Platelets promote inflammation in inflamed vessels. (A) Vessel permeability increases during inflammation. Vessels were cultured against Geltrex mix (i) or collagen I (ii) for 1 week and then stimulated overnight with 10 ng/mL TNF-α + 10 ng/mL IL-1β. FITC-dextran (250 kDa) was added for 45 minutes and then fluorescence imaged on an EVOS M5000. Dextran leakage was calculated by dividing the fluorescence in the ECM channel by the fluorescence in the vessel channel. (B) Platelets and neutrophils enhance permeability in inflamed Geltrex mix vessels. Plts and neuts were isolated from human blood and resuspended in EGM-FCS. They were then perfused through the vessels for 1.5 hours. FITC-dextran (250 kDa) was added as described in panel A. Dextran leakage was normalized to the media only control. (C) Platelets enhance neutrophil transmigration. Vessels were fixed, blocked, and stained with Hoechst-33342 and α-CD31. Z-stacks were taken on a Leica SP5 confocal microscope. Number of neutrophils transmigrated was analyzed using the Cell Counter plug in in FIJI. N = 5 independent experiments/blood donors (N = 4 in panel Cii), n = 2 chips/condition. Mean ± SEM; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001; ns, nonsignificant.