Patients with Sickle Cell Disease (SCD) demonstrate characteristics of chronic hemostatic activation including elevated baseline platelet activation. While it is well established that platelet activation is positively correlated with the magnitude of erythrocytic hemolysis in these patients, the mechanisms linking hemolysis to platelet activation remain unclear. In this study, we investigate the role of the platelet mitochondrion as the molecular link between hemolysis and downstream platelet activation. Using extracellular flux analysis, we demonstrate that platelets isolated from patients with SCD show a distinct alteration in mitochondrial function characterized by a decrease in the activity of the mitochondrial ATP Synthase (electron transport chain Complex V), which leads to enhanced mitochondrial membrane potential and elevated reactive oxygen species (ROS) production by the electron transport chain. Notably, levels of platelet mitochondrial ROS production was significantly correlated with markers of hemolysis (lactate dehydrogenase and plasma free hemoglobin concentration) as well as platelet activation in this cohort. Moreover, the mitochondrial dysfunction observed in platelets from SCD patients was recapitulated by exposing healthy platelets to cell-free hemoglobin ex vivo. Importantly, augmented mitochondrial ROS generation appears to initiate platelet activation, as scavenging of mitochondrial ROS inhibited cell free hemoglobin-induced activation of healthy platelets. Studies in the Berkley (BERK) transgenic murine model of SCD supported ex vivo data demonstrating that hemolysis-induced mitochondrial ROS production stimulates platelet activation. Homozygous BERK mice showed significantly inhibited platelet mitochondrial complex V activity and elevated mitochondrial ROS production, concomitant with enhanced baseline platelet activation compared to hemizygote mice. When subjected to a ferric chloride-induced vascular injury model of thrombosis, BERK homozygous mice showed a significantly shorter time to vascular occlusion due to thrombosis compared to hemizygote mice. Treatment of homozygous BERK mice with the mitochondrial ROS scavenger MitoTEMPO (supplemented in drinking water) significantly increased time to vascular occlusion in the ferric chloride thrombosis model. These ex vivo human platelet data and in vivo studies in murine models demonstrate that hemolysis induces platelet mitochondrial ROS production, which stimulates downstream platelet activation. Ongoing studies are focused on determining the molecular mechanisms by which cell free hemoglobin elicits an inhibition of platelet mitochondrial Complex V and implicate a role for activation of the platelet surface toll like receptor 4 (TLR4). Collectively, these data identify the platelet mitochondrion as an important signaling hub that links hemolysis to platelet activation in SCD patients. The potential of therapeutically modulating platelet mitochondrial ROS production to attenuate hemolysis induced thrombotic activation will be discussed.
Disclosures
No relevant conflicts of interest to declare.
ALCAT1 Overexpression Affects Supercomplex Formation and Increases ROS in Respiring Mitochondria
