The Reactive Oxygen Species Scavenger N‐Acetyl‐L‐Cysteine Reduces Storage‐Dependent Decline in Integrin α IIb β ₃ ‐Mediated Platelet Function, Inhibiting Pre‐Activation of Integrin and Its β ₃ Subunit Cleavage

Figures

• 

  Figure 1. Integrin β3 chain cleavage in the presence/absence of NAC (1 mM) or VAS2870 (30 μM). The representative western blots show the levels of β3 cleavage in non-treated platelets (control) (A) and platelets treated with NAC (1 mM) (B). Graphs (D) and (E) demonstrate the levels of β3 chain expression and β3 cleavage during 7 days of storage, respectively. As shown in (F), 1 mM NAC significantly reduced the levels of β3 cleavage compared to the control in 3- and 5-day-stored platelets. However, VAS2870 had no significant effect on the cleavage of the integrin β3 chain in 5-day-stored platelets (C). ns, not significant; NAC, N-acetyl-L-cysteine. ⁣∗p < 0.05, ⁣∗∗p < 0.01, n = 10.

• 

  Figure 2. The surface expression of αIIbβ3 integrin in the presence/absence of NAC (1 mM) or VAS2870 (30 μM). (A) and (B) show dotted plots and their corresponding histograms depict the levels of CD41 (αIIb) and CD61 (β3) surface expression in platelets treated with either NAC or VAS2870 compared to non-treated platelets (control) obtained from 3- and 5-day stored PCs. Graphs (C) and (D), respectively, also show the effects of either NAC or VAS2870 treatments on the levels of CD41 (αIIb) and CD61 (β3) surface expression compared to control during 5 days of platelet storage. ns, not significant; NAC, N-acetyl-L-cysteine. ⁣∗p < 0.05, ⁣∗∗p < 0.01, n = 10.

• 

  Figure 3. The level of PAC1 binding in the presence/absence of NAC (1 mM) or VAS2870 (30 μM). (A) and (B) show dotted plots and their corresponding histograms depict the levels of PAC1 binding in platelets treated with either NAC or VAS2870 compared to non-treated platelets (control) obtained from 3- and 5-day stored PCs. Bar charts (C) and (D), respectively, also show the effects of NAC and VAS2870 treatments on the basal level of PAC1 binding compared to control during 5 days of platelet storage. ns, not significant; NAC, N-acetyl-L-cysteine. ⁣∗p < 0.05, ⁣∗∗p < 0.01, n = 10.

• 

  Figure 4. The levels of platelet adhesion to the fibrinogen matrix in the presence/absence of NAC (1 mM) or VAS2870 (30 μM). (A) Demonstrates the levels of platelet adhesion to the fibrinogen matrix in platelets treated with either NAC or VAS2870 and non-treated platelets (control) on days 1 and 5 of storage. Graph (B) shows the number of adhered platelets to the fibrinogen matrix in non-treated platelets and platelets treated with either NAC or VAS2870 during 5 days of storage. Bar charts (C) and (D), respectively, also illustrate the effects of NAC and VAS2870 treatments on the levels of platelet adhesion to the fibrinogen matrix versus non-treated platelets (control) during storage. ns, not significant; NAC, N-acetyl-L-cysteine. ⁣∗p < 0.05, ⁣∗∗p < 0.01, n = 10.

• 

  Figure 5. The levels of platelet spreading on the fibrinogen matrix in the presence/absence of NAC (1 mM). (A) and (B), respectively, illustrate microscopic fields (100× magnification) of platelet spreading on the fibrinogen matrix in non-treated platelets (control) versus NAC-treated platelet products on days 1 and 5 of storage. Graph (C) shows the percentage of spread platelets on the fibrinogen matrix in non-treated platelets (control) and platelets treated with NAC during 5 days of storage. As shown in (D), 1 mM NAC significantly improved the levels of platelet spreading on the fibrinogen matrix compared to non-treated (control) in 5 days stored platelets. Bar chart (E) shows the platelet adhesion area on the fibrinogen matrix in the presence of NAC versus non-treated (control) during 5 days of storage. ns, not significant; NAC, N-acetyl-L-cysteine. ⁣∗p < 0.05, ⁣∗∗, p < 0.01, ⁣∗∗∗p < 0.001, n = 10.

• 

  Figure 6. The levels of platelet spreading on the fibrinogen matrix in the presence/absence of VAS2870 (30 μM). (A) and (B), respectively, show microscopic fields (100× magnification) of platelet spreading on the fibrinogen matrix in non-treated platelets (control) versus VAS2870-treated platelet products on days 1 and 5 of storage. Graph (C) demonstrates the percentage of spread platelets on the fibrinogen matrix in non-treated platelets (control) and platelets treated with VAS2870 during 5 days of storage. As illustrated in (D), 30 μM VAS2870 significantly reduced the percentage of spread platelets on days 0 and 1 of storage compared to the non-treated (control). Bar chart (E) shows the platelet adhesion area on the fibrinogen matrix in the presence of VAS2870 versus non-treated (control) during 5 days of storage. ns, not significant. ⁣∗p < 0.05, ⁣∗∗p < 0.01, ⁣∗∗∗p < 0.001, n = 10.

• 

  Figure 7. The correlation between platelet adhesion and spreading with the levels of the smaller band of β3 integrin in stored platelets. Graph (A) demonstrates a reverse correlation between both adhesion (r = −0.65; p=0.002) and spreading (r = −0.81; p < 0.001) with the smaller β3 integrin bands during platelet storage. Graph (B) also shows a reverse correlation between platelet adhesion area on fibrinogen and the smaller β3 integrin bands (r = −0.64; p=0.003) during storage.

• 

  Figure 8. The mechanism behind downregulation of platelet spreading following calpain-dependent cleavage of the β3 integrin cytoplasmic tail. (A) ROS production via NOX and mitochondrial pathways following platelet activation is associated with sustained calcium influx. (B) An increase in intracellular calcium leads to the activation of calpain, which cleaves the binding site of β3 integrin by c-Src and results in its disassociation from the integrin tail. Src plays a substantial role in the phosphorylation and activation of signaling proteins, including focal adhesion kinase (FAK), RhoGAP, and GEFs. (C) GAPs and GEFs regulate the activation status of Rac1, Cdc42, and RhoA, which play an important role in platelet spreading. In addition, tyrosine phosphorylation of FAK can also affect platelet spreading. Following calpain activation and disassociation of c-Src, a significant defect occurs in these downstream pathways which leads to a decrease in platelet spreading. (D) The activation of RhoA-GEFs following ligand binding to different GPCRs increases the RhoA activity by induction of inside out signals. On the other hand, the inhibitory effect of Rho GAP on RhoA is relieved due to the…