Generally, deleterious changes, so-called as platelet storage lesion (PSL) affects the quality and effectiveness of therapeutic platelets. The storage of PCs is associated with progressive platelet activation characterized by accumulating intra-platelet ROS, adhesive receptors shedding, platelet pro-coagulant activity and granule release leading to potential pro-inflammatory function of platelets [
34‐
36]. Pro-coagulant activity (monitored by PS exposure) is also associated with apoptotic events, including cytochrome C release, caspase 3 activation, and the loss of mitochondrial respiration capacity as specific markers of apoptosis, however the significant rises of these markers are usually detected in long-stored platelets, from day 5 or 7 of storage [
25]. Altogether, these are the stored-dependent changes of platelet which not only affect post-transfusion platelet survival but it may also attenuate platelet functional activity and effectiveness in circulation, while of note, some of these changes such as receptor shedding and the induction of pro-inflammatory or pro-coagulant phenotypes of platelets sound to be irreversible. Our previous studies on PCs indicated increasing levels of intra-platelet ROS during storage with the highest levels demonstrated in 5 days-stored platelets. We also showed that platelet storage can increasingly induce GPVI shedding [
1]. Therefore, in this study given the key role of ROS in receptors modulation, we tried to evaluate storage-dependent correlation of ROS generation with GPVI modulation and platelet spreading on collagen matrix. Considering different sources for intra-platelet ROS, the levels of either superoxide or mitochondrial ROS in stored platelets have been first evaluated here. For the baseline study, the lowest levels of ROS generation were detected in freshly prepared PCs (0 day-stored platelets) whereas these platelets showed an influx of both superoxide and mitochondrial ROS generation in response to PMA and CCCP respectively. The phorbol ester, PMA that activates protein kinase C (PKC), is an important agonist which significantly induces NOX activity in platelets [
37,
38] while as an uncoupler compound, CCCP induces mitochondrial lesion and apoptosis in platelets. CCCP is a protonophore which renders the mitochondrial inner membrane permeability to protons. This decreases proton gradient across the inner mitochondrial membrane and uncouples phosphorylation from oxidation while disrupting mitochondrial ATP synthesis. Therefore, CCCP treatment per se renders mitochondrial transmembrane potential (ΔΨm) and promotes Bax translocation to the mitochondria, leading to the release of apoptotic factors into the cytosol [
39]. On the other hand, CCCP augments mitochondrial ROS generation [
40,
41]. This elevated ROS can oxidize a main component of the inner mitochondrial membrane, cardiolipin and induce apoptosis through ΔΨm depolarization, mitochondrial Bax translocation, cytochrome C release, caspase-3 activation and PS exposure [
42,
43]. However, given the fact that ROS scavenger, NAC can inhibit CCCP-induced reduction of ΔΨm and Bax translocation, presumably here ROS generation precedes the loss of mitochondrial transmembrane potential [
39]. CCCP also showed to significantly promote GPVI shedding by ADAM17 rather than ADAM10 which is considered to be the specific sheddase for GPVI. Given the fact that ADAM 17 activity is modulated by ROS generation [
1], the CCCP induced shedding might be also affected by oxidant pathways. Based on these background studies here, for the first time we showed CCCP-induced ROS generation in platelets that makes a direct link between platelet mitochondrial lesion and induced oxidative stress in an experimental setup. We also applied specific probe, DHR123 to detect mitochondrial-originated source of ROS in platelets [
27]. In this study, a three-fold higher level of superoxide has been detected in 5 days-stored platelets. Intriguingly, this was even significantly higher than that induced by PMA as a potent NOX stimulating agent. Given this data, it seems that abovementioned pathways associated with mitochondrial lesion can be also involved in ROS generation here. To examine this theory, using DHR123, the similar patterns of mitochondrial ROS generation were also evaluated in stored platelets. Results showed 2.5 fold higher levels of ROS generation in 5 days-stored platelets at the levels comparable to that induced by CCCP which triggers serious mitochondrial damage and platelet apoptosis [
44] . Conclusively although, some other research also reported the increasing levels of ROS generation during platelet storage [
13,
14], our findings first highlighted the fact that ROS accumulation in 5 days-stored platelets is comparable to the highest levels of ROS generation experimentally induced with either potent non-physiological activator of NOX or mitochondrial damaging compound. In addition our results suggest a critical role for aged-dependent mitochondrial lesion of platelets in the augmentation of ROS elevation in longer stored platelets. This may indicate the significance of platelet lesion during storage that raises question about the quality of 5 days-stored PCs. The oxidant stress is generally indicated to be associated with different arrays of cellular damage and dysfunction. Augmented levels of ROS have been shown to disturb either natural proteomic or genomic materials in the cells [
45,
46]. Several line of evidence indicated that a well-tuned physiologic concentration of ROS can act as an important modulator of platelets adhesive capacity. However, as a general rule, the oxidant stress induced by unleashed accumulation of ROS in platelets can seriously affect adhesion receptors shedding and their function [
1]. We already showed the increasing levels of GPVI shedding in stored platelets with highest levels detected in the 5 days of storage [
11]. Studies indicated an intense experimental shedding of GPVI induced by mitochondrial-targeting reagent CCCP that mimics platelet aging. PMA also activates protein kinase C (PKC) which triggers downstream ADAMs and significantly induces the shedding of platelet adhesion receptors including GPVI [
47‐
49]. Results presented here also showed significant shedding of GPVI in fresh PCs treated with either PMA or CCCP while more interestingly, 5 days storage of platelets induces two folds higher level of GPVI shedding compared to that induced by PMA. GPVI shedding in 5 days-stored platelets was also statistically comparable to what induced by CCCP. This may indicate the levels of stored-dependent damage that affect platelets. In addition, the much higher shedding level in 5 days-stored platelets compared to that induced by PMA suggests the significant role of mitochondrial lesion rather than protein kinase- dependent pathways in these events. So far, several studies using different methods have indicated platelet adhesion loss to collagen during storage. In addition, we already showed that total adhesion of platelets to collagen is reversely correlated to GPVI shedding [
11]. However, it seems that the evaluation of platelet spreading on collagen provides more information about the platelet signaling competence and functional quality beyond that seen for the simple adhesion [
50]. In addition, since platelet firm adhesion and spreading is mainly mediated by GPVI receptor [
1], platelets adhesion area seems to be a better indicator of GPVI competence rather than simple adhesion which might be also modulated by other less important receptors or be affected by artifacts. As presented here, in 5 days-stored PCs, the reduction of platelet spreading on collagen (calculated by platelet surface area) is much more significant than simple adhesion loss during storage. Such a decline in platelet adhesion surface area indicates that platelets profoundly lose their functional quality during long storage. On the other hand, platelet adhesion area correlates with GPVI shedding with the significance much higher than that was observed in simple adhesion. This may also suggest that platelet spreading on collagen is extremely GPVI-dependent. Notably, unlike our previous research [
11], in current study we also evaluated the relevance of GPVI expression and adhesion capacities of platelet during storage. In general, GPVI shedding of stored platelets can be a more sensitive indicator of platelet activation than its expression, as to some extent the loss of GPVI expression by shedding might be compensated by platelet receptors re-expression and their dimerization due to continuous activating signals during storage [
51,
52]. However, if the aim of GPVI evaluation is to examine platelet adhesive function, GPVI expression seems to be a better choice. Intriguingly here unlink shedding, while GPVI expression was directly relevant to platelet surface adhesion area (spreading), it did not show a significant correlation with simple adhesion. This confirms the fact that platelet firm adhesion is mainly modulated with GPVI expression while the earlier staged of adhesion to platelets (stable or unstable simple adhesion) might be orchestrated by other collagen receptors such as integrin α
2β
1 [
2] or be due to nonspecific binding. Of note, whereas CCCP reduced GPVI expression to the levels which was comparable with those of 5 days-stored platelet, as an important shedding inducer, PMA did not show any significant effects. Two reasons might be considered to explain this event. First reason can be a lower effect of PMA on GPVI shedding compared to CCCP, which cannot overcome GPVI re-expression. The second one is PMA-induced GPVI dimerization that may compensate PMA-induced ADAM10 activity in GPVI shedding [
52,
53]. Considering direct relevance between GPVI expression/shedding and ROS generation, we also evaluated any correlation of platelet adhesive capacity with ROS in stored platelets. Our data indicated that superoxide accumulation in PCs reversely correlates with both platelet adhesion number and spreading on collagen. Nonetheless, mitochondrial generated ROS was only relevant to platelet spreading on collagen while had no significant correlation with the number of adhered platelets. This finding suggests the key role of superoxide in earlier stage of platelet adhesion to collagen. This may also confirm other studies that have already highlighted the critical role of superoxide-induced oxidation of Cys residue (an unpaired thiol) located on the cytoplasmic tail of receptor, which results in rapid disulfide-dependent homodimerization of GPVI, igniting platelet adhesion to collagen [
19,
20]. Collagen-induced platelet aggregation is also considered as another relevant functional assay which has shown to be reversely correlated with GPVI shedding [
11]. PSL seriously affects collagen-induced platelet aggregation which significantly correlated with ROS generation. The significant reverse correlation of ROS with this observed platelet aggregation also highlights the potential involvement of stored-dependent oxidant stress in abnormal aggregation and thrombus growth.