BackgroundNitrite is a nitric oxide (NO) metabolite in tissues and blood, which can be converted to NO under hypoxia to facilitate tissue perfusion. Although nitrite is known to cause vasodilation following its reduction to NO, the effect of nitrite on platelet activity remains unclear. In this study, the effect of nitrite and nitrite+erythrocytes, with and without deoxygenation, on platelet activity was investigated.Methodology/FindingPlatelet aggregation was studied in platelet-rich plasma (PRP) and PRP+erythrocytes by turbidimetric and impedance aggregometry, respectively. In PRP, DEANONOate inhibited platelet aggregation induced by ADP while nitrite had no effect on platelets. In PRP+erythrocytes, the inhibitory effect of DEANONOate on platelets decreased whereas nitrite at physiologic concentration (0.1 µM) inhibited platelet aggregation and ATP release. The effect of nitrite+erythrocytes on platelets was abrogated by C-PTIO (a membrane-impermeable NO scavenger), suggesting an NO-mediated action. Furthermore, deoxygenation enhanced the effect of nitrite as observed from a decrease of P-selectin expression and increase of the cGMP levels in platelets. The ADP-induced platelet aggregation in whole blood showed inverse correlations with the nitrite levels in whole blood and erythrocytes.ConclusionNitrite alone at physiological levels has no effect on platelets in plasma. Nitrite in the presence of erythrocytes inhibits platelets through its reduction to NO, which is promoted by deoxygenation. Nitrite may have role in modulating platelet activity in the circulation, especially during hypoxia.

Summary Thromboembolic complications have been documented in thalassaemia patients. The aggregability of abnormal red blood cells and the high level of membrane‐derived microparticles (MPs) stemming from blood cells are thought to be responsible for the associated thrombotic risk. We investigated the number of MPs, their cellular origin and their procoagulant properties in β‐thalassaemia. Fresh whole blood was simultaneously stained for annexin V, cellular antigens and the known density beads. The procoagulant properties of these phosphatidylserine (PS)‐bearing MPs were also measured by assessing the platelet factor‐3‐like activity in the blood. Flow cytometric results showed that splenectomised β‐thalassaemia/HbE patients had significantly higher levels of PS‐bearing MPs than non‐splenectomised β‐thalassaemia/HbE patients and normal individuals (P < 0·0001). There was a good correlation between PS‐bearing MPs and PS‐bearing platelets, reflecting the existence of chronic platelet activation in β‐thalassaemia/HbE patients (rs = 0·511, P < 0·001). The cellular origin of PS‐bearing MPs showed mostly activated‐platelet origin with adhesion (CD41a/CD62P/CD36). Moreover, the platelet procoagulant activity was higher in splenectomised β‐thalassaemia/HbE patients when compared with non‐splenectomised (P < 0·05) and normal individuals (P < 0·01), and the amount correlated with PS‐bearing MPs (rs = 0·560, P < 0·001). These findings suggest that MPs originate from activated platelets with a potential to aggravate thrombotic events when the numbers are excessive, as is commonly seen in splenectomised β‐thalassaemia/HbE patients.

Background: Thalassemia is a hereditary hemolytic anemia caused by mutations in the globin gene complex. Circulatory disturbances including arterial and venous thrombosis have also been noted in these patients. Aggregability of abnormal RBC and the high level of membrane-derived microparticles stemming from activated platelets and other blood cells are thought to be responsible for the associated thrombotic risk. Destruction of RBC is also thought to be an important pathophysiological consequence, particularly through the formation of circulating vesicles. To our knowledge, there has been no attempt to quantitatively evaluate the number of RBC vesicles in thalassemia. This prompted us to study the level of RBC vesicles in the peripheral blood of thalassemia patients using quantitative flow cytometry.Methods: Whole blood from each subject was doubly stained for RBC and platelet or annexin V markers, together with the known density TruCount™ beads. RBC vesicles were gated according to their forward/side scatter and RBC marker. Percentage of RBC vesicles and their absolute number were analyzed by flow cytometry.Results: Our data indicated that RBC vesicles were annexin V-positive. The number of annexin V-positive events was higher than their intact RBCs. RBC vesicles were present in both normal and thalassemic blood samples, but the numbers of RBC vesicles were significantly higher in thalassemia. Both the percentage and the absolute number of RBC vesicles were especially marked in splenectomized subjects with ␤-thalassemia/ Hemoglobin E. When clinical and hematological indices were compared with RBC vesicles, there was an inverse relationship between the degree of severity in thalassemia patients and the number of RBC vesicles.Conclusion: Flow cytometric quantitation of RBC vesicles is simple, reliable and may offer new insights in to study of the relationship between defective hemoglobin synthesis, RBC perturbation and pathophysiological complications in thalassemia.