Oxidative Status of Platelets in Normal and Thalassemic Blood.

Thromboembolic complications, possibly involving chronic platelet activation, are an important cause of morbidity and mortality in beta-thalassemia. Oxidative stress, with the generation of reactive oxygen species (ROS), has been suspected to play a role in the pathophysiology of thalassemia and cardiovascular disorders. Previous investigations demonstrated that ROS profoundly affect platelet function and promote platelet activation. Other studies have shown that platelets themselves produce ROS upon activation.

In the present study, we adapted flow cytometric techniques to measure oxidative-state markers, ROS generation and reduced glutathione (GSH), using 2′-7′-dichlorofluorescin diacetate and mercury orange, respectively, in platelets. GSH is the major intracellular antioxidant - an important scavenger of ROS. To exclude non-platelets from analysis, a two-parameter (side light scatter and forward light scatter) gate was set. The identity of the gated cells was verified by immunofluorescence staining for CD41 - a platelet-specific antigen. Using these techniques, the average Mean Fluorescence Channel (MFC) values of platelets from 46 normal donors and 22 beta-thalassemic donors were 176 ± 99 vs. 314 ± 81, respectively, for ROS and 319 ± 87 vs. 113 ± 47, respectively, for GSH. These results show that thalassemic platelets contain higher ROS and lower GSH levels than do normal platelets, indicating a state of oxidative stress. The relationship between platelet activation and oxidative status was determined by treating platelets with thrombin (0.1 U/ml), calcium ionophore (5 μM) or phorbol myristate acetate (400 ng/ml). All these treatments caused platelet activation as well as ROS generation; thalassemic platelets were more responsive than platelets from normal controls. In the absence of any known inherent abnormality in thalassemic platelets, the increased oxidative status was attributable to continuous exposure to oxidative insults from extra-platelet sources. Indeed, further investigation indicated that the oxidative status of normal platelets was increased by thalassemic plasma and was inhibited by the iron-chelator Desferoxamin. Iron and hemin, whose levels are increased in thalassemic plasma, stimulated the platelets’ oxidative stress. This was also affected by RBC: it was higher in normal platelets incubated with thalassemic RBC than when incubated with normal RBC. Normal RBC stimulated with hydrogen peroxide, a treatment which results in an elevated oxidative status, increased platelet ROS to a greater extent (3.3-fold) than did unstimulated RBC. These results suggest that thalassemic RBC, having higher than normal ROS, mediate oxidative stress in platelets directly, probably by contact or close proximity. Platelet oxidative stress was ameliorated by antioxidants such as N-acetyl-L-cysteine and vitamin C. Treatment with these agents of oxidant-stimulated platelets reduced ROS and enhanced the GSH level.

The present results indicate that in thalassemia, platelets are in a state of oxidative stress, causing their chronic activation and possibly thromboembolic consequences. This situation may also prevail in other RBC anomalies, such as sickle cell anemia, Polycythemia Vera and Paroxysmal Nocturnal Hemoglobinuria, which are also associated with thromboembolic phenomena. Our findings raise the possibility of using antioxidants in addition to antithrombotic drugs as prophylactic treatment in these diseases.