Figure 5.

NOX1-and NOX2-dependence of collagen and thrombin aggregation and superoxide generation tested in transgenic mice. Platelets were isolated for wild-type (C57BL6/J), NOX1^−/− or NOX2^−/− mice exsanguinated via intracardiac puncture and resuspended at 2×10⁸ platelets/mL density. Platelets were stimulated with 3 μg/mL collagen (A) or 0.1 unit/mL thrombin (C). Aggregation (left) and superoxide anion formation (right) were measured as described for 5 and 10 minutes (min), respectively. (B) The functional role of NOX1 and NOX2 in collagen-dependent platelet activation was also assessed in a whole blood flow assay. Platelets were stained with DiOC6 and the Bioflux platform (Fluxion, San Francisco, CA, USA) was utilized to assess the thrombus formation induced by collagen under physiological flow (1,000 sec-1). Images were taken at 10 min of flow and are representative of 4 independent experiments. They were quantified by assessing the surface area coverage by platelets with Image J. Data are representative of 4 independent experiments. Statistical analysis was performed by one-way ANOVA with Bonferroni post-hoc test. *P<0.05. N=4 for (A-C).