Filamin A regulates platelet shape change and contractile force generation via phosphorylation of the myosin light chain

Abstract

Platelets are critical mediators of hemostasis and thrombosis. Platelets circulate as discs in their resting form but change shape rapidly upon activation by vascular damage and/or soluble agonists such as thrombin. Platelet shape change is driven by a dynamic remodeling of the actin cytoskeleton. Actin filaments interact with the protein myosin, which is phosphorylated on the myosin light chain (MLC) upon platelet activation. Actin-myosin interactions trigger contraction of the actin cytoskeleton, which drives platelet spreading and contractile force generation. Filamin A (FLNA) is an actin cross-linking protein that stabilizes the attachment between subcortical actin filaments and the cell membrane. In addition, FLNA binds multiple proteins and serves as a critical intracellular signaling scaffold. Here, we used platelets from mice with a megakaryocyte/platelet-specific deletion of FLNA to investigate the role of FLNA in regulating platelet shape change. Relative to controls, FLNA-null platelets exhibited defects in stress fiber formation, contractile force generation, and MLC phosphorylation in response to thrombin stimulation. Blockade of Rho kinase (ROCK) and protein kinase C (PKC) with the inhibitors Y27632 and bisindolylmaleimide (BIM), respectively, also attenuated MLC phosphorylation; our data further indicate that ROCK and PKC promote MLC phosphorylation through independent pathways. Notably, the activity of both ROCK and PKC was diminished in the FLNA-deficient platelets. We conclude that FLNA regulates thrombin-induced MLC phosphorylation and platelet contraction, in a ROCK- and PKC-dependent manner.

Keywords: Filamin A; cytoskeleton; myosin; phosphorylation; platelets; protease-activated receptor signaling.

Figure 1.. FLNA-deficient platelets have diminished stress fiber formation compared to control platelets.

(A) Lysates from floxed (control) FLNA-deficient (platelet-specific KO) platelets were resolved by SDS–PAGE and probed with an anti-FLNA antibody. Beta-actin is shown as a loading control. (B and C) Floxed (control, B) and FLNA-deficient (platelet-specific KO, C) Platelets were allowed to spread on fibrinogen-coated coverslips for 45 min prior to labeling with Alexa-488-phalloidin. Insets: higher magnification images of the floxed (control) and FLNA-deficient (platelet-specific KO) allowed to spread for 45 min on immobilized fibrinogen. (D) Bar graph depicts the quantification of the presence of visible stress fibers in floxed (control, blue bar) and FLNA-deficient (platelet-specific KO, pink bar) platelets. Calculations were based on the mean percentage (%) of stress-fiber positive platelets per field of view. Data are mean ± SEM and represent three independent experiments. *P < 0.05, based on Student's t-test.

Figure 2.. Reduced contractile force generation by FLNA-deficient platelets.

(A) Floxed (control) and FLNA-deficient (platelet-specific KO) platelets were seeded onto the black dots and labeled with phalloidin (top panels; middle panels depict the cells on the black dots). The bottom panels depict the traction forces exerted by individual platelets (cyan arrows) that were calculated based on the displacement of the black dots. (B) Dot-plot depicts the forces generated by control platelets (blue, n = 251 at 20 min, n = 270 at 45 min) and FLNA-deficient platelets (platelet-specific KO, red, n = 242 at 20 min, n = 283 at 45 min). Gray dots represent all of the individual platelets measured. *P < 0.05; **P < 0.01; ****P < 0.0001, based on one-way ANOVA and Sidak's multiple comparisons tests. Data were obtained from six independent experiments. (C) Line graph depicts linear regression analysis of force generation by floxed (control, blue line) and FLNA-deficient (platelet-specific KO, red line) platelets. ****P < 0.001. (D) Photographs depict fibrin clot retraction by floxed (control) and FLNA-deficient (platelet-specific KO) platelets in fibrinogen (1 mg/ml), stimulated with thrombin (1 U/ml) after 45 min. (E) Bar graph depicts clot retraction by floxed platelets (control, blue bar) and FLNA-deficient platelets (platelet-specific KO, pink bar), based on the percentage (%) of the retracted clot area. Data are mean ± SEM and represent three independent experiments. ***P < 0.001, based on Student's t-test.

Figure 3.. FLNA-null platelets exhibit diminished RhoA activation and MLC phosphorylation compared to control platelets.

(A) RhoA activation in response to thrombin stimulation was measured in floxed (control, blue bars) platelets and FLNA-deficient (platelet-specific KO, pink bars) platelets. The level of active, GTP-bound RhoA was measured using a G-LISA activation assay, and represented as relative fold change. Data are mean ± SEM and represent three independent experiments. ***P < 0.001, based on two-way ANOVA and Tukey post hoc multiple comparison tests. (B) Immunoblot depicts MLC phosphorylation at Ser19 in control platelets (control) and FLNA-null platelets (platelet-specific KO) after thrombin treatment for the indicated times. Total MLC is shown as a loading control. (C) Bar graph represents the densitometry quantification of phospho-MLC2 (Ser19) relative to total MLC protein in floxed platelets (control, blue bars) and FLNA-deficient platelets (platelet-specific KO, pink bars). Data are mean ± SEM, analyzed by two-way ANOVA and Tukey post hoc multiple comparison tests, and represent a minimum of three independent experiments. ****P < 0.0001, based on two-way ANOVA and Tukey post hoc multiple comparison tests. (D) Immunoblot depicts MYPT1 phosphorylated at Thr853 in floxed platelets (control) and FLNA-null platelets (platelet-specific KO) after thrombin treatment for the indicated times. Total MYPT1 is shown as a loading control. (E) Bar graph represents the densitometry quantification of phospho-MYPT1 (Thr853) relative to total MYPT1 in floxed platelets (control, blue bars) and FLNA-null platelets (platelet-specific KO, pink bars). Data are mean ± SEM, analyzed by two-way ANOVA and Tukey post hoc multiple comparison tests, and represent a minimum of three independent experiments. ***P < 0.001, ****P < 0.0001, based on two-way ANOVA and Tukey post hoc multiple comparison tests. NT, no treatment.

Figure 4.. PKC and ROCK regulate MLC phosphorylation.

(A) Immunoblot depicts MLC phosphorylation at Ser19 in wild-type mouse platelets pre-incubated with vehicle, the PKC inhibitor BIM (15 µM), and/or the ROCK inhibitor Y27632 (50 µM) for 30 min prior to stimulation with thrombin (0.1 U/ml) for 10 min. Total MLC is shown as a loading control. (B) Bar graph represents the densitometric quantification of p-MLC2 (Ser19) relative to total MLC in thrombin-activated platelets pre-treated with vehicle (TR, orange), BIM (BIM, green bar), Y27632 (Y27632, blue bar), or BIM plus Y27632 (BIM + Y27632, purple bar). Resting platelets serve as the negative control (NT, red bar). Data are mean ± SEM and represent a minimum of four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, based on one-way ANOVA and Tukey post hoc multiple comparison tests. (C) Immunoblot depicts MYPT1 phosphorylation at Thr853 in wild-type mouse platelets pre-incubated with vehicle, the PKC inhibitor BIM (15 µM), and/or the ROCK inhibitor Y27632 (50 µM) for 30 min prior to stimulation with thrombin (0.1 U/ml) for 10 min. Total MYPT1 is shown as a loading control. (D) Bar graph represents the densitometric quantification of p-MYPT1 (Thr853) relative to total MYPT1 in thrombin-activated platelets pre-treated with vehicle (TR, orange), BIM (BIM, green bar), Y27632 (Y27632, blue bar), or BIM plus Y27632 (BIM + Y27632, purple bar). Resting platelets serve as the negative control (NT, red bar). Data are mean ± SEM and represent a minimum of four independent experiments. *P < 0.05, ****P < 0.001, based on one-way ANOVA and Tukey post hoc multiple comparison tests.

Figure 5.. FLNA regulates the function of PKC in platelets.

(A) Immunoblot depicts phospho-serine PKC substrates in floxed platelets (control) and FLNA-null platelets (platelet-specific KO) after treatment with thrombin or the phorbol ester PMA for the times indicated. Beta-actin is shown as a loading control. (B) Bar graphs depict the densitometric quantification of phospho-serine PKC substrates relative to beta-actin in floxed (control, blue bars) and FLNA-null platelets (platelet-specific KO, pink bars). Data are mean ± SEM, and represent a minimum of three independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001, based on two-way ANOVA and Tukey post hoc multiple comparison tests. (C) Immunoblot depicts MLC phosphorylation at Ser19 in floxed platelets (control) and FLNA-null platelets (platelet-specific KO) after treatment with thrombin and/or PMA for 10 min. Total MLC is shown as the loading control. (D) Bar graph depicts the densitometric quantification of phospho-MLC2 (Ser19) relative to total MLC in floxed (control, blue bars) and FLNA-null platelets (platelet-specific KO, pink bars). Platelets were untreated (NT), treated with thrombin (TR), treated with PMA (PMA), or treated with thrombin and PMA (TR + PMA). Data are mean ± SEM and represent a minimum of five independent experiments. *P < 0.05, ****P < 0.0001, based on two-way ANOVA and Tukey post hoc multiple comparison tests.

Figure 6.. Proposed model of FLNA as a regulator of MLC phosphorylation.

Schematic diagram illustrating a proposed model in which FLNA regulates MLC phosphorylation and cell contraction in platelets. (A) FLNA acts as a major scaffolding protein for both RhoA and ROCK to facilitate the phosphorylation (and therefore inhibition) of MLC phosphatase after thrombin activation. (B) FLNA modulates the activity of PKC which also contributes to MLC phosphorylation.