Involvement of platelet CD47 in the adhesion of resting platelets on the inflammatory vascular endothelium under flow conditions. EA endothelial cells were grown onto permanox Lab-Tek 1 chamber slides and stimulated or not stimulated with 25 ng/mL rhTNF-α for 18 hours. Then the slides were placed in a parallel plate flow chamber that produces a linear variable shear rate. (A) Human platelets were labeled with calceine in the PRP and were incubated or not incubated with 40 μg/mL anti-CD47 mAb B6H12 for 10 minutes at 37°C. Whole blood was reconstituted and perfused through the chamber at 37°C. Single-frame images were obtained after perfusion for 3 minutes at a shear rate of 100 seconds–1 on EA cells treated or not treated with TNF-α. Platelet adhesion, expressed as percentage of surface covered with platelets, is the average ± SEM of 10 random fields per coverslip. ***P < .001. These experiments are representative of at least 8 experiments performed using blood from different donors. For each single-frame image shown in the panels, the magnification of the objective was × 10. (B) Mouse platelets from 10 wild-type or 10 CD47–/– C57BL/6 mice were labeled with DiOC6 in the PRP (see “Materials and methods”). Whole blood was reconstituted and perfused through the chamber at 37°C. Single-frame images were obtained after perfusion for 3 minutes at a shear rate of 100 seconds–1 on EA cells treated or not treated with TNF-α. Platelet adhesion, expressed as percentage of surface covered with platelets, is the average ± SEM of 10 random fields per coverslip. ***P < .001. These experiments are representative of 4 different experiments. For each single-frame image shown in the panels, the magnification of the objective was × 10.
