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D. Vara et al.
stimulus, while the potentiation of thrombin responses could not be tested in NOX2-/-, for the same reason.
Discussion
The study of platelet function is critical to understanding vascular homeostasis and disease. A thorough understand- ing of the redox-dependent regulation of platelet function has been hampered by the lack of a reliable technique to measure intracellular ROS.16,17 We have resolved this prob- lem by optimizing an EPR-based technique for the detec- tion of oxygen radicals and combining it with a classical tur- bidimetric assay for the simultaneous measurement of platelet aggregation. Although EPR has previously been used for the analysis of ROS formation in live cells;16,17 this technique has never been applied to the study of platelet redox signaling. We utilized the assay developed here to
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clarify several unknown aspects of the regulation of platelet activity by endogenous oxidants.
The molecular mechanisms underlying the redox dependence of platelet activation in response to collagen, thrombin, and oxidized LDL are summarized in Online Supplementary Figure S12. Although, the dependence of platelet activation on the generation of endogenous ROS has been described,1-6 here for the first time we elucidated the chemical nature of the ROS involved in the signaling of different platelet agonists and modulators. O2•− are generat- ed in response to all tested agonists and modulators, but although these ROS are directly involved in the signaling of collagen, oxLDL and Aβ1-42, their dismutation to hydrogen peroxide is necessary for the signaling of thrombin. The lit- erature on this aspect of platelet biology is quite inconclu- sive because of the use of different techniques and condi- tions. Although previous studies reported the generation of hydrogen peroxide in response to thrombin (and leading to
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C
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 intracar- diac puncture and resuspended at 2x108 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 plat- form (Fluxion, San Francisco, CA, USA) was utilized to assess the thrombus for- mation induced by collagen under phys- iological flow (1,000 sec-1). Images were taken at 10 min of flow and are representative of 4 independent experi- ments. They were quantified by assess- ing the surface area coverage by platelets with Image J. Data are repre- sentative of 4 independent experi- ments. Statistical analysis was per- formed by one-way ANOVA with Bonferroni post-hoc test. *P<0.05. N=4 for (A-C).
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haematologica | 2019; 104(9)