Adhesion of platelets to arterial endothelial cells under physiologic flow conditions. (A) Arterial endothelial cells were allowed to grow to confluence in μ-slides and stimulated (closed bars) or not (open bars) with TNF-α and IFN-γ for 20 hours. Human blood platelets in reconstituted blood were perfused at the indicated wall shear rate as described in Figure 1. The experiment was performed 3 times, and results presented here are derived from one representative experiment and are presented as mean ± SEM for that experiment performed in pentaplicate. (B) Endothelial cells were preincubated with vehicle alone (closed bars), a neutralizing anti-FKN antibody (open bars), or isotype-matched control (hatched bars) as indicated for 2 hours before perfusion initiation. Shown is the percentage of platelets adhering to stimulated endothelial cells at the indicated shear rate as percentage of the number of platelets adhering to resting endothelial cells with the respective treatments (dotted line). The results are the mean ± SEM of 3 independent experiments with blood from different donors.*P < .05 in one sample t test versus 100%, which represents the adhesion rate of platelets to resting endothelial cells. (C) Adhesion of platelets to the endothelium of intact human arteries. Blood was perfused at a wall shear rate of 600 s−1 over human artery, and the number of adherent platelets after 15-minute perfusion was recorded. Platelet adhesion rates to unstimulated arteries were set to 100% (dotted line). Shown are the relative adhesion rates to TNF-α- and IFN-γ-stimulated arteries with and without a neutralizing anti-FKN mAb. The results are the mean ± SEM of 3 independent experiments with blood and arteries from different donors.*P < .05 in one sample t test versus 100%, which represents the adhesion rate of platelets to resting arteries. (D) Immunohistochemical detection of FKN in sections of human internal mammary arteries. Representative photomicrographs of the endothelium immunostained for FKN (brown staining) in resting (left panel) and TNF-α- and IFN-γ-stimulated (middle panel) artery segments, counterstained blue with hematoxylin. Also shown is immunohistochemical detection of VWF (right panel). Bar represents 200 μm in the upper row and 50 μm in the lower row. Images were recorded with a Zeiss AxioObserver Z1 using a 5×/0.16 NA (top row) and a 20×/0.8 NA objective (bottom row) with an AxioCam MR3 camera (Zeiss) and processed in AxioVision LE 4.8.2.0 and ImageJ 1.45d software.
