Fig 1: Platelet procoagulant membrane dynamics analysis reveals IL-4 and TNF-β as platelet primers and activators, respectively. Whole blood was preincubated/treated (30 min) with increasing concentrations of IL-4 (0, 50, 100 and 300 pg/mL) or TNF-β (0, 1, 3, 9, 30,100 and 300 pg/mL). PRP was isolated by centrifugation at 180 g for 17 min. To visualise platelets, detect degranulation, phosphatidylserine exposure and integrin αIIbβ3 activation, PRP was labelled with immunofluorescent dyes: Alexa Fluor 405 GP1b, Alexa Fluor 488 anti-human CD62P (P-selectin), Alexa Fluor 568 Annexin-V and Alexa Fluor 647 anti-human CD41/CD61 (PAC-1). Plasma platelets were allowed to adhere to glass-bottom dishes coated with BSA or fibrillar collagen. (A) A box plot summarising the quantified mean fluorescent intensity of P-selectin, Annexin-V and PAC-1 on platelet membranes. (B) Representative confocal images of unstimulated platelets treated with 50 pg/mL and 300 pg/mL of IL-4 (two leftmost columns). The next two columns show untreated, then treated (50 pg/mL of IL-4) collagen-activated platelets, respectively. (C) Confocal images of platelets treated with 0, 3 and 300 pg/mL of TNF-β and allowed to adhere to BSA-coated dishes. (D) Box plots of mean P-selectin, Annexin-V and PAC-1 intensity from TNF-β treated platelets under BSA. Box plots show the minimum to maximum values. The medians and interquartile data ranges are represented by the horizontal line through the box and the height of the box, respectively. Data and statistical analysis were performed using GraphPad Prism V.10.4 (San Diego, California, USA), and one-way ANOVA and Bonferroni post hoc test determined statistical significance. p<0.05 (*) or p<0.01 (**) were considered significant. Images were taken using a Nikon A1R laser scanning confocal microscope (objective magnification, (×60). Image analysis was conducted using Volocity software (Quorum Technologies, Canada). The data is representative of 10 healthy participants. ANOVA, analysis of variance; BSA, bovine serum albumin; IL, interleukin; TNF, tumour necrosis factor; PRP, platelet-rich plasma.