Agonist-dependent increase in platelet adhesion, aggregation, and ATP release in exposed animals. (A-F) Platelets were isolated from both control and hypoxia-exposed rats, fluorescently labeled, and allowed to adhere to collagen or fibrinogen precoated plates for up to 60 minutes at 37°C. (A,D) Representative images of fluorescence microscopy-based platelet adhesion on type 1 collagen-coated (10 µM) and fibrinogen-coated (20 µM) plates, using fluorogenic dye calcein. Images were captured using a fluorescein isothiocyanate filter on a Motic Inverted Microscope AE31(×200 original magnification). (B-C,E-F) Quantitation of platelet adhesion data was expressed as average area or size of adhered platelet clumps and percentage area covered by adhered platelets on the collagen-coated plate (top) and the fibrinogen-coated plate (bottom) after a 60-minute incubation. Quantitation was performed by Motic ImagePlus 2.0 software. (G) For the platelet aggregation assay, platelet-rich plasma from rats of indicated groups, incubated at 37°C for at least 3 minutes, was induced by ADP with stirring at 1200 rpm and optically monitored. The rate and extent of ADP-induced platelet aggregation was higher in hypoxia-exposed animals compared with control animals. Representative aggregation curves are shown in response to ADP (2.5 and 5 µM). (H) A bar graph shows aggregation results expressed as maximal amplitude of aggregation. (I) Representative ATP release curve in response to ADP analyzed using a luciferase assay. (J) Quantitation of aggregation and ATP release were expressed as maximum amplitude. Data are presented (mean ± SEM) as average results of at least 3 independent experiments (n = 6). *P < .05, **P < .01 vs control. See the supplemental Methods for details.