Mice from each group or human platelets (normal or glycoprotein (GP)* IIb-IIIa deficient; reference 6 (link)) were labeled with CFSE (DCF). The DCF-labeled platelet suspension was adjusted to a final concentration of 50 × 106 platelets per 250 μl and infused intravenously. Where indicated, fluorescent wild-type platelets were preincubated with 50 μg/ml anti-GPIbα (p0p/B-Fab) or anti-GPIIb-IIIa (JON/A-F(ab)2 (14 (link)) for 10 min before infusion. Leukocytes were stained in vivo by intravenous injection of 100 μl 0.02% rhodamine 6G (Molecular Probes). Subsequently, the carotid artery was visualized at low (250-fold) magnification using a Zeiss Axiotech microscope (water immersion objective: 20×, W 20×/0.5; Zeiss) with a 100 W HBO mercury lamp for epi-illumination. As illustrated in Fig. 1 A, two perpendicular axes were dropped through the origin of the internal and the external common carotid artery. A third line was drawn connecting the two perpendicular axes, where they crossed the vessel wall. Platelet and leukocyte adhesion were determined at high magnification (500-fold) in a 200 μm × 100 μm area adjacent to the third line. This implicates that platelet recruitment was assessed exactly at the carotid bifurcation, which is a predilection site for the development of atherosclerotic lesions.
All images were videotaped and evaluated off-line using a computer-assisted image analysis program (Cap Image 7.1; Dr. H. Zeintl, Ingenieurbüro Dr. Zeintl, Heidelberg, Germany) (references 5 (link) and 6 (link)). Transiently adherent platelets were defined as cells crossing an imaginary perpendicular through the vessel at a velocity significantly lower than the centerline velocity; their numbers are given as cells per mm2 endothelial surface. The number of adherent cells (leukocytes or platelets) was assessed by counting the cells that did not move or detach from the endothelial surface within 20 s.
In a subset of experiments, we determined whether or not platelet adhesion is restricted to lesion-prone sites within the carotid bifurcation or rather occurs throughout the entire carotid artery. To address this issue, platelet adhesion was analyzed at the carotid bifurcation (lesion-prone site) and in the proximal portion of the common carotid artery, ∼500 μm upstream of the carotid bifurcation (nonlesion-prone site; see Fig. 1 A) in young (10 wk, n = 8) and old ApoE−/− mice (n = 8, 22 wk).