Figure 1.

Platelet CLEC-2 delays inflammatory BMDM phagocytic capacity and reduces TNF-α secretion (A–C) pH sensitive Alexa Fluor-488 conjugated Escherichia Coli bioparticles (3x10⁶ beads/condition) were added to LPS-treated BMDM (Mϕ) in the absence or presence of (A) WT platelets or (B) CLEC-2-deficient platelets (100 platelets: 1 Mϕ) for 4h. (A, B) Phagocytosis was visualised and quantified by time lapse-imaging using an Incucyte Live-cell analysis system. (C) Phagocytosis profiles were quantified at 4h by detecting fluorescence/mm³ using area under the curve (AUC; a.u. = arbitrary units; n=3). (D) TNF-α and (E) IL-10 secretion from control Mϕ or in the presence of WT or CLEC-2-deficient platelets was quantified in the supernatant by ELISA (n=4). (F–H) Scratch wound migration of Mϕ was monitored every 2h for 96h using an Incucyte ZOOM system. Following wound scratch, (F) WT or CLEC-2-deficient platelets (100 platelets:1 Mϕ) were added to Mϕ. (G) Wound closure was quantified as percentage of closure using ImageJ. (H) Total wound closure was quantified by AUC at 96h (n=3). The statistical significance between 2 groups was analyzed using a student’s paired t-test and the statistical difference between multiple groups using one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05 **p < 0.01. NS, non-stimulated.