Fig 2: The interaction of zyxin with VASP is required for zyxin-mediated GPIbα expression and platelet production.A Cartoons for the constructs of zyxin mutants lacking α-actinin and NMMHC-IIA (Zyx43–564), and VASP (Zyx4F > A) binding sites. NES, nuclear export signals. B Zyx−/− FL HPCs were infected with adenoviruses expressing WT zyxin (Zyx1–564), zyxin mutants Zyx43-564 and Zyx4F > A, and vehicle vector (control). The expression levels of WT zyxin and zyxin mutants were determined by Western blot with an anti-flag tag antibody. The green fluorescence (GFP) indicates the introduction of the plasmids into HPC-derived MKs. Blots are representative of five independent experiments. C–F The expression of CD41 (C) and GPIbα (D) on culture-derived MKs was determined on day 4 by flow cytometry. The presence of CD41+ (E) and GPIbα+ (F) platelet-sized particles released from culture-derived MKs was determined on day 5 by flow cytometry. FC, fold-change. Data are from five independent experiments, and means are indicated by horizontal lines in (B–F). *P < 0.05, **P < 0.01, compared with control by one-way ANOVA followed by Dunnett’s post hoc test. NS, not significant. G Representative fluorescent microscopy images of proplatelet formation from five independent experiments (original magnification ×200). Scale bar: 40 μm. Proplatelet protrusions were indicated with arrows.

Fig 3: Summary results of immunohistochemical staining of CD41, TGF-β1, NK1R, α-SMA, collagen I, as well as Masson trichrome staining in endometriotic lesions from 3 groups of mice. Boxplots showing the density of CD41 + platelets (A), lesional staining of TGF-β1 (B), NK1R (C), α-SMA (D), and collagen I (E), and the extent of lesional fibrosis as evaluated by Masson trichrome staining (F). Linear regression analysis was used, with the TMP concentration as the co-variable. The red arrow indicates the linear trend of concentration dependency. Symbols of statistical significance levels: ***: p < 0.001. Untreated: mice receiving no TMP treatment; L-TMP: mice receiving low-dose TMP treatment; H-TMP: mice receiving high-dose TMP treatment

Fig 4: Platelets accumulate in response to demyelination.(A) LPC induced demyelinating lesions in spinal cord white matter of WT mice at 1, 3, 7, and 14 dpl, stained for platelets (CD41+). Scale bar 100 μm. (B) Quantification of CD41+ signal within the demyelinated lesion at 1 (n=6), 3 (n=5), 5 (n=5), 7 (n=6), 10 (n=4), and 14 dpl (n=4), and in NAWM (n=3). (C) Platelet staining (CD41+) in spinal cord white matter injected with PBS/DAPI. Scale bar 50 μm. (D) Upper left panel: localization of platelets within blood vessels (ColIV+) and in close proximity with OPCs (Olig2+) at 5 dpl. Upper right panel: IMARIS 3D projection shows the spatial distribution of platelets. Scale bar 10 μm. Lower panels: magnification of the IMARIS projection showing platelet aggregation within the blood (left panel) and penetration into the parenchyma (right panel). Scale bars: 5 μm (left panel) and 7 μm (right panel). Data were analysed using a Kruskal Wallis test. Data represent the mean ± SD. ** p<0.01; ns (not significant), p>0.05.

Fig 5: Histologic Changes Produced by AD13 TreatmentHistology from the aortic root at the level of the aortic sinuses in LDL-R−/− on WSD at 30 weeks of age comparing untreated and AD13-treated (2 weeks by osmotic minipump), include quantitative measurement of plaque area on Masson trichrome staining (A), plaque macrophage area by Mac-2 staining (B), plaque collagen area by Masson trichrome (C), and platelet number by CD41 staining (D) seen per aortic root section. Values are mean ± SEM. ∗p < 0.05 versus WT and LDL-R−/− by unpaired Student's t-tests. n = 6 for each animal group. (E) Examples illustrating Masson trichrome (left), fluorescent immunohistochemistry for Mac-2 (center), and CD41 for platelets (right) are shown for comparison to Figure 4. Bars = 150 μm. Abbreviations as in Figures 1, 3, and 4.