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D. Vara et al.
by EPR, we utilized cell-permeant pegylated superoxide dismutase (PEG-SOD), which scavenges O2•− by dismuting it into H202. The EPR signal detected in human platelets stimulated by collagen and thrombin was abolished by PEG-SOD, suggesting that O2•− is the oxygen radical species generated under these conditions (Online Supplementary Figure S4A and C, respectively). The genera- tion of O2•− radicals appeared necessary for platelet activa- tion by collagen, as the scavenging of this oxygen radical by PEG-SOD significantly inhibited aggregation in response to this agonist (Online Supplementary Figure S4B). On the other hand, thrombin-dependent aggregation was not affected by PEG-SOD, suggesting that O2•− is not an essential component of the signaling cascades induced by this agonist. Similarly to collagen, both EPR and aggrega- tion signals in response to CRP were abolished by PEG- SOD (Online Supplementary Figure S3C and D). The data obtained with PEG-SOD were confirmed using another O2•− scavenger, 4-hydroxy-2,2,6,6-tetramethylpiperidin-1- oxyl (or TEMPOL). TEMPOL abolishes the EPR signal in response to either collagen or thrombin (Online Supplementary Figure S5A and C, respectively), while only the aggregation response to collagen but not thrombin (Online Supplementary Figure S5C and D, respectively) is inhibited. Interestingly, the non-selective antioxidant and cell permeable amino acid N-acetyl cysteine (NAC) inhibits aggregation induced by both collagen and thrombin, sug- gesting that the thrombin responses depend on oxidative reactions, but that O2•−, per se, is not the oxidant species involved in this response (Online Supplementary Figure S5C and D). In order to confirm that the CMH-based EPR did not detect H202 (another major ROS) in our hands, we per- formed experiments in the presence of catalase (CAT, non- cell permeable) and pegylated catalase (PEG-CAT, cell per- meable), which convert H202 to water (Online Supplementary Figure S6). No effect of either enzymes on the collagen- or thrombin-induced EPR signal was observed, proving the specificity of the CMH-based EPR measurements for oxygen radicals and O2•− in particular.
Low levels of superoxide anion are released by platelets although they are not required for platelet activation
We demonstrated that the CMH-based EPR spectroscopy predominantly measures intracellular but not extracellular platelet O2•− in response to either collagen or thrombin (Online Supplementary Figure S7), as the non-pegylated and thus non-cell permeable version of the enzyme superoxide dismutase (SOD) did not affect the responses. Interestingly, we also utilized a non-cell permeable EPR probe called 1- Hydroxy-4-phosphono-oxy-2,2,6,6-tetramethylpiperidine (or PPH) to detect extracellular O2•− released by platelets (Online Supplementary Figure S8A-D). Noticeably, the scav- enging of extracellular O2•− with SOD was effective in abol- ishing the PPH-based signal (Online Supplementary Figure S8E and F), but did not affect collagen- or thrombin-induced aggregation (Online Supplementary Figure S8G and H), sug- gesting that extracellular O2- does not participate in the process of platelet activation. In addition, we confirmed the formation of H202 in response to collagen or thrombin using the H2O2-specific probe Amplex Red (Online Supplementary Figure S9A and B) and that catalase effectively quenched the signal. More importantly, the degradation of H202 by cata- lase or pegylated catalase inhibited platelet aggregation stimulated by thrombin, but not collagen (Online Supplementary Figure S9C-F). This suggests that H202 is a crit-
ical component of the signaling triggered by thrombin but not collagen.
Differential role of NADPH oxidases 1 and 2 in collagen- and thrombin-dependent activation of human platelets
We utilized the combinatorial EPR/aggregometry that we developed to assess the role of NOX1 and NOX2 in platelet activation by collagen and thrombin. In experi- ments illustrated in Figure 2, we used the NOX1-specific inhibitory peptide NoxA1ds to assess the role of this enzyme in platelet activation.26 NOX1 inhibition almost completely inhibited oxygen radical formation in response to collagen (Figure 2A), but not thrombin (Figure 2B). In parallel, collagen- (Figure 2C) but not thrombin-dependent (Figure 2D) aggregation was inhibited by NoxA1ds. Importantly a scrambled version of the peptide was used as control. Taken together, these data suggest that NOX1 is activated and participates in the signaling of platelet activation in response to collagen but not thrombin. We also tested the role of NOX2 using the specific inhibitory peptide Nox2ds-tat.27,28 Both collagen- and thrombin- dependent oxygen radical formation are significantly impaired by NOX2 inhibition (Figure 3A and B, respec- tively). Interestingly, although the inhibition of the throm- bin response by Nox2ds-tat reduces oxygen radical forma- tion to basal levels, the inhibition of collagen-induced oxy- gen radical levels is only partial (i.e. in the presence of Nox2ds-tat the oxygen radical levels induced by collagen are significantly higher than resting controls). This is reflected in the aggregation data, which show complete inhibition by Nox2ds-tat of the aggregation induced by thrombin but only marginal inhibition of the response to collagen (Figure 3D and C, respectively). This is consistent with NOX1 playing a larger role than NOX2 in collagen- induced platelet aggregation. The data obtained with col- lagen were essentially confirmed using the synthetic lig- and for GPVI CRP (Online Supplementary Figure S10), which suggests that GPVI is the key receptor linking col- lagen-dependent platelet activation to oxygen radical gen- eration and determines the redox patterns triggered by collagen in platelets. The active engagement of NOX1 and NOX2 in collagen and thrombin signaling was confirmed by co-immunoprecipitation of these two core subunits with respective essential cytosolic components of each complex. NOX1 is co-immunoprecipitated with its canon- ical activating subunit NOXA129 in the presence of colla- gen, which is consistent with NOX1 being post-transla- tionally activated in response to collagen (Figure 4A). NOX2 appears to be co-immunoprecipitated with its canonical organizing subunit p47phox in the presence of thrombin and weakly in the presence of collagen (Figure 4B). This aligns with the conclusions reached using our EPR and aggregation experiments, i.e. that collagen acti- vates a primarily NOX1-dependent response, while thrombin activates primarily NOX2. Accordingly, whole blood thrombus formation experiments on collagen showed that NOX1 inhibition with NoxA1ds abolishes thrombus formation (Figure 4C), while NOX2 inhibition by Nox2ds-tat induces only a marginal inhibition (Figure 4D). Experiments in platelets from wild-type, NOX1-/- or NOX2-/- mice confirmed the centrality of NOX1 for colla- gen signaling with marginal involvement of NOX2 in the aggregation response (Figure 5A and B), while NOX2 is critical for thrombin signaling (Figure 5C).
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haematologica | 2019; 104(9)