L-arginine levels are diminished in adult acute vaso-occlusive sickle cell crisis in the emergency department

Paediatric studies have demonstrated that l-arginine (l-arg), the precursor to nitric oxide, is diminished in vaso-occlusive crisis (VOC). This study aimed to determine whether l-arginine levels are altered in adult VOC in the emergency department. Plasma l-arg and nitric oxide metabolite (NOx) levels were obtained in adult VOC patients presenting to the emergency department. Fifty patients had significantly low plasma l-arg (29.78 micromol/l +/- 11.21, P < 0.05 vs steady-state control = 41.16 micromol/l +/- 5.04) and significantly low plasma NOx (12.33 micromol/l +/- 10.28, P < 0.05 vs steady-state control = 25.2 +/- 2.6 micro mol/l). Neither l-arg nor NOx levels could predict VOC clinical course.     

Nitric oxide and cyclic GMP levels in sickle cell patients receiving hydroxyurea

Recent studies suggest that nitric oxide (NO) may partly be responsible for the beneficial effect of hydroxyurea (HU) in sickle cell disease (SCD) patients. NO stimulates cyclic guanosine monophosphate (cGMP) production, which mediates vasodilatation. We investigated the association between NO, cGMP and fetal haemoglobin (HbF) levels after HU administration. Our data showed that chronic HU significantly increased NO, cGMP, and HbF levels in SCD. Recently it was shown that HbF production was stimulated by cGMP-dependent protein kinase. Our results suggest that NO stimulates cGMP production, which then activates a protein kinase and increases the production of HbF.     

Severity of pulmonary hypertension during vaso-occlusive pain crisis and exercise in patients with sickle cell disease

Pulmonary hypertension is associated with sudden death and is a risk factor for mortality in adult patients with sickle cell disease. The high mortality despite only mild-to-moderate increases in pulmonary vascular resistance remains an unresolved paradox. Accordingly, little is known about the cardiovascular effects of stressors, such as vaso-occlusive pain crisis (VOC) and exercise, which may acutely increase pulmonary pressures and impair right heart function. We therefore evaluated pulmonary artery pressures by echocardiogram in 25 patients with sickle cell disease in steady-state and during VOC, and by right heart catheterisation with exercise in a second cohort of 21 patients to determine whether pulmonary hypertension worsens during acute cardiopulmonary stress. TRV increased during VOC (P < 0.001), and the increased pulmonary pressures during VOC were associated with decreases in haemoglobin levels (P < 0.001), and increases in lactate dehydrogenase (P < 0.001) and plasma haemoglobin levels (P = 0.03). During exercise stress performed during cardiac catheterisation, mean pulmonary artery pressures (P < 0.001) and pulmonary vascular resistance increased (P < 0.001) in all subjects. These data suggest that acute elevations in pulmonary pressures during VOC or exercise may contribute to morbidity and mortality in patients with sickle cell disease.     

Plasma histamine elevation in a large cohort of sickle cell disease patients

The role of mast cells has been questioned in sickle cell disease (SCD). We performed a prospective study evaluating plasma histamine and tryptase levels in a cohort of paediatric and adult patients, in steady state (n = 132) and during vaso-occlusive crisis (VOC) (n = 121). Histamine level was elevated in 18% of patients in steady state and in 61% during VOC. Median histamine level was significantly higher during VOC than in steady state (24·1 [7·0–45·0] vs 9·6 [6·2–14·4] nmol/l, P < 0·0001). Tryptase level was slightly increased during VOC without reaching pathological values. These results suggest a role of mast cell activation in SCD pathophysiology.     

Biological action of nitric oxide donor compounds on platelets from patients with sickle cell disease

Several lines of evidence point to the potential role of nitric oxide (NO) in the pathophysiology, as well as in the therapy, of sickle cell disease (SCD). In this study, we compared the effects of NO on platelets from normal individuals and from patients with SCD. Three NO donors were used to deliver NO to platelets: sodium 2-(N, N-diethylamino)-diazenolate-2-oxide (DEANO), S-nitrosocysteine (CysNO) and sodium trioxdintrate (OXINO or Angeli's salt). ADP-induced platelet aggregation, CD62P expression, PAC-1 binding and calcium elevation were evaluated in paired studies of normal and SCD subjects. DEANO significantly reduced aggregation in SCD platelets compared with normal platelets. DEANO similarly reduced the extent of CD62P expression in SCD platelets. All NO donors reduced PAC-1 binding, but there were no significant differences between platelets from normal or SCD subjects. Calcium elevation, as induced by ADP, was not altered by the presence of NO donors. However, when platelets were stimulated with thrombin, there was an increased initial response of SCD platelets compared with normal platelets. Taken together, these data suggest that the mode of NO delivery to platelets may produce various physiological responses and the optimization of NO delivery may contribute to reducing platelet aggregation in sickle cell disease.     

Decreased exhaled nitric oxide in sickle cell disease: relationship with chronic lung involvement

A deficiency in airway nitric oxide (NO) could contribute to pulmonary vaso-occlusion in sickle cell disease (SCD). We measured the fractional expired concentration of NO (FE(NO)) by chemiluminescence during a slow vital capacity maneuver against a positive pressure of 16 cm H(2)O at an expiratory flow rate of 50 mL/sec in 44 stable ambulatory adults with SCD and 30 healthy controls. A history of acute chest syndrome was present in 29 patients, and 22 complained of dyspnea. Mean +/- SD FE(NO) was significantly reduced in the SCD group compared with controls (14.8 +/- 8.4 vs. 24.9 +/- 13.5 ppb, P < 0.001). SCD patients with dyspnea had lower FE(NO) than those without dyspnea (10.1 +/- 5.7 vs. 19.6 +/- 8 ppb, P < 0.001) and those with a history of ACS had lower values than those no episodes of ACS (13.0 +/- 8.3 vs. 18.4 +/- 7.6 ppb, P < 0.05). There was a weak correlation between FE(NO) and percent-predicted DLCO (r = 0.4, P = 0.02) among the SCD patients. We conclude that exhaled NO is reduced in adults with SCD, and this may play a role in the pathogenesis of acute chest syndrome and chronic sickle cell lung disease.     

Effect of low-dose warfarin on D-dimer levels during sickle cell vaso-occlusive crisis: a brief report

OBJECTIVE: To evaluate the activation of clotting systems in patients with sickle cell disease (SCD) by measuring the plasma D-dimer level and to determine the effect of low-dose warfarin on D-dimer level during vaso-occlusive crisis. METHODS: Plasma D-dimer level was measured in 65 blood samples of 37 adult patients with SCD who were hospitalized for vaso-occlusive painful crisis. D-dimer level of patients who were on low-dose warfarin was compared with those patients who were not on any anticoagulation treatment. Analysis of variance (anova) was carried out to determine factors significantly associated with low D-dimer level in patients with SCD. The following factors were included in the anova model; warfarin, homozygous hemoglobin S, history of blood transfusion in past 3 months, hydroxyurea, hemoglobin S%, hemoglobin F%, white blood cell counts, hemoglobin level, platelet count, and plasma fibrinogen level. RESULTS: Overall median D-dimer level in 65 samples was 2.7 microg fibrinogen equivalent units (FEU)/mL (0.34-4). Patients who were on low-dose warfarin had a median D-dimer level of 0.81 microg FEU/mL (0.34-1.8) compared with 3.1 microg FEU/mL (0.94-4) in those patients who were not on anticoagulation treatment. Using anova to model D-dimer levels, only warfarin was significantly correlated with low D-dimer levels after controlling for other variables. CONCLUSIONS: Patients with SCD during vaso-occulsive painful crisis have an elevated D-dimer level. Low-dose anticoagulation treatment is associated with a significant reduction in the D-dimer levels.     

Nitric oxide donor properties of hydroxyurea in patients with sickle cell disease

Hydroxyurea therapy reduces the rates of vaso-occlusive crisis in patients with sickle cell anaemia and recent data suggest that hydroxyurea treatment can generate nitric oxide (NO). Nitric oxide has been proposed as a novel therapy for sickle cell disease via a number of pathways. We therefore sought to determine whether hydroxyurea has NO donor properties in patients with sickle cell anaemia and explore potential mechanisms by which NO production could be therapeutic. Venous blood was collected from 19 fasting sickle cell anaemia patients, on chronic hydroxyurea therapy, at baseline and 2 and 4 h after a single morning dose of hydroxyurea, as well as 10 patients not taking hydroxyurea. The plasma and red cell NO reaction products nitrate, nitrite and nitrosylated- haemoglobin were measured using ozone-based chemiluminescent assays (using vanadium, KI and I3- reductants respectively). Consistent with NO release from hydroxyurea, baseline levels of total nitrosylated haemoglobin increased from 300 nmol/l to 500 nmol/l (P = 0.01). Plasma nitrate and nitrite levels also significantly increased with peak levels observed at 2 h. Glutathionyl-haemoglobin levels were unchanged, while plasma secretory vascular cellular adhesion molecule-1 levels were reduced in patients taking hydroxyurea (419 +/- 40 ng/ml) compared with control patients with sickle cell anaemia (653 +/- 55 ng/ml; P = 0.003), and were inversely correlated with fetal haemoglobin levels (r = -0.72; P = 0.002). These results demonstrate that hydroxyurea therapy is associated with the intravascular and intraerythrocytic generation of NO. The role of NO in the induction of fetal haemoglobin and possible synergy between NO donor therapy and classic cytostatic and differentiating medications should be explored.     

Modulation of erythrocyte arginase activity in sickle cell disease patients during hydroxyurea therapy

An elevated erythrocyte arginase activity with a corresponding decrease in nitric oxide (NO) level has been implicated in the pathophysiology of sickle cell disease (SCD). Recent studies have shown that hydroxyurea (HU) increases the production of NO, which increases the soluble guanylate cyclase activity and fetal haemoglobin (HbF) synthesis. To study the effects of HU on the arginase and nitric oxide synthase (NOS) activities in SCD patients, we compared levels of arginase activity and NO metabolites in red blood cells and plasma, respectively, from 23 patients with SCD (HbSS) receiving HU therapy, with those of 12 SCD patients not receiving HU treatment. Patients on HU therapy showed significantly lower arginase activity than that of HbSS patients not on HU therapy (1.36+/-0.2 U/10(8) cells vs. 3.31+/-0.29 U/10(8) cells). NOS activity was higher in patients on HU therapy than in untreated patients (0.72+/-0.4 nmol/ml/min vs. 0.35+/-0.15 nmol/ml/min, P<0.05). Among the HU-treated patients, the decreased level of arginase activity correlated (r=0.71) with HbF level as well as the mean corpuscular haemoglobin content. These data suggest that one of the beneficial effects of HU in vivo may involve the regulation of arginase activity and a concomitant induction of NOS activity, which may lead to an increased production of NO. The outcome of this study may lead to the development of improved NO-based treatments for SCD.     

Matched-related donor transplantation for sickle cell disease: report from the Center for International Blood and Transplant Research

We report outcomes after myeloablative haematopoietic cell transplantation (HCT) from human leucocyte antigen (HLA)-matched sibling donors in 67 patients with sickle cell disease transplanted between 1989 and 2002. The most common indications for transplantation were stroke and recurrent vaso-occlusive crisis in 38% and 37% of patients respectively. The median age at transplantation was 10 years and 67% of patients had received >10 red blood cell transfusions before HCT. Twenty-seven percent of patients had a poor performance score at transplantation. Ninety-four percent received busulfan and cyclophosphamide-containing conditioning regimens and bone marrow was the predominant source of donor cells. Most patients achieved haematopoietic recovery and no deaths occurred during the early post-transplant period. Rates of acute and chronic graft-versus-host disease were 10% and 22% respectively. Sixty-four of 67 patients are alive with 5-year probabilities of disease-free and overall survival of 85% and 97% respectively. Nine patients had graft failure with recovery of sickle erythropoiesis, eight of who had recurrent sickle-related events. This report confirms and extends earlier reports that HCT from HLA-matched related donors offers a very high survival rate, with few transplant-related complications and the elimination of sickle-related complications in the majority of patients who undergo this therapy.     

The management of crisis in sickle cell disease

Abstract: The symptoms and signs of sickle cell disease are exacerbated in times of crisis, characterized by tissue infarction or worsening anaemia. Prompt medical intervention is required in these distressing situations to provide relief and comfort to the patient. Effective analgesia is crucial in treating the painful crisis of sickle cell disease. The haemoglobinopathy may cause hyposthenuria with reduced ability to excrete the sodium load in normal saline. A 5% dextrose solution or 5% dextrose in 25% normal saline is therefore recommended for intravenous hydration. As the leading cause of morbidity and mortality in sickle cell disease, infections call for vigorous antibiotic therapy. Oxygen administration should be reserved for hypoxic patients, and blood transfusion given only when really indicated. Acute chest syndrome and cerebrovascular accidents are life-threatening complications of sickle cell disease whereas priapism can cause important long-term sequelae; all deserve urgent attention. In the long term, comprehensive care is cost-effective in reducing the frequency and adverse effects of sickle cell crisis.     

Cerebrovascular disease associated with sickle cell pulmonary hypertension

In patients with sickle cell disease, anemia is a recognized risk factor for stroke, death, and the development of pulmonary hypertension. We have proposed that hemolytic anemia results in endothelial dysfunction and vascular instability and can ultimately lead to a proliferative vasculopathy leading to pulmonary hypertension. Consistent with this mechanism of disease, we now report a case series of six patients with obliterative central nervous system vasculopathy who also have pulmonary hypertension and high hemolytic rate. These patients, identified in the course of a prospective screening study for pulmonary hypertension, presented with neurological symptoms prompting neuroimaging studies. Compared to 164 other patients of similar age in the screened population, those with newly diagnosed or clinically active cerebrovascular disease have significantly lower hemoglobin levels and higher levels of lactate dehydrogenase. A review of the literature suggests that many clinical, epidemiological, and physiological features of the arteriopathy of pulmonary hypertension closely overlap with those of stroke in sickle cell disease, both known to involve proliferative vascular intimal and smooth muscle hypertrophy and thrombosis. These cases suggest that cerebrovascular disease and pulmonary hypertension in sickle cell disease share common mechanisms, in particular, reduced nitric oxide bioactivity associated with particularly high-grade hemolysis. Clinicians should suspect occult cerebrovascular disease in sickle cell patients with pulmonary hypertension.     

Acute chest syndrome in sickle cell disease

Acute chest syndrome is a common cause of death among patients with sickle cell disease, and an unfamiliar condition to most Australian medical practitioners. We present a case of acute chest syndrome successfully treated with inhaled nitric oxide and exchange transfusion. In the discussion we review current and future management options of acute chest syndrome.     

A pilot study of the short-term use of simvastatin in sickle cell disease: effects on markers of vascular dysfunction

Sickle cell disease (SCD) is characterized by progressive vascular injury and its pathophysiology is strikingly similar to that of atherosclerosis. Statins decrease inflammation and improve endothelial function in cardiovascular disease, but their effect in SCD is not known. In this pilot study, we examined the safety and effect of short-term simvastatin on biomarkers of vascular dysfunction in SCD. We treated 26 SCD patients with simvastatin, 20 or 40 mg/d, for 21 d. Plasma nitric oxide metabolites (NOx), C-reactive protein (CRP), interleukin-6 (IL-6), vascular cell adhesion molecule-1 (VCAM-1), tissue factor (TF) and vascular endothelial growth factor (VEGF) were analyzed and responses to simvastatin were compared between the two treatment groups. Simvastatin increased NOx levels by 23% in the low-dose (P =0·01) and 106% in the moderate-dose (P =0·01) groups, and by 52% overall (P=0·0008). CRP decreased similarly in both dose groups and by 68% overall (P =0·02). Levels of IL-6 decreased by 50% (P=0·04) and 71% (P<0·05) in the low- and moderate-dose groups, respectively. Simvastatin had no effect on VEGF, VCAM1 or TF. Simvastatin was well-tolerated and safe. Our preliminary findings showing a dose-related effect of simvastatin on levels of NOx, CRP and IL-6 suggest a potential therapeutic role for simvastatin in SCD.     

Pathophysiology of stroke in sickle cell disease

Stroke affects both motor and cognitive function in patients with sickle cell disease (SCD). Symptomatic stroke is associated with intimal disease of the large cerebral arteries. Silent stroke, defined as cerebral infarction in the absence of overt clinical neurologic symptoms, is often due to microinfarcts suggestive of microvascular disease. While the natural history of stroke in SCD is well described, the pathophysiology remains poorly understood and probably varies with the site of vascular injury. Increased red cell adhesion, oxidative injury of the vessel wall, inflammation, abnormal vasomotor tone regulation, and increased activity of the coagulation system all may contribute to cerebral vasopathology in SCD.     

Sildenafil therapy in patients with sickle cell disease and pulmonary hypertension

Pulmonary hypertension is a frequent complication of sickle cell disease that is associated with haemolysis, impaired nitric oxide bioavailability and high mortality. We sought to evaluate the safety and efficacy of selective pulmonary vasodilators and antiproliferative agents in this at-risk population. After optimising sickle cell disease therapy to stabilise haemoglobin and fetal haemoglobin levels, we evaluated the safety and efficacy of sildenafil in 12 patients with sickle cell disease and pulmonary hypertension. Sildenafil therapy (mean duration 6 +/- 1 months) decreased the estimated pulmonary artery systolic pressure [50 +/- 4 to 41 +/- 3 mmHg; difference 9 mmHg, 95% confidence interval (CI): 0.3-17, P = 0.043] and increased the 6-min walk distance (384 +/- 30 to 462 +/- 28 m; difference 78 m, 95% CI: 40-117, P = 0.0012). Transient headaches occurred in two patients and transient eye-lid oedema in four patients. No episodes of priapism occurred in the three men in the study; two of them were on chronic exchange transfusions and one had erectile dysfunction. In conclusion: (1) sickle cell disease patients with anaemia and pulmonary hypertension have significant exercise limitation; (2) the 6-min walk distance may be a valid endpoint in this population; (3) therapy with sildenafil appears safe and improves pulmonary hypertension and exercise capacity. Additional phase I studies in males with sickle cell disease followed by phase II/III placebo controlled trials evaluating the safety and efficacy of sildenafil therapy in sickle cell disease patients with pulmonary hypertension are warranted.     

High red blood cell nitric oxide synthase activation is not associated with improved vascular function and red blood cell deformability in sickle cell anaemia

Human red blood cells (RBC) express an active and functional endothelial‐like nitric oxide (NO) synthase (RBC‐NOS). We report studies on RBC‐NOS activity in sickle cell anaemia (SCA), a genetic disease characterized by decreased RBC deformability and vascular dysfunction. Total RBC‐NOS content was not significantly different in SCA patients compared to healthy controls; however, using phosphorylated RBC‐NOS‐Ser¹¹⁷⁷ as a marker, RBC‐NOS activation was higher in SCA patients as a consequence of the greater activation of Akt (phosphorylated Akt‐Ser⁴⁷³). The higher RBC‐NOS activation in SCA led to higher levels of S‐nitrosylated α‐ and β‐spectrins, and greater RBC nitrite and nitrotyrosine levels compared to healthy controls. Plasma nitrite content was not different between the two groups. Laser Doppler flowmetric experiments demonstrated blunted microcirculatory NO‐dependent response under hyperthermia in SCA patients. RBC deformability, measured by ektacytometry, was reduced in SCA in contrast to healthy individuals, and pre‐shearing RBC in vitro did not improve deformability despite an increase of RBC‐NOS activation. RBC‐NOS activation is high in freshly drawn blood from SCA patients, resulting in high amounts of NO produced by RBC. However, this does not result in improved RBC deformability and vascular function: higher RBC‐NO is not sufficient to counterbalance the enhanced oxidative stress in SCA.