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D. Vara et al.
reducing collagen-dependent aggregation and O2•− forma- tion, in both human (Figure 3A and B) and mouse (Figure 5A) platelets. The secondary role of NOX2 in collagen responses is further demonstrated by thrombus formation experiments under physiological flow, which is marginally but significantly reduced by NOX2 inhibition/silencing in human but not mouse blood (compare Figure 4D and Figure 5B). This discrepancy between thrombus formation in human and mouse blood could be suggestive of a higher involvement of NOX2 in collagen responses of human platelets. In any case, the dominant role of NOX1 in colla- gen response is clear in our experiments both in human and mouse platelets. Similarly, NOX2 is essential for the signal- ing of thrombin without NOX1 involvement in human platelets (Figure 2C and D) and limited yet significant involvement in mouse platelets (Figure 5C). This is in agree- ment with previous indications from our and other groups,14,40 but in essential disagreement with a recent study by Delaney et al. in mouse platelets reaching opposite con- clusions (i.e. NOX1 involvement in thrombin signaling and NOX2 involvement in collagen signaling).13 Some differ- ences can be expected between signaling pathways and redox dependence of human and mouse platelets, which may explain this discrepancy. Overall, combining human and mouse platelet data, it is safe to state that thrombin- dependent aggregation depends very heavily on NOX2 activity (although a role of NOX1 in thrombin was detected in mouse platelets, which we did not observe in human platelets), while collagen-induced aggregation was predom- inantly NOX1-dependent with marginal involvement of NOX2 (as shown in our human platelet data). So, although mouse platelets display some engagement of NOX1 in thrombin responses, species-specific differences cannot fully explain the discrepancy between our report and Delaney et al.’s work. The fact that Delaney et al. used male animals for NOX1 studies and females for NOX2 studies may explain some of the differences with our study (per- formed entirely on female animals). Other potentially important differences are in the platelet isolation procedure, the concentration of agonists (very low concentration of thrombin used), and the use of collagen-related peptide instead of collagen.
In addition to physiological stimuli, in this study we ana- lyzed the effect of oxLDL and Aβ1-42, platelet modulators associated with the thrombotic complications of athero- sclerosis41 and cerebrovascular amyloid angiopathy (CAA),42 respectively. Both oxLDL and Aβ1-42 have been shown to activate platelets and act as positive modula- tors.15,43 We confirmed the ability of these molecules to induce partial platelet aggregation. On the other hand, they were able to significantly increase the responses to low lev- els of physiological agonists. This mode of action is consis- tent with the definition of positive modulators or primers,44 which are characterized by the ability to trigger an unwant- ed hemostatic response and clot formation leading to thrombosis. The involvement of platelet positive modula- tors in thrombotic complications associated with diseases is particularly important for vascular health and relative phar- macotherapy.45 Interestingly, the aggregation induced by these primers was also redox-dependent and inhibited by the O2•− scavenger PEG-SOD. This is in agreement with previous literature on oxLDL15,46 and Aβ1-42,47 and sup- ports the hypothesis that platelet primers may act in a redox-dependent manner.48
Also intriguing were our conclusions regarding the involvement of NOX1 and NOX2 in the signaling of oxLDL and Aβ1-42. We provide compelling evidence in human platelets with NOX-selective inhibitors Nox2ds- tat and NoxA1ds or in genetically modified mouse platelets (NOX1-/- or NOX2-/-) that both NOX1 and NOX2 are activated by oxLDL and Aβ1-42 and that they are both required for the functional effects of these pathology-asso- ciated modulators on platelets. These conclusions were confirmed by experiments with transgenic mouse platelets, both in thrombus adhesion and aggregation experiments. Only adhesion to Aβ1-42 under low shear seemed exclusively NOX1-dependent. This is a low level adhesion response incapable of properly triggering throm- bus formation (Online Supplementary Figure S11). These data may, therefore, suggest a differential involvement of NOX1 and NOX2 in different molecular events triggered by Aβ1-42. This hypothesis merits further study for satis- factory elucidation. The involvement of NOX2 in the sig- naling of oxLDL has been suggested previously,15,46 while the involvement of NOX1 is novel. The possibility of abolishing the effect of oxLDL and Aβ1-42 on platelets by inhibiting only one of the two NOX may suggest that both enzymes are required for reaching a threshold in the superoxide anion levels leading to platelet stimulation. The similarities between the activity and redox-depen- dence of oxLDL and Aβ1-42 may suggest that they act on similar receptors. As suggested by literature for both mol- ecules, the receptors for these platelet modulators are like- ly to be CD36.15,49 Importantly, the fact that both platelet NOX are required for the modulatory activity of oxLDL or Aβ1-42 offers the opportunity of targeted intervention without the complete inhibition of the response to physi- ological agonists. In other words, the inhibition of only one NOX isozyme could reduce the pro-thrombotic ten- dencies associated with vascular inflammation without completely impairing the hemostatic response (i.e. NOX1 inhibition will not impair thrombin response, while NOX2 inhibition will not impair collagen responses). This is the ultimate goal of modern antiplatelet drug develop- ment and may help to resolve the persisting problem of bleeding risks associated with all existing antiplatelet treatment.50
In summary, herein we describe the development and application of a novel approach to simultaneously monitor the generation of oxygen radicals and platelet aggregation. This technique has the potential to become a standard tech- nique to assess platelet responsiveness and thrombotic risks associated to vascular conditions in a clinical setting. In addition, this study allowed the identification of patterns of platelet regulation and the clarification of the mode of action of physiological agonists and pathological modula- tors of platelets. The results of this study also highlight the potential of NADPH oxidase targeting for the development of novel antiplatelet drugs with better pharmacodynamic profiles (i.e. limited bleeding side effects).
Acknowledgments
The authors would like to thank the British Heart Foundation for sponsoring this research (PG/15/40/31522), the Clinical Research Facility (CRF) of the University of Exeter and in particular Dr Bridget Knight for blood collections and Dr Bruno Fink from Noxygen Science Transfer & Diagnostics GmbH for the technical support.
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haematologica | 2019; 104(9)