Fig 2: Inhibition of Wnt/β-catenin signaling enhanced platelet binding to TNF-α-stimulated endothelial cells. Cells were treated with either 0.05% DMSO vehicle control or 25 μM inhibitor of β-catenin-responsive transcription (iCRT) in the presence or absence of 10 ng/mL recombinant human TNF-α stimulus for 18 h. BCECF-AM-labeled, thrombin-activated platelets were co-cultured with human umbilical vein endothelial cells (HUVECs) (A) and HCAECs (B) for 10 min, bound platelets lysed, and the fluorescent signal quantified. Data are expressed as optical density (n = 8 and 4, respectively). (C) Representative Western blots for integrins αv and β3 in whole cell lysates, and vWF and ADAMTS13 in conditioned media, and stain-free controls in HUVECs. (D) Segments of human saphenous vein were co-cultured with BCECF-AM-labeled, thrombin-activated platelets for 10 min, and the number of bound platelets quantified (n = 4). (E) In HUVECs, integrin αv from whole cell lysates was quantified by Western blotting, normalized to stain-free controls and expressed as a fold change from control (n = 4). Quantification and representative images of immunofluorescence (green) for vWF in permeabilised (F,G) and non-permeabilised (H,I) HUVECs, to detect intracellular and membrane-tethered vWF, respectively (n = 5 and 4, respectively). Nuclei were stained with DAPI (blue). Scale bars represent 10 μm and apply to all panels. From cultured HUVECs, ADAMTS13 (J), and soluble vWF (K) in conditioned culture medium were quantified by Western blotting, normalized to stain-free controls and expressed as a fold change from control (n = 7 and 3, respectively). *Indicates p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. NS denotes not significant.

Fig 3: Final PLS-SEM model incorporating the following latent constructs: NETs, VWF, cardiac dysfunction, overall leukocyte levels, blood glucose, discharge NIHSS, vitamin B12. Overall leukocyte levels are effectively represented by neutrophils, lymphocytes and eosinophils. Blood glucose is represented by fasting blood glucose and random blood glucose. Cardiac dysfunction is represented by CKMB and Myoglobin. VWF and NETs are represented by the AOD measured in five different fields of view. PLS-SEM defines the relationships between latent variables and observed variables and among latent variables in the form of a path model. The directions of the explained paths are indicated by connecting arrows. Solid lines denote statistically significant paths, whereas dashed lines denote non-significant paths. Numbers on the arrows correspond to path coefficients, indicating the strength of each association. VWF, Von Willebrand factor; NETs, neutrophil extracellular traps; Hcy, Homocysteine; NIHSS, National Institutes of Health Stroke Scale.

Fig 4: Immunohistochemical analysis of thrombi specimens. Representative images showing VWF and NETs staining in thrombi of different origins. These images illustrate the presence and spatial distribution of the two markers within thrombus sections. Immunohistochemically positive areas are shown in brownish-yellow. (A) Represents NETs, (B) represents VWF. 400x magnification.