FIGURE 3.

Activation of iNOS-dependent NO production by platelet agonists. A and B, equal numbers of washed mouse platelets isolated from wild-type mice (WT) or iNOS^–/– mice were stimulated with 0.1 unit/ml thrombin. NO release was measured in real time with a porphyrinic microsensor. Shown are representative traces (A) and quantitative data from four experiments (B). C, platelet lysates from wild-type (WT), iNOS knockout (iNOS^–/–), or eNOS knock-out (eNOS^–/–) mice were incubated with ADP-agarose beads. The bead-bound proteins were then immunoblotted with a monoclonal anti-eNOS antibody as described (13). To confirm that equal numbers of platelets were used in the assay, platelet lysates were also immunoblotted with an anti-integrin β3 antibody. D, washed mouse platelets isolated from wild-type (WT) or iNOS^–/– mice were stimulated with or without 0.1 unit/ml thrombin. iNOS was then detected by Western blotting as described under “Experimental Procedures.” Equal loading was monitored by immunoblotting with an anti-tubulin antibody. E, Western blotting results from each of three experiments as shown in D were scanned and quantitated using National Institutes of Health ImageJ for uncalibrated optical density. The ratio of optical density of iNOS band in thrombin-stimulated wild-type platelets to that of resting wild-type platelets is expressed as percentage of wild type.