Non-Transferrin Plasma Iron in β-Thalassaemia/Hb E and Haemoglobin H Diseases

Non-transferrin plasma iron concentrations were determined in 45 normal controls and in 37 patients with Hb H disease and 104 patients with β-thalassaemia/Hb E disease. This revealed that non-transferrin plasma iron exists in cases with severe iron overload, more striking in β-thalassaemia/Hb E than in Hb H disease. Non-transferrin plasma iron is associated with higher transferrin iron saturation and higher plasma ferritin levels. The most striking finding was the significantly higher non-transferrin plasma iron in splenectomized patients with β-thalassaemia/Hb E disease than in the non-splenectomized patients. In view of the potential toxicity of non-transferrin iron, this fraction of iron may be responsible for tissue damage in these patients especially after splenectomy.     

Activated platelet-derived microparticles in thalassaemia

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 beta-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 beta-thalassaemia/HbE patients had significantly higher levels of PS-bearing MPs than non-splenectomised beta-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 beta-thalassaemia/HbE patients (r(s) = 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 beta-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 beta-thalassaemia/HbE patients.     

Vitamin E reduces platelet adhesion to human endothelial cells in vitro

Although it has been reported that vitamin E (alpha-tocopherol) can reduce platelet adhesiveness and aggregation in vivo, the mechanism is still unknown. Therefore, the aim of the present study was to determine whether incubations of platelet-rich plasma (PRP) with vitamin E influence platelet adhesion to cultured endothelial cells. To exclude blood plasma involvement, also washed platelets were pretreated with alpha-tocopherol. Vitamin E (0.5-1.0 mM) was added to PRP or washed platelets. Endothelial cells in monolayer were incubated with thrombin-activated platelets (1 or 2 U/ml). After 1 hr of incubation, non-adhered platelets were removed and counted. Treating of PRP with alpha-tocopherol inhibited platelet adhesion to endothelial cell monolayer. This effect was dose dependent on concentrations of alpha-tocopherol and thrombin. In our experiments PRP was treated with alpha-tocopherol and endothelial cell monolayer was used as test surface. These findings agree with previous observations on the adhesivity of platelets to synthetic surfaces after dietary vitamin E in healthy volunteers. When washed platelets were incubated with alpha-tocopherol, no significant reduction of adhesion was detectable. As preincubation of washed platelets with alpha-tocopherol does not inhibit platelet adhesion, it may be supposed that the effect of vitamin E does not occur in a directly cellular mechanism. The data suggest that alpha-tocopherol may reduce platelet adhesiveness probably after incorporation by plasma lipoproteins.     

Alpha-tocopherol, an effective inhibitor of platelet adhesion

Platelet adhesiveness was tested ex vivo in a group of six normal individuals receiving varying doses of alpha-tocopherol. Adhesion to glass slides coated with fibronectin, collagen, fibrinogen, or plasma proteins was studied by perfusing platelet-rich plasma through a flow chamber that allowed time- and space-resolved observations of platelet adhesion. Platelet adherence was measured in an area of parallel flow lines and low shear rate under standardized conditions before and after dietary supplementation with vitamin E at doses of 200 and 400 IU/d. Platelet adherence differed in magnitude depending on the adhesive surface. There was a distinct preference of platelets to adhere to sites that had been previously occupied. A remarkable decrease in platelet adherence was observed after vitamin E supplementation. The average decrease in adhesion after 2 weeks of 200 IU vitamin E was 75%. After 2 weeks of 400 IU vitamin E, platelet adhesion was reduced by 82%. The inhibitory activity of alpha-tocopherol was dose dependent and correlated well with the increase in alpha-tocopherol concentration in platelets after supplementation. Scanning electron microscopy revealed a striking decrease of pseudopodium formation in alpha-tocopherol- enriched platelets. Our results suggest that vitamin E may also be an effective antiadhesive agent in vivo.     

Serum Ferritin in Patients with Hereditary Spherocytosis

S ummary . Serum ferritin was measured in 61 patients with autosomal dominant hereditary spherocytosis (HS), 44 splenectomized and 17 with intact spleens. In the majority (78%) the serum ferritin concentration was not elevated. Thirteen (22%) had mildly elevated levels, including five splenectomized females (range 181-236 μg/l), and eight males, two with intact spleens (range 236-436 μg/l). The serum ferritin was not raised in 15 of the 17 non-splenectomized individuals. This group included one female who had been venesected for iron overload and further investigations have shown that both the genes for HS and haemochromatosis are present in her family.
These results demonstrate that iron stores are usually normal in HS, and that prevention of iron overload alone is not an indication for splenectomy. The rare report of a patient with HS and severe iron overload may perhaps be explained by the fact that the gene frequency for haemochromatosis is common in the population.     

Vitamin E (alpha-tocopherol) does not inhibit platelet stimulation by oxidized low density lipoprotein in vitro

Platelet-rich plasma were treated with increasing concentrations of vitamin E (alpha-tocopherol). Washed platelets were exposed to oxidized low density lipoprotein (LDL) and examined by aggregometry and electron microscopy. The treatment of washed platelets by oxidized LDL induced morphological signs of activation like pseudopodia formation and an increase in light transmission. Alpha-tocopherol in a range of 0.001-1.0 mmol had no inhibiting influences on platelet activation by oxidized LDL. These results indicate that the free radical scavenger vitamin E cannot directly inhibit platelet activation by oxidized LDL. It may be supposed that platelet activation by oxidized LDL does not occur in a radical-dependent mechanism.     

Zinc-induced platelet aggregation is mediated by the fibrinogen receptor and is not accompanied by release or by thromboxane synthesis

We demonstrate that zinc (0.1 to 0.3 mmol/L) induces aggregation of washed platelet suspensions. Higher concentrations (1 to 3 mmol/L) of zinc were needed to aggregate platelets in platelet-rich plasma obtained from blood anticoagulated with low-molecular-weight heparin, probably due to the binding of zinc to the plasma proteins. Zinc- induced aggregation of normal washed platelets required added fibrinogen and no aggregation occurred with thrombasthenic platelets or with normal platelets pretreated with a monoclonal antibody (10E5) that blocks the platelet fibrinogen receptor. These data indicate that the platelet membrane fibrinogen receptor-glycoproteins IIb and IIIa mediate the effect of zinc. Zinc-induced aggregation was blocked by the agent TMB-8, which interferes with the internal calcium flux, and by prostacyclin, which elevates platelet cyclic adenosine monophosphate levels. Zinc-induced aggregation was not accompanied by thromboxane synthesis or by the secretion of dense-body serotonin and was not affected by preexposure of platelets to acetylsalicylic acid. Experiments with creatine phosphate/creatine phosphokinase showed that the zinc effect on platelets was independent of extracellular adenosine diphosphate (ADP). Zinc had an additive effect when platelet aggregation was stimulated with subthreshhold concentrations of collagen or ADP. Together with the known effects of nutritional zinc on in vivo bleeding, on platelet aggregation, and on lipid metabolism, the results suggest that zinc may have an important bearing on normal hemostasis, thrombosis, and atherosclerosis.     

Activation of monocytes for the immune clearance of red cells in β°-thalassaernia/HbE

Summary We have recently provided evidence that IgG antibodies play a role in the destruction of red cells in thalassaemia syndromes. In order further to delineate factors involved in the clearance of thalassaemic cells, monocytes of 30 Thai patients with β°-thal/HbE (17 non-splenectomized and 13 splenectomized) and 16 normal controls were examined for their ability to bind and phagocytose normal red cells coated with IgG anti-Rh(D). In β°-thal/HbE. the mean number of red cells attached to the monocytes was approximately 3-fold greater than in normal controls and the number ingested 30% higher. Among the non-splenectomized patients, the number of red cells attached to and ingested by the monocytes, correlated inversely with mean basal Hb levels, suggesting that activation of mononuclear phagocytes for the immune clearance of red cells is a factor in determining the severity of the anaemia. As Fc-gamma-RI is of primary importance in the recognition of IgG-coated red cells by monocytes, leucocytes from 10 β°-thallHbE patients (four non-splenectomized and six splenectomized) and five normal controls were investigated for their expression of Fc-gamma-RI by flow cytometry. In β°-thallHbE there was an approximately 3-fold increase in the percentage of leucocytes expressing this receptor: the receptor was up-regulated on monocytes and induced on granulocytes. The up-regulation of Fc-gamma-RI in β°-thallHbE is likely to be an important component in the activation of monocytes and in mediating their enhanced effector function towards antibody-coated cells.     

Pulmonary Arterial Hypertension in Previously Splenectomized Patients with β-Thalassemic Disorders

Our aim was to study the cause and describe the clinical features of pulmonary arterial hypertension (PHT) in splenectomized beta-thalassemia (beta-Thal) patients. Ten splenectomized beta-Thal patients with systolic pulmonary artery (PA) pressure >30 mm Hg were evaluated by echocardiography, right-heart catheterization, and pulmonary angiography. Five of these patients later underwent hemodynamic studies. Echocardiography and pulmonary angiography on the 10 patients showed normal values of left ventricular systolic function and no findings of acute or chronic pulmonary embolism. Hemodynamic evaluation showed very high PA pressures associated with markedly increased pulmonary vascular resistance indices (PVRIs). Hematological evaluation of the 10 patients showed marked anemia, markedly increased numbers of nucleated red blood cells (nRBCs), and serum ferritin. Mean platelet count, plasma beta2 thromboglobulin, and thrombin-antithrombin III complex levels were significantly increased. It was concluded that PHT can be found in splenectomized beta-Thal patients. Features associated with PHT were female sex, hemoglobin E/beta-Thal, status many years postsplenectomy, marked anemia, markedly increased nRBC count, thrombocytosis, and very high serum ferritin levels. PHT was not due to pulmonary emboli. Our findings suggested that severe PHT was due to increased PVRI from thrombotic pulmonary arteriopathy, likely from chronic low-grade hypercoagulability and platelet activation after splenectomy.     

Red cell and plasma calcium, copper and zinc in beta-thalassemia/hemoglobin E

Beta-thalassemia/Hb E is a genetic disease prevalent in Thailand. This study has used atomic absorption spectroscopy to evaluate red cell and plasma calcium, copper and zinc in patients with beta-thalassemia/Hb E, both splenectomized and non-splenectomized. The levels of these trace elements in both red cells and plasma were different between the non-thalassemic controls and the disease patients. The most prominent result was that calcium concentration in red cells increased significantly in thalassemia subjects, particularly in splenectomized cases. These results might reflect the abnormal trace element metabolism and defects in the calcium transport system of the red cell membrane in thalassemia.     

Double-faced cell-mediated immunity in β-thalassemia major: stimulated phenotype versus suppressed activity

In this study, the immunologic abnormalities of Iranian β-thalassemia major patients were characterized, considering their clinical parameters including splenectomy status and iron overload. Serum samples and peripheral blood mononuclear cells were collected from 28 patients and 30 age- and sex-matched healthy individuals. Patients with thalassemia showed significantly increased absolute lymphocyte counts compared with the control group. An increased number of activated T cells and higher levels of serum neopterin were also observed in thalassemia patients, which suggest chronic stimulation of immune system. On the contrary, T-cell proliferation and interleukin 2 (IL-2), interferon gamma (IFN-γ), and IL-4 production were suppressed in patients compared to controls. Patients with high serum ferritin levels produced significantly less IFN-γ and IL-2, indicating the immunosuppressive effect of iron overload in β-thalassemia patients. The serum levels of tumor necrosis factor alpha and absolute counts and percentages of B and T cells were higher in splenectomized patients; however, serum levels of neopterin significantly decreased in splenectomized patients compared to the non-splenectomized group. Taken together, T lymphocytes express activated phenotype in polytransfused β-thalassemia major patients, while T cell proliferation and effector function are significantly suppressed. Multiple blood transfusion and continuous immune stimulation could be responsible for making such a double-faced immune response.     

Moderate supplementation with natural alpha-tocopherol decreases platelet aggregation and low-density lipoprotein oxidation.

Previous studies have shown that oral administration of 300 mg alpha-tocopherol/day to healthy volunteers decreases platelet function and enhances their sensitivity to the platelet inhibitor, prostaglandin E(1), when full dose-response curves to a range of agonist concentrations are made. In this study, the effects of oral doses of natural alpha-tocopherol (75, 200 and 400 IU/day) were studied in order to determine whether the same effects might be achieved with lower intakes of vitamin E and whether inhibition is related to the platelet levels of the antioxidant in platelet membranes. Twenty two subjects undertook the supplementation regime, divided into three units of 2 weeks, each cycling through each of the dosages. The results show that uptake of vitamin E by the platelets was optimal at 75 IU/day, correlating with the maximal influence on platelet aggregation and platelet responsiveness to inhibition by PGE1, increased supplemental levels exerting no greater effects.     

Effects on the buoyant density of rabbit platelets of ADP and agents that increase the concentration of cyclic AMP

Rabbit platelets were aggregated by adenosine diphosphate (ADP), allowed to deaggregate and then separated into density subpopulations by centrifugation through discontinuous Stractan density gradients. Although ADP causes little or no release of the contents of the amine storage granules of rabbit platelets, ADP caused a decrease in platelet density as compared with control platelets subjected to the same procedures except for exposure to ADP. The density change persisted for at least four hours. The apparent size of platelets stimulated with ADP increased initially, but returned to control values during a one-hour period. A similar decrease in platelet density was observed with an albumin density gradient. Under conditions in which aggregation did not occur in response to ADP with ethylenediaminetetraacetic acid (EDTA) in the medium, little or no decrease in platelet density was observed. Agglutination with polylysine did not change platelet density. Thus, not only agents such as thrombin and plasmin that cause the release of the contents of the platelet granules decrease platelet density, but ADP also has this effect. Platelets would be exposed to all of these stimuli during thromboembolic processes, and their effect on platelets may account for the decrease in platelet density observed previously in experiments with rabbits with indwelling aortic catheters. Agents that increase the concentration of cyclic AMP (cAMP) in platelets (PGE1, adenosine, dibutyryl cAMP, forskolin, and papaverine) also decreased platelet density. This effect persisted when the platelets were washed and resuspended in fresh medium and was also demonstrable in plasma. Platelet size was gradually increased by prostaglandin E1 (PGE1) which maintains platelets in a disc shape and does not cause the release of granule contents, indicating that the decrease in platelet density caused by PGE1 may be attributable to platelet swelling.     

Changes in vitamin E status during obesity treatment

The effects of vitamin E on platelet function and erythrocyte membrane rigidity are extensively described. Little is known, however, about the vitamin E status in an obese population and about the effect of weight loss on it. This study evaluates the changes in vitamin E status during obesity treatment in 8 morbidly obese females. They received a protein-sparing modified fast (PSMF) diet for a period of 5 weeks; mean vitamin E supplementation did not exceed the recommended daily allowance (8 mg of alpha-tocopherol equivalents). During the investigated period plasma vitamin E levels increased (p less than 0.02), while there was a slight decrease in plasma cholesterol. The rise in total tocopherol/total cholesterol ratio was highly significant (p less than 0.002). Both the experimental design and the results are comparable with previously reported data in hypothalamic obese mice. It is, therefore, suggested that the hypothalamic obese mouse is a convenient animal model for the study of vitamin E nutritional status in obesity.     

In vitro platelet abnormality in adenosine deaminase deficiency and severe combined immunodeficiency.

The platelets of an infant with severe combined immune deficiency and adenosine deaminase deficiency showed markedly diminished responses to ADP-induced aggregation in vitro. This abnormality was corrected by the addition of purified adenosine deaminase in vitro. Exogenous adenosine added to platelet-rich plasma caused markedly prolonged inhibition of ADP-induced aggregation. This was shown by isotopic studies to be due to slow clearance of adenosine and hence persistence of this nucleoside. Direct assay for adenosine deaminiase in plasma and platelet lysates of the patient confirmed the very low activity of this enzyme. Raised cAMP levels were demonstrated in his platelets. The deranged adenosine metabolism and raised cAMP in the platelets of this child with severe combined immunodeficiency may explain the altered response to ADP. Despite the in vitro platelet aggregation abnormality, the patient had no clinical evidence of impaired hemostasis.     

Platelet aggregation is affected by nitrosothiols in patients with chronic hepatitis： In vivo and in vitro studies

AIM： To investigate the relationship among the number of platelets and plasma levels of S-nitrosothiols （S-NO）, nitrite, total non-protein SH （NPSH）, glutathione （GSH）, cysteine （CYS）, malondialdehyde （MDA）, 4-hydroxininenal （4HNE）, tumor necrosis factor-alpha （TNFα） and interleukin （IL）-6 in patients with chronic hepatitis C （CH）.
METHODS： In vitro the aggregation of platelets derived from controls and CH patients was evaluated before and after the addition of adenosine diphosphate （ADP） and collagen, both in basal conditions and after incubation with nitrosoglutathione （GSNO）.
RESULTS： In vivo, S-NO plasma levels increased significantly in CH patients and they were significantly directly correlated with platelet numbers. Patients with platelet counts 〈 150000/μL, had a smaller increase in S-NO, lower levels of GSH, CYS, NPSH, TNFα, and IL-6, and higher levels of nitrite, MDA, and 4-HNE relative to those of patients with platelet counts 〉 150000/μL. In vitro, the ADP and collagen aggregation time was increased in platelets from patients and not from controls; in addition, platelets from CH patients but not from controls also showed a latency time after exposure to collagen.
CONCLUSION： The incubation of platelets with GSNO improved the percentage aggregation and abolished the latency time.     

The Exchangeable Splenic Platelet Pool Studied with Epinephrine Infusion in Idiopathic Thrombocytopenic Purpura and in Patients with. Splenomegaly

S ummary . The exchangeable splenic platelet pool (ESPP) was studied with epinephrine infusion and platelet labelling with ⁵¹Cr in five healthy students, 10 patients with idiopathic thrombocytopenic purpura (ITP) and 10 patients with splenomegaly. Five of the ITP-patients were studied after splenectomy. Platelet recovery of infused labelled platelets was calculated in all subjects and also in nine healthy volunteers who had been splenectomized for traumatic rupture of the spleen. Spleen size was determined by gamma camera scintigraphy. It was shown that the spleen is the only site of an exchangeable platelet pool in ITP and that this pool was of the same size in ITP-patients as in the normal controls, viz ∼30% of the total body platelet mass.
In patients with splenomegaly the ESPP was larger than that in controls and ITP-patients. A highly significant correlation was found between the ESPP and the spleen volume. In splenectomized and in non-splenectomized ITP-patients platelet recovery was significantly less than in their respective control groups, indicating that a proportion of the labelled platelets was immediately removed from the circulation after infusion into an ITP recipient and that the recovery of labelled platelets cannot be used as a measure of the ESPP in ITP.
It is suggested that the early destruction of platelets may be due to slight damage to the platelets during the labelling procedure. These damaged platelets can survive in a normal recipient, but are destroyed when infused into the'milieu' of an ITP-patient.     

Effects of sarpogrelate hydrochloride on adenosine diphosphate- or collagen-induced platelet responses in arteriosclerosis obliterans.

To evaluate the effects of the 5-HT2 receptor antagonist sarpogrelate hydrochloride (sarpogrelate) on platelet responses in arteriosclerosis obliterans (ASO), we examined platelet aggregation and its relationships to platelet-derived growth factor (PDGF), soluble P-selectin (sP-selectin), and transforming growth factor-beta 1 (TGF-beta1). Circulating plasma levels of PDGF and sP-selectin in 13 patients with ASO after 1 week of medication with sarpogrelate were significantly lower than those before medication. In contrast, circulating plasma levels of TGF-beta1 after medication were significantly higher than those before medication. When platelet-rich plasma obtained from ASO patients after medication was stimulated with adenosine diphosphate (ADP) or collagen, platelet aggregation was suppressed compared with rates before medication. Significant decreases in levels of PDGF, sP-selectin and TGF-beta1 released from platelets in response to 5 micromol/l ADP and 1 microg/ml collagen after taking of sarpogrelate were found. There were close correlations between platelet aggregation and respective molecules released from platelets. In conclusion, since platelet activation is involved in pathogenesis of thrombotic disease, sarpogrelate may suppress the development of obstructive arteriosclerosis. PDGF and TGF-beta1, as well as sP-selectin, appear to be useful markers for clinical evaluation of anti-platelet drugs.     

Non-Specific Iron in Patients with Beta-Thalassaemia Trait and Chronic Active Hepatitis

A non-specific iron fraction, not bound to transferrin, has been looked for in the sera of 42 never-transfused patients with beta-thalassaemia trait, 17 of whom had chronic active hepatitis, negative for HBV infection or alcohol abuse. Non-specific iron was found only in the sera of those patients with beta-thalassaemia trait plus chronic active hepatitis who had complete transferrin saturation, high serum ferritin levels and urinary iron excretion and a high degree of hepatic siderosis. In view of the known toxicity of non-transferrin iron, we suggest that this non-transferrin iron fraction may be responsible for the liver damage in these patients. Furthermore, the positive correlation between the presence and the amount of non-transferrin iron and the levels of serum ferritin suggests that this fraction is a sensitive indicator of iron-induced toxicity when severe iron overload slowly develops in patients with beta-thalassaemia trait even in the absence of any iron administration.