Deferiprone increases endothelial nitric oxide synthase phosphorylation and nitric oxide production

Iron chelation can improve endothelial function. However, effect on endothelial function of deferiprone has not been reported. We hypothesized deferiprone could promote nitric oxide (NO) production in endothelial cells. We studied effects of deferiprone on blood nitrite and blood pressure after single oral dose (25 mg/kg) in healthy subjects and hemoglobin E/β-thalassemia patients. Further, effects of deferiprone on NO production and endothelial NO synthase (eNOS) phosphorylation in primary human pulmonary artery endothelial cells (HPAEC) were investigated in vitro. Blood nitrite levels were higher in patients with deferiprone therapy than those without deferiprone (P = 0.023, n = 16 each). Deferiprone increased nitrite in plasma and whole blood of healthy subjects (P = 0.002 and 0.044) and thalassemia patients (P = 0.003 and 0.046) at time 180 min (n = 20 each). Asymptomatic reduction in diastolic blood pressure (P = 0.005) and increase in heart rate (P = 0.009) were observed in healthy subjects, but not in thalassemia patients. In HPAEC, deferiprone increased cellular nitrite and phospho-eNOS(Ser1177) (P = 0.012 and 0.035, n = 6) without alteration in total eNOS protein and mRNA. We conclude that deferiprone can induce NO production by enhancing eNOS phosphorylation in endothelial cells.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Increased platelet activation and lower platelet-monocyte aggregates in COVID-19 patients with severe pneumonia.

BackgroundThe increased procoagulant platelets and platelet activation are associated with thrombosis in COVID-19. In this study, we investigated platelet activation in COVID-19 patients and their association with other disease markers.MethodsCOVID-19 patients were classified into three severity groups: no pneumonia, mild-to-moderate pneumonia, and severe pneumonia. The expression of P-selectin and activated glycoprotein (aGP) IIb/IIIa on the platelet surface and platelet-leukocyte aggregates were measured prospectively on admission days 1, 7, and 10 by flow cytometry.ResultsP-selectin expression, platelet-neutrophil, platelet-lymphocyte, and platelet-monocyte aggregates were higher in COVID-19 patients than in uninfected control individuals. In contrast, aGPIIb/IIIa expression was not different between patients and controls. Severe pneumonia patients had lower platelet-monocyte aggregates than patients without pneumonia and patients with mild-to-moderate pneumonia. Platelet-neutrophil and platelet-lymphocyte aggregates were not different among groups. There was no change in platelet-leukocyte aggregates and P-selectin expression on days 1, 7, and 10. aGPIIb/IIIa expression was not different among patient groups. Still, adenosine diphosphate (ADP)-induced aGPIIb/IIIa expression was lower in severe pneumonia than in patients without and with mild-to-moderate pneumonia. Platelet-monocyte aggregates exhibited a weak positive correlation with lymphocyte count and weak negative correlations with interleukin-6, D-dimer, lactate dehydrogenase, and nitrite.ConclusionCOVID-19 patients have higher platelet-leukocyte aggregates and P-selectin expression than controls, indicating increased platelet activation. Compared within patient groups, platelet-monocyte aggregates were lower in severe pneumonia patients

Decreased nitrite reductase activity of deoxyhemoglobin correlates with platelet activation in hemoglobin E/ß-thalassemia subjects.

Nitric oxide (NO) can be generated from nitrite by reductase activity of deoxygenated hemoglobin (deoxyHb) apparently to facilitate tissue perfusion under hypoxic condition. Although hemoglobin E (HbE) solutions have been shown to exhibit decreased rate of nitrite reduction to NO, this observation has never been reported in erythrocytes from subjects with hemoglobin E/ß-thalassemia (HbE/ß-thal). In this study, we investigated the nitrite reductase activity of deoxyHb dialysates from 58 non-splenectomized and 23 splenectomized HbE/ß-thal subjects compared to 47 age- and sex-matched normal subjects, and examined its correlation with platelet activity. Iron-nitrosyl-hemoglobin (HbNO) was measured by tri-iodide reductive chemiluminescence as a marker of NO generation. HbNO produced from the reaction of nitrite with deoxyHb dialysate from both non-splenectomized and splenectomized HbE/ß-thal subjects was lower than that of normal (AA) hemoglobin subjects. P-selectin expression, a marker of platelet activation, at baseline and in reactivity to stimulation by adenosine diphosphate (ADP), were higher in HbE/ß-thal subjects than normal subjects. HbNO formation from the reactions of nitrite and deoxyHb inversely correlated with baseline platelet P-selectin expression, HbE levels, and tricuspid regurgitant velocity (TRV). Nitrite plus deoxygenated erythrocytes from HbE/ß-thal subjects had a lower ability to inhibit ADP-induced P-selectin expression on platelets than erythrocytes from normal subjects. We conclude that deoxyHb in erythrocytes from HbE/ß-thal subjects has a decreased ability to reduce nitrite to NO, which is correlated with increased platelet activity in these individuals

Platelet inhibition by nitrite is dependent on erythrocytes and deoxygenation.

Nitrite 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.Platelet 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.Nitrite 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

Platelet aggregation in whole blood showed inverse correlation with the nitrite levels in whole blood and erythrocytes.

<p>Platelet aggregation in whole blood (20% hematocrit) was induced by 10 µM ADP and monitored for 5 min using the impedance aggregometry. The degree of platelet aggregation was correlated with the nitrite levels in whole blood (A) and erythrocytes (B). Each dot represents mean of duplicate measurement (n = 15).</p

Nitrite+erythrocytes inhibited platelet aggregation.

<p>(A) ADP induced platelet aggregation in PRP+erythrocytes (20% hematocrit) in the concentration dependent manner. (B) Erythrocytes at 0, 1, 10 and 20% hematocrit did not have effect on ADP-induced platelet aggregation in the absence of nitrite. (C) Nitrite+erythrocytes (20% hematocrit) inhibited ADP-induced platelet aggregation. Nitrite was incubated in PRP or PRP+erythrocytes in the presence or absence of 200 µM C-PTIO for 5 min before induction of aggregation by ADP. ^*<i>P</i><0.05 compared with PRP and PRP+erythrocytes+C-PTIO (ANOVA). (D) The inhibitory effect of 0.1 µM nitrite was dependent on hematocrits. PRP and PRP+erythrocytes (1, 10, and 20% hematocrit) were incubated with 0.1 µM nitrite for 5 min before induction of platelet aggregation by 20 µM ADP. ADP-induced platelet aggregation in the absence of nitrite was shown in the control group. ^*<i>P</i><0.05 compared with PRP+nitrite at 0% hematocrit (ANOVA). (E) Nitrite+erythrocytes inhibited collagen-induced platelet aggregation. PRP or PRP+erythrocytes samples (20% hematocrit) were incubated with nitrite in the presence and absence of 200 µM C-PTIO for 5 min and then the aggregation was induced by 2.5 µg/mL collagen. ^*<i>P</i><0.05 compared with PRP and PRP+erythrocytes+C-PTIO (ANOVA). (F) Nitrite+erythrocytes inhibited U46619-induced platelet aggregation. 0.1 µM nitrite was incubated in PRP or PRP+erythrocytes (20% hematocrit) in the presence or absence of 200 µM C-PTIO for 5 min. Then, the aggregation was induced by 1 µM U46619. ^*<i>P</i><0.05 compared with PRP+erythrocytes and PRP+erythrocytes+C-PTIO (ANOVA). All experiments were performed at 37°C. Data are means ± SEM (n≥3).</p

Erythrocytes abolished the effect of DEANONOate on platelet aggregation.

<p>(A) Inhibition of platelet aggregation by DEANONOate decreased in the presence of erythrocytes. PRP and PRP+erythrocytes (20% hematocrit) were incubated with DEANONOate for 5 min. (B) Dependence of platelet aggregation in the presence of 0.1 µM DEANONOate on hematocrit. PRP and PRP+erythrocytes (1, 10, and 20% hematocrit) were incubated with 0.1 µM DEANONOate for 5 min. Platelet aggregation was induced by 20 µM ADP. ADP-induced platelet aggregation in the absence of nitrite was shown in the control group ^*<i>P</i><0.05 compared with 0% hematocrit (ANOVA). All experiments were performed at 37°C. Data are means ± SEM (n≥3).</p