Exaggerated Intra-platelet ROS generation in 5 days-stored PCs
Non-physiological agents including PMA and CCCP induce intra-platelet ROS generation from different sources. For this study, CCCP has been used as a typical mitochondrial uncoupler that disrupts oxidative phosphorylation inducing mitochondrial dysfunction and augmented ROS generation which here detected in a DHR123- dependent assay. On other hand, we alternatively treated fresh platelets (obtained from 0 day-stored PCs) with PMA to induce NOX activity with the highest levels of cytosolic ROS detected by DHE assay. As shown in figure 1A & C, treatment of 0 day-stored platelets (fresh platelets) with CCCP increased ROS to ~ 2.5 folds higher than that observed in non-treated one. In addition, platelets incubation with PMA elevated O2-- to levels that were two times more than those observed in non-treated 0 day-stored platelets (figure 1E & F). Previous studies showed increasing levels of ROS generation in platelets during storage with the highest levels of ROS accumulation in 5-day stored platelets. However, the significance of this elevation was under question. As showed in figure 1C, the levels of mitochondrial ROS accumulated in 5-day stored platelets were comparable to that induced by CCCP. Interestingly, O2-- generation in 5-day stored platelets was significantly (p<0.05) higher than that achieved by PMA while the levels of O2-- formed in 3-day stored platelets was comparable to those observed in PMA stimulated fresh platelets (figure 1F).
Shedding patterns of platelet collagen receptor, GPVI in response to non-physiological stimuli versus storage
Platelet storage showed to be associated with the increasing levels of GPVI shedding with the highest levels observed in 5 days-stored PCs [10]. Figure 2A presents a blot image illustrating the highest levels of GPVI shedding in 5 days-stored platelet with that of day 0 which is at the lowest levels ever tested here. Figure 2A & B also showed the significant increments of GPVI shedding levels in response to CCCP (100µM) and PMA (10µM) in 0 day-stored platelets (freshly prepared PCs). The treatment of platelet with the CCCP indicated to induce higher levels of GPVI shedding compared to that obtained by PMA. Of note, the increased levels of shedding induced by PMA are still lower than those observed in 3 or 5 days-stored PCs in which the shedding levels are comparable to those induced by CCCP. With further evaluation (figure 2C), a direct correlation was found between GPVI shedding and either mitochondrial ROS (r = 0.81; p < 0.001) or cytosolic superoxide generation (r = 0.8; p < 0.001).
Platelet spreading on collagen in stored platelets
In a dynamic process platelet adhesion to collagen is followed by spreading processes which reflects platelet metabolic and functional abilities. Previous studies showed decreeing adhesion capacity of platelet during storage; however there were no data to evaluate platelets spreading to collagen as an important functional marker of storage-dependent lesion. Figure 3A presents a demonstrative image depicting fresh platelets spreading capacities which seriously attenuated in 5-day stored PCs. Graph 3B demonstrates tremendous reduction in platelet adhesion surface area(p= 0.0022) in 5 days-stored platelets compared to fresh one(one day-stored). Of note, the percentage of spread platelets (p= 0.003) and even with less significance (p= 0.01) the number of adhered platelets also decreased in 5 days-stored platelets (figure 3 C&D). However, as showed in figures the significant reductions in platelet spreading and adhesion surface area have been actually started from day 3 of storage, while the numbers of adhered platelets have not shown significant changes.
The correlation of platelet GPVI shedding with adhesion capacities
GPVI is a main receptor involved in platelet firm adhesion and spreading to the site of vascular injury. We already showed the significant reverse correlation between platelet simple adhesion to collagen matrix and GPVI shedding [10]. Here, in addition to simple adhesion we also evaluated the correlation between platelet spreading to collagen (platelet adhesion area) and GPVI shedding in stored PCs. As shown in figure 2D, a prominent reverse correlation between GPVI shedding and platelet adhesion area was observed here, which according to its correlation indexes (r = - 0.91; p = 0.0002) is more potent than that observed for simple adhesion (r = - 0.58; p = 0.011).
The correlation of platelet spreading (platelet adhesion area) on collagen with either mitochondrial ROS or cytosolic superoxide generation in stored platelets
As showed in figure 3E, whereas DHR123 as an indicator of mitochondrial ROS was not significantly correlated with the simple adhesion of platelets to collagen, this adhesive pattern showed to be reversely relevant to platelet superoxide generation detected by DHE expression (r = - 0.80; p < 0.001). However, platelet spreading showed to be significantly correlated with either mitochondrial ROS (r = - 0.82; p = 0.004) or cytosolic superoxide generation (r = - 0.89; p < 0.001) (figure 3F).
Collagen-induced platelet aggregation
Figure 4A demonstrates the representative images of platelet aggregation curve in response to collagen on day0 and 5 of storage. Platelet aggregation attenuated during storage with a significant (p<0.05) drop started from day 3 reaching to lowest levels in day 5 of storage (figure 4B). With further evaluation (figure 4C), a direct correlation was found between collagen-dependent platelet aggregation and either mitochondrial ROS (r = - 0.81; p < 0.001) or cytosolic superoxide generation (r = - 0.85; p < 0.001).