Thymosin β4 is essential for thrombus formation by controlling the G-actin/F-actin equilibrium in platelets

Abstract

Coordinated rearrangements of the actin cytoskeleton are pivotal for platelet biogenesis from megakaryocytes but also orchestrate key functions of peripheral platelets in hemostasis and thrombosis, such as granule release, the formation of filopodia and lamellipodia, or clot retraction. Along with profilin (Pfn) 1, thymosin β4 (encoded by Tmsb4x) is one of the two main G-actin-sequestering proteins within cells of higher eukaryotes, and its intracellular concentration is particularly high in cells that rapidly respond to external signals by increased motility, such as platelets. Here, we analyzed constitutive Tmsb4x knockout (KO) mice to investigate the functional role of the protein in platelet production and function. Thymosin β4 deficiency resulted in a macrothrombocytopenia with only mildly increased platelet volume and an unaltered platelet life span. Megakaryocyte numbers in the bone marrow and spleen were unaltered, however, Tmsb4x KO megakaryocytes showed defective proplatelet formation in vitro and in vivo. Thymosin β4-deficient platelets displayed markedly decreased G-actin levels and concomitantly increased F-actin levels resulting in accelerated spreading on fibrinogen and clot retraction. Moreover, Tmsb4x KO platelets showed activation defects and an impaired immunoreceptor tyrosine-based activation motif (ITAM) signaling downstream of the activating collagen receptor glycoprotein VI. These defects translated into impaired aggregate formation under flow, protection from occlusive arterial thrombus formation in vivo and increased tail bleeding times. In summary, these findings point to a critical role of thymosin β4 for actin dynamics during platelet biogenesis, platelet activation downstream of glycoprotein VI and thrombus stability.

Figure 1.

Thrombocytopenia and impaired proplatelet formation in thymosin β4 knockout mice. (A) Protein levels of thymosin β4 and integrin β1 in wild-type (WT) and Tmsb4x knockout (KO) platelets were analyzed by an automated quantitative capillary-based immunoassay platform; Jess (ProteinSimple). Platelet count (B) and volume (C) were determined using an automated blood cell analyzer (ScilVet). Mean ± standard deviation (SD) (n= 4, 3 independent experiments). Unpaired, two-tailed Student’s t-test. ***P<0.001. (D) Representative transmission electron microscopic images of 1 WT mouse and 3 Tmsb4x KO mice: (2) platelets comparable to WT size, (3) big roundish platelets, (4) platelets with increased size. Scale bars: 2 mm. (E and F) Hematoxylin-eosin stainings of femur paraffin sections of WT and Tmsb4x KO mice (E) and quantification of megakaryocyte (MK) numbers (F). Arrow heads indicate the MK. Scale bars: 100 mm. Values are mean ± SD (n=3). (G and H) Proplatelet formation of bone marrow MK after lineage depletion and culturing in rHirudin- and TPO-conditioned medium. On day 4, proplatelet-forming MK were counted. Average of 5 analyzed visual fields per MK culture of 3 animals/genotype is shown. Values are mean ± SD. Unpaired, two-tailed Student’s t-test. *P<0.05. (I) Proplatelets were visualized using an α-tubulin antibody and phalloidin and analyzed by confocal microscopy (40x objective, Leica TCS SP8) using a 40x objective. Scale bar: 20 mm.

Figure 2.

Impaired actin equilibrium and assembly in thymosin β4 knockout platelets. (A and B) Relative F-actin content of resting and activated platelets was determined by flow cytometry. Values are mean ± standard deviation (SD) of 4 mice per group. The values are displayed as the ratio of MFI: mean fluorescence intensity from activated and resting platelets. Unpaired, two-tailed Student’s t-test. *P<0.05, **P<0.005, ***P<0.001. (C and D) The actin cytoskeleton was isolated by ultracentrifugation, immunoblotted with an anti-β-actin antibody and analyzed for the content of monomeric vs. filamentous actin using densitometry. GAPDH served as loading control. Values are mean ± SD (n=3). P: pellet, S: supernatant, T: total protein. Unpaired, two-tailed Student’s t-test. **P<0.005. (E and F) Visualization of the cytoskeleton of spread platelets (15 minutes) on fibrinogen, which were stained with DNase I-AlexaF488 (green) to label G-actin and Phalloidin-atto647N (red) for visualization of F-actin and analyzed by confocal microscopy. Scale bar: 20 mm. Values are mean ± SD (n=3). Unpaired, two-tailed Student’s t-test. **P<0.005.

Figure 3.

Accelerated spreading of thymosin β4 knockout platelets. (A and B) Washed platelets were stimulated with 0.01 U mL thrombin and allowed to spread (5, 15, 30 minutes [min]) on fibrinogen (100 mg mL^-1 ). DIC pictures were taken (A) and phase abundance was determined (B). Images are representatives of at least 6 animals per group. Scale bar: 3 mm. (C and D) Representative images of the platelet cytoskeleton ultrastructure of wild-type (WT) and Tmsb4x knockout (KO) mice on fibrinogen after 5 min (C) and 15 min (D). Scale bar: 500 nm.

Figure 4.

Accelerated clot retraction of thymosin β4 knockout platelets. (A) Clot retraction of wild-type (WT) and Tmsb4x knockout (KO) platelet-rich plasma (PRP) was determined in response to 4 U mL^-1 thrombin and monitored over time. (B) Residual volume at the end of the experiment. Values are mean ± standard deviation (SD) (n=6 per group). Unpaired, two-tailed Student’s t-test. *P<0.05. (C) Analysis of the fibrin meshwork of WT and Tmsb4x KO clots. Washed platelets were labeled with an anti-GPIX Alexa 647 derivative and added to a mix of unlabeled fibrinogen (2 mg mL^-1) and Alexa Fluor A488-labeled fibrinogen (50 mg mL^-1 f.c.). Platelets were stimulated with 0.1 U mL^-1 thrombin and clotting was initiated by addition of 5 mM CaCl₂. The mixture was immediately transferred to an uncoated 8-well chamber slide (Ibidi), and allowed to clot. Images were obtained using a Leica SP8 inverted microscope with a 63x oil immersion lens. Optical z-stacks were deconvolved and are shown as maximum projection. Images are representatives of at least 2 z-stacks per mouse and 4 animals per group.

Figure 5.

Altered αIIbβ3 integrin activation, degranulation and aggregation of thymosin β4 knockout platelets. (A and B) Activation of platelet αIIbβ3 integrin (JON/A-PE) (A) and degranulation (α-P-selectin-FITC) (B) in wild-type (WT) and Tmsb4x knockout (KO) platelets upon stimulation with the indicated agonists was determined by flow cytometry (n=12). U46: U46619; CRP: collagen-related peptide; Rhd: rhodocytin. Unpaired, two-tailed Student’s t-test. *P<0.05, **P<0.005. (C) Dense granule secretion was assessed by luminometric measurement of released ATP of activated WT and Tmsb4x KO platelets. Results are given as mean ATP concentration [mM] ± standard deviation (SD) (n=12 per group). Unpaired, two-tailed Student’s t-test. *P<0.05, **P<0.005, ***P<0.001. (D) Aggregation responses of washed platelets or platelet-rich plasma (PRP) in turbidometric aggregometry (n=6). (E) Western blot analysis of phosphotyrosine levels in resting and CVX-stimulated WT and Tmsb4x KO platelets using the 4G10 antibody. GAPDH served as loading control. CVX: convulxin; K: Tb4^-/-; W: WT. (F and G) Phosphorylation and total protein levels of Syk in resting and CVX-stimulated WT and Tmsb4x KO platelets were analyzed (E) and quantified (F) by an automated quantitative capillary-based immunoassay platform. Values are mean ± SD (n = 3). Unpaired, two-tailed Student’s t-test. *P<0.05, **P<0.005, ***P<0.001. MFI: mean fluorescence intensity.

Figure 6.

Thymosin |34 is required for thrombus formation and stability in vitro and in vivo. (A-C) Assessment of platelet adhesion (A, B) and aggregate formation (A, C) on Horm collagen (200 mg mL^-1) under flow (150, 1,000 and 1,700 s-) in heparinized whole blood or platelet-count adjusted blood of WT and Tmsb4x knockout (KO) mice. Values are mean ± standard deviation (SD) (n = 12). Scale bar, 50 mm. (D) Representative graph of blood flow of one WT and two Tmsb4x KO mice after mechanical injury of the abdominal aorta. (E) Occlusion times after mechanical injury of the abdominal aorta. Data are mean ± SD of at least 8 mice per group. Fisher’s exact test. **P<0.005. (F) Tail bleeding times in WT and Tmsb4x KO mice (filter paper method). Each symbol represents one individual. Unpaired, two-tailed Student’s t-test. **P<0.005. plt.: platelet.