Figure 1.

Filamin A (FLNA) is required for platelet δ- and α-granule secretion. (A) Equal amounts of platelet lysate from control and platelet-specific conditional Flna-knockout (KO) mice were resolved by SDS-PAGE and immunoblotted to confirm the knockout of FLNA in the platelets. Beta-tubulin is shown as a loading control. (B–D) Platelets were isolated from whole blood of control mice (red bars) and platelet-specific Flna-KO mice (platelet-specific KO, blue bars). Washed platelets (2 × 10⁸/mL) were stimulated at 37°C for 6 minutes with (B) thrombin, (C) collagen-related peptide (1 μg/mL), or (D) the thromboxane mimetic U46619 (1 μM) plus 10 μM ADP. Bar graphs represent the resultant ATP secretion, as measured based on luminescence using the Chronolume luciferase reagent. Data are presented as mean ± SEM (∗∗∗P < .001, based either on the Student’s t-test or analysis of variance and Bonferroni multiple comparison tests, as appropriate) and represent 4 independent experiments. (E–G) Bar graphs depict the release of platelet factor 4 (aka CXCL4) from control (red bars) platelets and FLNA-null (platelet-specific KO, blue bars) mice in response to (E) thrombin, (F) collagen-related peptide (1 μg/mL), or (G) U46619 (1 μM) plus 10 μM adenosine diphosphate. Data are presented as mean ± SEM (∗P < .05, ∗∗∗P < .001, ∗∗∗∗P < .0001, based either on the Student’s t-test or analysis of variance and Bonferroni multiple comparison tests, as appropriate) and represent 4 independent experiments. (H) The bar graph depicts the total platelet factor 4 content in control (red bars) platelets and FLNA-null (platelet-specific KO, blue bars) platelets based on enzyme-linked immunosorbent assay of platelet lysates. Data are presented as mean ± SEM (not significant, based on the Student’s t-test). ADP, adenosine diphosphate; CRP, collagen-related peptide; ns, not significant.