PB0424 - The critical role of filamin A in the platelet shape change reaction

Background: Platelets circulate in the blood as quiescent disks that change shape upon activation by soluble agonists or vascular injury. The shape change reaction is therefore a critical aspect of hemostasis and is orchestrated by a dynamic remodeling of the platelet’s actin cytoskeleton. Upon platelet activation, the actin filaments interact with myosin; this involves phosphorylation of the myosin light chain (MLC) protein. This actin-myosin interaction triggers the contraction of the actin cytoskeleton, which initiates the platelet shape change reaction. Filamin A (FLNA) is an actin crosslinking protein that stabilizes the attachment between the subcortical actin filaments and the cell membrane. In addition, FLNA binds multiple proteins and serves as a critical intracellular signaling scaffold. However, the role of FLNA in relation to MLC phosphorylation (and the platelet shape change reaction) is not defined.

Aims: To determine the role of FLNA in modulating MLC phosphorylation in thrombin-activated platelets.

Methods: Since the deletion of FLNA in mice is embryonic lethal, we used conditional knockout (cKO) mice in which FLNA is specifically deleted in the megakaryocyte/platelet cell lineage. Platelets were then isolated from control (floxed) mice and conditional KO (cKO) mice to characterize the role of FLNA in MLC phosphorylation and platelet shape change. Thrombin-induced phosphorylation of MLC was determined by immunoblotting; shape change was assessed by assays of platelet spreading.

Results: Following activation by thrombin, control (floxed) platelets exhibited an increase in MLC phosphorylation; this response was attenuated in FLNA cKO platelets. In addition, platelet spreading was perturbed in FLNA cKO platelets relative to controls. FLNA-deficient platelets also showed increased myosin light chain phosphatase (MLCP) activity relative to controls, suggesting that FLNA regulates MLC phosphorylation by constraining phosphatase activity.

Conclusion(s): FLNA is required for normal MLC phosphorylation and shape change in thrombin-stimulated platelets.