Fig 2: Immunohistochemical analysis of platelet interaction with neurovascular niche cellular components in the brain of APP-PS1 mice. Brain sections from APP-PS1 mice (n = 3, including two females and one male) were co-stained for platelets (CD41), (A–E, green), and one of the following cell types: astrocytes (GFAP) (A, white); pericytes (PDGFRβ) (B, red), microglia (Iba-1) (C, red), neurons (NeuN) (D, red), or oligodendrocytes (Oligo2) (E, red). DAPI (blue) was used as nucleus stain. Representative confocal images were selected for 3D surface projection and co-localization analysis. In APP-PS1 mice, platelets were found in tight association with astrocytes (A). Co-localization of CD41 and GFAP signals is depicted in magenta (A, zoom in). No further interactions were observed between CD41+ platelets and other immunolabeled cells (B). Scale: 5 μm (A,B), 2 μm (B—zoom in).

Fig 3: Normal MK differentiation but impaired maturation, DMS development, and proplatelet formation in Zyx-/- MKs.A MK counts in mouse BM and spleen. Left panels, representative confocal images of MKs in femoral BM and spleen sections immunostained with anti-CD41 antibody. MKs (CD41) are in green, and nuclei are in blue. Original magnification × 200 for BM sections and × 100 for spleen sections. Scale bar: 100 µm. Right panel, the quantification of MK numbers per visual field; n = 15 visual fields from five mice per genotype. Data are expressed as means ± SD. B Percentage of CD41+ cells differentiated from WT and Zyx-/- FL HPCs on day 4 was analyzed by flow cytometry. Data are from five independent experiments. C Upper panels, representative microscopy images of MKs in BM smears stained with Wright–Giemsa stain from WT and Zyx-/- mice (original magnification × 400). Scale bar: 20 µm. Lower panel, the quantification of different stages of MKs using BM smears from WT (n = 4) and Zyx-/- mice (n = 7). No less than 100 MKs were analyzed for each mouse. D Representative TEM images of MKs isolated from WT and Zyx-/- mouse BM (original magnification, ×3,400 for left panels, and ×13,500 for right panels). Images were obtained from five mice in each genotype. Scale bar: 1 µm. E Percentage of CD41+ platelet-sized particles released from culture-derived MKs was analyzed by flow cytometry. Data are from four independent experiments. F Representative microscopy images of proplatelet formation from culture-derived MKs (E) on day 4 (original magnification ×200). Scale bar, 20 µm. Proplatelet protrusions were indicated with arrows. Means are indicated by horizontal lines in (B), (C), and (E). **P < 0.01, ***P < 0.001, compared with WT mice by unpaired Student’s t-test in (A) and (B), and by two-way ANOVA followed by Bonferroni’s post hoc test in (C) and (E). NS, not significant.

Fig 4: PSL2 is an adhesion receptor.(A) Activated T cells expressing PSGL1 and/or PSL2. P-selectin binding profiles of activated T cells generated using donor/recipient combinations specified expressing PSGL1 and PSL2, PSL2 alone, or neither. Staining with P-selectin was performed on untreated cells or on cells after EDTA pre-washing revealing the independent contributions of PSGL1 (EDTA-resistant) and PSL2 (EDTA-strippable). Independent triplicate staining profiles are shown. Fig 4A is representative of five independent analyses. (B) Activated platelets expressing P-selectin can bind activated T cells via PSGL1 and/or PSL2. Platelets from P-selectin+/+ and P-selectin-/- mice were isolated, labeled with anti-CD41-APC, activated with thrombin, paraformaldehyde fixed, and washed. Cells shown in (A) were then assessed for capacity to bind CD41-APC labeled platelets by flow cytometry. Free platelets were excluded from analysis by forward/side light scatter characteristics. Platelets from P-selectin-/- mice were assayed in parallel to resolve P-selectin dependent platelet adhesion to responding T cells. Expected P-selectin ligand displayed in parentheses. Mean and standard deviation of percentage CD41-APC+ (platelet bound) cells indicated for P-selectin+/+ platelets and P-selectin-/- platelets. P-values for differences in P-selectin+/+ platelet binding determined by Student’s T test, with confidence levels reaching 2, 3 or 4 SD as indicated by **,***,**** respectively. Fig 4B is representative five independent analyses. (C) PSL2 supports adhesive interaction with immobilized P-selectin. Tracking dye-labeled donor T cells responding in OT1? PSGLnull, OT1? C2GnT1null, OT1 C2GnT1null? PSGLnull, and OT1 C2GnT1null ? C2GnT1null adoptive transfers yielded activated cells expressing PSGL1 and PSL2, PSGL1, PSL2, or neither ligand respectively. These cells were compared for adhesion to P-selectin immobilized on V-bottom wells under shear generated by centrifugation. Wells were coated with blocking agent alone (no P-selectin) or with PSel-hIgG chimera (immobilized P-selectin). Cells were also centrifuged in wells pre-coated with P-selectin but in the presence of EDTA to prevent selectin mediated binding (immobilized P-selectin + EDTA). Non-adherent cells pelleting to the nadir were harvested directly and cells adhering to the tapered well sides were subsequently harvested using EDTA elution (Adherent). Donor derived activated T cells in non-adherent vs adherent fractions were quantified by flow cytometry-based counting and each normalized to input cell numbers. Triplicate counts of pooled quadruplicate samples were used to generate mean and standard deviation values of cell yields as shown. The significance of differences in cell yields were determined by applying two-tailed Student’s T-tests with equal variance. P-values reaching 1–5 sigma levels of significance were indicated by *, **, ***, ****, ***** respectively. The results shown are representative of four independent analyses.

Fig 5: Histamine ablation facilitates thrombocytopoiesis and granulopoiesis after AMI. A, Fraction of HDC-expressing cells in the bone marrow. B, Histamine level in the serum (Bld) and bone marrow (BM) lavage fluid. C-G, Flow cytometry analysis of haematopoietic stem and progenitor cells (HSPCs) in the bone marrow. C, Gating strategy of long-term HSCs (LT-HSCs, CD48-CD150+Lin-Sca-1+C-kit+ [LSK]), short-term HSCs (ST-HSCs, CD48+CD150+LSK) and multipotent progenitors (MPPs, CD48+CD150-LSK). D, Fraction of LT-HSCs, ST-HSCs and MPPs in LSK. E, Gating strategy of common myeloid progenitors (CMPs, CD34+Fc?R-Lin-IL-7Ra-Sca-1-C-kit+ [LK]), granulocyte-macrophage progenitor (GMP, CD34+Fc?R+LK), megakaryocyte-erythroid progenitors (MEPs, CD34-Fc?R-LK) and megakaryocyte progenitor cell-like cells (MkPLCs, CD41+CD34-Fc?R-LK). Fraction of (F) CMPs, GMPs, MPPs and (G) MkPLCs in LK. H, Fraction of neutrophils in the bone marrow and peripheral blood. I, Representative images of Wright-Giemsa staining. Arrowheads show platelets (white, scattered platelets, and red, highly concentrated platelets). Arrows indicate neutrophils (white, mature neutrophils, and red, immature neutrophils). Bar: 20 µm. Graphs show mean ± SEM. P-values were determined by one-way ANOVA with Bonferroni-Dunn correction