane A2 secretion

Background: TXA2 (thromboxane A2) is an eicosanoid lipid secreted by activated platelets that serves as a critical mediator of hemostasis. Ligation of platelet protease-activated receptors (PARs) by thrombin triggers the activation of the mitogen-activated protein kinase (MAPK) signaling cascade, notably the MEK/ERK and the p38 MAPK pathways. MAPK activation in turn leads to the generation of TXA2. Filamin A (FLNA) is an actin-binding protein that also serves as a critical intracellular signaling scaffold but the role of FLNA in platelet MAPK activation and TXA2 generation is unknown.

Aims: To determine the role of FLNA in (1) MAPK activation and (2) TXA2 secretion in thrombin-activated platelets.

Methods: Since FLNA-knockout mice do not survive birth, conditional knockout (KO) mice were generated using the Cre/lox system in which FLNA is specifically deleted in the megakaryocyte/platelet cell lineage. Control (floxed) and FLNA-null (conditional KO) platelets were isolated from whole blood and washed by centrifugation. Resting platelets were stimulated with thrombin. Following isolation of the releasate fraction (supernatant), the secretion of TXA2 in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA). In some experiments, the platelets were solubilized in lysis buffer; the phosphorylation of ERK1/2 and p38 in the control and FLNA-KO platelets was determined by Western blotting.

Results: Control (floxed) platelets exhibited a marked increase in ERK1/2 and p38 phosphorylation. However, this increase in ERK1/2 and p38 phosphorylation was not observed in FLNA-deficient platelets. Moreover, control (floxed) platelets showed a ~15-fold increase in TXA2 generation following thrombin stimulation. In contrast, FLNA-deficient platelets failed to generate TXA2 in response to thrombin.

Conclusion(s): FLNA regulates thrombin-induced TXA2 generation by modulating the activation of MAPK signaling in platelets.