DO MITOCHONDRIA-DERIVED REACTIVE OXYGEN SPECIES MODULATE PLATELET ACTIVATION AND AGGREGATION: COMPARISON BETWEEN SICKLE CELL PATIENTS AND HEALTHY INDIVIDUALS

Previous studies have shown that platelets play an important role in the pathophysiology of sickle cell disease (SCD). Increased platelet activation has been reported and would contribute to the occurrence of clinical complications such as vaso-occlusive crises. In addition, increased production of mitochondrial reactive oxygen species (ROS) in platelets from SCD patients have been found to correlate with bioenergetic dysfunction and mitochondrial hyperpolarization. Our study investigated whether mitochondria-derived ROS could play a role in platelet activation and aggregation in SCD patients. Blood samples were collected in 3.6% citrate tubes from 25 SCD and 12 healthy individuals to obtain washed platelets. Flow cytometry was used to assess mitochondrial ROS, total ROS, P-selectin and intracellular calcium (Ca2+) levels. Platelet aggregation was assessed in a transmitted light aggregometer. We compared baseline values between the two groups and also conducted experiments by using TRAP6 (25µM, a platelet activator), antimycin A (AA, an inhibitor of complex III of mitochondrial respiration), or N-acetylcysteine (NAC, an antioxidant). SCD platelets were more activated at baseline than platelets from controls (P < 0.05). No difference was observed between the two groups for platelet Ca2+ levels. TRAP-6 was able to further activate platelets in both groups but the activation was higher in controls (P < 0.05). SCD platelets had higher levels of mitochondria-related ROS compared to platelets from healthy volunteers (P < 0.001). The use of AA increased mitochondrial ROS in platelets, as well as platelet activation, in the two groups (P < 0.001) but the increase for these two parameters was higher or tended to be higher in controls (P < 0.05; P = 0.1). The use of NAC was able to decrease P-selectin exposure in patients and controls at a dose of 200mg/mL (P < 0.01 and P < 0.001, respectively). NAC tended to decrease total ROS and decreased mitochondrial ROS (P < 0.01) in the SCD group, and decreased platelet aggregation in the control group only (P < 0.05). Our results suggest that the higher mitochondria ROS is responsible for the higher platelet activation in SCD individuals. The lower platelet aggregation in patients, confirmed by our study, may be a consequence of a refractory state of platelets to continuous activation in vivo. Dysfunction in platelet mitochondrial respiration could lead to alterations in platelet bioenergetics, which could contribute to a worsening of the patients' clinical condition. We are currently exploring the activity of the entire mitochondrial respiratory chain in healthy and SCD platelets and plan to investigate associations with clinical severity.