Urinary β-Thromboglobulin in Essential Hypertension

Urinary concentrations of the platelet-specific protein beta-thromboglobulin (beta-TG) were measured in patients with essential hypertension. Values obtained for patients with normal renal function fell within the same population as those for a control group of normal individuals, but nevertheless 21% of these patients had urinary beta-TG values above the upper limit of the normal range. Values for patients with renal insufficiency were significantly different from the other groups, and 44% of these individuals had abnormally elevated urinary beta-TG concentrations. These elevated values may reflect an increased plasma concentration of beta-TG in patients with renal impairment or they could be an indicator of renal damage.     

Evidence for the Platelet Specificity of β-Thromboglobulin and Studies on its Plasma Concentration in Healthy Individuals

The concentration of normal human platelet beta-thromboglobulin (beta-TG) was measured in various washed organ samples by a radioimmunoassay. As only trace amounts were detected, beta-TG appears to be a platelet specific protein. Assay of beta-thromboglobulin in plasma samples from 180 normal individuals gave a range of 10--65 mg/ml. In the 10 subjects studied, plasma beta-TG concentration was related to platelet lifespan but not to turnover. The plasma beta-TG concentration rose with increasing age but did not correlate with the whole blood platelet count or the percentage of megathrombocytes. These results provide further substantial evidence that measurement of plasma beta-TG concentration is useful for assessing the participation of platelets in various disease processes.     

Three Approaches to the Radioimmunoassay of Human β-Thromboglobulin

Three radioimmunoassays for the measurement of beta-thromboglobulin are described. The standard method, using antiserum in solution, could be used to measure plasma concentrations of beta-thromboglobulin with the results available after 2-3 d. The use of a greater dilution of antiserum and tracer, with delayed addition of tracer, resulted in a more sensitive assay suitable for measuring b-thromboglobulin in urine. The use of a solid-couples antiserum under non-equilibrium conditions allowed the measurement of plasma levels of beta-thromboglobulin after an assay incubation time of 1 h. These three radioimmunoassay systems for beta-thromboglobulin cover the likely clinical requirements for the measurement of this platelet specific protein.     

Studies on the Liberation of β-Thromboglobulin from Human Platelets in Vitro

A platelet specific protein, beta-thromboglobulin, is liberated during the preparation of platelet poor plasma. Using combinations of different anticoagulant and anti-platelet compounds, this release can be significantly reduced. The best results were obtained when native blood was collected as soon as possible into a mixture of EDTA, prostaglandin E1 and theophylline and maintained and processed at a temperature between 0 and 4 degrees C. These technical innovations have permitted the use of a radioimmunoassay for beta-thromboglobulin on plasma samples in clinical practice.     

Radioimmunoassay of Platelet Factor 4 and β-Thromboglobulin: Development and Application to Studies of Platelet Release in Relation to Fibrinopeptide A Generation

Platelet and fibrinogen survival and turnover studies have shown that platelet activation and fibrin formation may occur to different degrees in different thrombotic disorders. More direct evidence of differential involvement of platelet activation and fibrin formation should be provided by specifically measuring the products of these reactions, i.e. released platelet proteins and fibrinopeptide A. Two platelet proteins, platelet factor 4 (PF4) and beta-thromboglobulin (betaTG), were isolated and characterized, and sensitive and specific radioimmunoassays were developed to measure them. These assays were employed, along with the radioimmunoassay for fibrinopeptide A (FPA), to study the release of PF4 and betaTG in relation to FPA cleavage. PF4 and betaTG were released by ADP and collagen with time course and concentration dependence similar to that of [14C]serotonin release. FPA was not cleaved from fibrinogen during ADP or collagen-induced platelet release. Thrombin caused release of PF4 and betaTG as well as cleavage of FPA. Cleavage of FPA occurred with concentrations of thrombin about 100 times less than did release of PF4 and betaTG, and release of [14C]serotinin required still higher thrombin concentrations. Release of [14C]serotonin and platelet proteins was similar as a function of time. Sodium citrate was found to inhibit platelet release induced by thrombin.     

Release of β-Thromboglobulin from Human Platelets by Therapeutic Intensities of Ultrasound

The effects of therapeutic intensities of ultrasound on human platelets in whole blood were investigated by monitoring the release of the platelet specific protein beta-thromboglobulin (beta-TG). More beta-TG was released as the intensity of the ultrasound was increased and also as the driving frequency was decreased from 3.0 to 0.75 MHz. Some beta-TG was released at spatially-averaged intensities as low as 0.6 W/cm2 at 0.75 MHz, a value significantly lower than that observed for the onset of aggregation of platelet rich plasma (obtained from the same volunteer) in the same exposure system. Liberation of beta-TG by ultrasound was diminished but not abolished in the presence of inhibitors which rendered the platelets functionally inert. Our data suggests that beta-TG is liberated in two ways, firstly as a result of platelet disruption by cavitation, and subsequently by potent aggregating agents, liberated in parallel with beta-TG, inducing the physiological release reaction in adjacent platelets. The low therapeutic intensities and short exposure times (30 s or less) necessary to liberate beta-TG from normal human platelets in vitro, suggests that patients with abnormally sensitive platelets and/or 'hypercoagulable state' could be at risk if subjected to high therapeutic intensities of ultrasound.     

Platelet and Plasma β Thromboglobulin in Mycloproliferative Syndromes and Secondary Thrombocytosis

Elevated plasma beta thromboglobulin (betatg) and decreased intracellular platelet betatg were found in patients with various myeloproliferative disorders. In secondary thrombocytosis, similar, but less marked changes were found. The abnormalities were increased during episodes of clinical thrombosis and could be suppressed in two-thirds of cases by aspirin therapy.     

Circulating Soluble Interleukin-2 Receptor α and β Chain in Inflammatory Bowel Disease

Objectives : Inflammatory bowel disease is characterized by T cell activation. Activated T cells shed interleu-kin-2 receptors (IL-2R) in a soluble form. A positive correlation between sIL-2Rα (CD25) and disease activity in inflammatory bowel disease has been shown previously, whereas IL-2Rβ (CD122) has never before been investigated in this respect. Serum from 27 patients with ulcerative colitis (UC), 31 with Crohn's disease (CD), and 29 healthy volunteers was obtained. Methods : Disease activity was scored according to a semiquantitative score for UC and by Crohn's disease activity index for CD. sIL-2Rα and -β chains were assessed by a sandwich ELISA technique using monoclonal antibodies specific for CD25 and CD122, respectively. Results : The median concentration of sIL-2Rα was 4424 pg/ml in healthy controls, 6460 in UC ( p < 0.004), and 6371 in CD ( p < 0.01). The corresponding value of sIL-2Rβ in healthy volunteers was 605 pg/ml; in active UC, significantly lower levels were found at 233 pg/ml ( p < 0.01), whereas in inactive UC, no such difference was observed at 725 pg/ml ( p > 0.05). In CD, the levels were 839 pg/ml in inactive and 920 pg/ml in active disease stages ( p > 0.05 vs controls). A positive and significant correlation existed between sIL-2R levels of α and β chains in CD ( r = 0.64; p < 0.01) but not in UC ( r = -0.32; p > 0.05) or in healthy volunteers ( r = 0.16; p > 0.05). Conclusion : Future longitudinal studies will be necessary to learn whether this newly assessed sIL-2Rβ (CD122), which may interfere with IL-15R, could be used to predict disease exacerbation and to monitor anti-inflammatory therapy in UC.     

Platelet Production Time in Surgery

A study of platelet production time in major surgery is described. This was equated to the time taken for renewal of maximum malondialdehyde (MDA) activity in blood platelets following suppression by a single dose of aspirin (Stuart et al 1975). The method was simplified by using EDTA disodium salt as an anticoagulant instead of citrate, resuspending platelets with small bar magnets, and reducing time of incubation with thiobarbituric acid reagent from 1 h to 15 min. Results for maximum platelet renewal taken to be the time to achieve a pre-aspirin baseline level were median in surgery 6.3 (3.8–9.2) d and controls 7.0 (6.2–12.5) d. Using the modified method, average baseline malondialdehyde (MDA) levels were 6.2 ± 1.4 nmol/10⁹ platelets in surgical patients and 6.34 ± 1.2 nmol/10⁹ platelets in controls. Platelet production time determination using this method is not strictly analogous compared with survival of ⁵¹Cr-labelled platelets and it however warrants further study.     

β-ENDORPHIN AND β-MSH IN HUMAN PLASMA

The aim of this study was to establish whether or not a peptide with chromatographic and immunological properties of beta-endorphin exists in human plasma. Using direct chromatography under conditions designed to minimize generation of beta-endorphin and beta-MSH from beta-LPH, we invariably found a peptide with beta-endorphin immunoreactivity eluting in the position of beta h-endorphin on gel chromatography in samples of plasma from patients with elevated ACTH and LPH levels. beta-MSH was only found in the plasma of one patient with the ectopic ACTH syndrome.     

Increased β-Thromboglobulin in Essential Hypertension: Interactions between Arterial Plasma Adrenaline,Platelet Function and Blood Lipids

ABSTRACT Twenty-three 50-year-old men with untreated, essential hypertension had elevated plasma concentrations of the platelet release product β-thromboglobulin (BTG) compared to 14 age-matched control men (p<0.01). BTG correlated with arterial plasma adrenaline concentrations in the hypertensive (r=0.44, p<0.05), normotensive (r=0.73, p<0.01) and combined group (r=0.51, p<0.01). Significant correlations (p<0.05) between BTG and cholesterol (LDL + VLDL fraction) were observed both in the hypertensive and the normotensive group. In the hypertensive group arterial adrenaline correlated with cholesterol (LDL + VLDL) (p<0.05). These findings are consistent with increased platelet activity in middle-aged men with essential hypertension, and may indicate that plasma adrenaline influences platelet function. The risk factors for coronary artery disease (blood pressure, lipid status, stress as evidenced by catecholamine release and platelet function) were positively related. Measurement of arterial instead of venous adrenaline is essential for the demonstration of the associations presented.     

Effect of α-Chain Precipitates on Bone Marrow Function in Homozygous β-Thalassaemia

S ummary . An autoradiographic study of protein synthesis by erythropoietic cells in liomozygous β-thalassaemia has shown that a high proportion of non-dividing, late polychromatic erythroblasts fail to become labelled when incubated with radioactive amino acids. It is possible that this abnormality and the previously described disturbance of cell proliferation in the early polychromatic erythroblasts result from damage by intracellular α-chain precipitates. This possibility has been investigated by correlating the extent of α-chain precipitation with protein or DNA synthetic activity in individual cells. Over 80% of late polychromatic cells with the largest α-chain inclusions failed to label with ³H-leucine or ³H-phenylalanine but 10–23% of cells with no inclusions were also unlabelled. None of the dividing, early polychromatic cells with moderate or large quantities of insoluble α-chains incorporated ³H-thymidine. These results support the hypothesis that a-chain precipitates are associated with and possibly responsible for the ineffectiveness of erythropoiesis in thalassaemia, provided it is assumed that such precipitates are continuously degraded or extruded. The latter assumption is necessary to account for the presence of several metabolically abnormal cells with no α-chain precipitates.     

Stimulation of thrombocytopoiesis decreases platelet β2 but not β1 or β3 integrins

The expression of CD29, CD61, CD18 and CD11a on platelets was examined by flow cytometry in mice treated with leukaemia inhibitory factor (LIF) or megakaryocyte growth and development factor (PEG-rHuMGDF or mpl-ligand). Treatment for 7–14 d with PEG-rHuMGDF or LIF increased the number of platelets in peripheral blood from 0.9 up to <2.0×10⁶/μl. These treatments decreased the expression of CD11a and CD18, whereas that of CD29 or CD61 was not markedly changed. Study after various doses or times of PEG-rHuMGDF administration indicated that a decrease of CD18 expression occurred when platelet counts started to rise. Platelet RNA content was increased in mice treated with PEG-rHuMGDF but double staining indicated that expression of CD18 was not correlated with RNA content. To evaluate integrin expression as a function of time in circulation, platelets were biotinylated in vivo . In normal or PEG-rHuMGDF-treated mice, the expression of CD29 or CD61 did not change, whereas that of CD18 decreased significantly as a function of time in circulation. These findings indicate, firstly, that stimulation of thrombocytopoiesis leads to the release of platelets with a low content of β2 integrin and, secondly, that this integrin is also selectively lost while in the circulation.     

β-LIPOTROPHIN AND β-ENDORPHIN PLASMA LEVELS DURING PREGNANCY

β-lipotrophin (β-LPH) and β-endorphin (β-EP) plasma levels were measured by radioimmunoassay after glass powder extraction and Sephadex G-75 column chromatography in plasma samples from controls (ten healthy males and twenty-six young women in early follicular phase), from eighty-two pregnant women in weeks 9–40 after their last menstrual period, from nine women just after delivery and the cord blood of their neonates, in fifteen mixed cord blood samples and in seven amniotic fluid samples obtained by amniocentesis. No sex differences were found in β-LPH (120·6 ± 8·5 pg/ml) or β-EP (31·1 ± 2·4 pg/ml) plasma levels or in their molar ratio (1·34 ± 0·09) (MR). β-LPH plasma levels increased in early pregnancy (13–16 weeks) (185·0 ± 27·1 pg/ml) and remained high until weeks 21–24, then declining to levels similar to those of controls. β-EP plasma levels were significantly depressed in weeks 9–12 (20·7 ± 5·3 pg/ml), subsequently increasing to a maximum at weeks 36–37 (42·7 ± 6·8 pg/ml). β-LPH/β-EP molar ratio was about double normal in early pregnancy and decreased to normal in the second half. The present data indicate that β-LPH and β-EP present different patterns throughout pregnancy and that β-EP levels increase progressively, reaching the highest concentrations at term. At delivery, both β-LPH and β-EP showed maximum values (β-LPH: 230·2 ± 20·4 pg/ml; β-EP: 78·0 ± 7·4 pg/ml) and a MR of 1·02 ± 0·10 indicating that stressful situations, such as labour, stimulate a simultaneous rise in β-LPH and β-EP plasma levels. Cord blood specimens showed a wide range of values (β-LPH:75–347 pg/ml; β-EP: 16–287 pg/ml) with a MR of 1·21 ± 0·14. Amniotic fluid samples obtained late in the third trimester of pregnancy were characterized by β-LPH levels of 119·4 ± 26·4 pg/ml and β-EP levels of 29·6 ± 7·5 pg/ml.     

Homozygous β-thalassaemia resulting in the β-thalassaemia carrier state phenotype

Summary. This paper describes the phenotypic manifestations of a very mild β-thalassaemia mutation detected in several members of two families of Italian descent. The molecular defect, defined by denaturing gradient gel electrophoresis analysis and direct sequencing. consists of a C G substitution at position 844 of IVSII of the β-globin gene within the consensus sequence of IVSII acceptor splice site. Heterozygotes for this mutation show a haematological phenotype ranging in severity from silent β-thalassaemia to that of a mild β-thalassaemia carrier silent β-thalassaemia to that of a mild β-thalassaemia carrier state, whereas homozygotes have the typical manifestations commonly resulting from heterozygosity for a β-thalassaemia mutation. Compound heterozygotes for the IVSII nt844 (C G) mutation and a severe β-thalassaemia mutation have the phenotype of thalassaemia intermedia.
This paper indicates that the presence of borderline red blood cell indices or HbA₂ values should make one suspect the presence of a very mild or silent β-thalassaemia.     

Splenic Platelet Kinetics and Platelet Production after Major Reconstructive Vascular Surgery

ABSTRACT By using ¹¹¹In-labelled platelets and dynamic gamma camera scintigraphy, platelet production rate and intrasplenic platelet kinetics were determined in 13 patients at 1 and 4 months after aortic reconstructive vascular surgery with implantation of dacron prostheses. A significant decrease in platelet production rate and venous platelet count was recorded over time after surgery. Irrespective of whether the exchangeable splenic platelet pool was estimated from initial recovery of platelet-bound radioactivity or from compartmental analysis, the size of this pool was significantly lower at the first study; a change in intrasplenic platelet transit time accounted for the observed difference. Platelet mean life-span increased over time after surgery but the difference between the duplicate studies was not statistically significant. It can be concluded that there is a reduction of platelet production rate and venous platelet count over time after major reconstructive vascular surgery. The early postoperative elevation in the platelet count is mainly the result of an increased platelet production and to a lesser degree due to redistribution of platelets between the splenic platelet pool and general circulation.     

β2-Microglobulin: Structure, Function and Significance

beta 2-Microglobulin is a low molecular weight protein with sequence homology to immunoglobulins. As a portion of the HLA complex this protein is an important cell-surface structure. Under normal conditions beta 2-microglobulin is synthesized and shed by many cells, particularly lymphocytes, and is detectable in the circulation of normal individuals. Because of its small size it is normally filtered readily at the glomerulus and is catabolized by proximal tubular cells of the kidney. Impaired renal function and hyperproduction of beta 2-microglobulin are both associated with increased serum levels. A function for beta 2-microglobulin as a modulator of lymphocyte surface and as a potential regulator of the immune system is proposed.     

Discrimination between β-endorphin and β-lipotrophin in human plasma using two-site immunoradiometric assays

OBJECTIVE We wished to discriminate between the opioid peptide β-endorphin (β-EP) and its non-opioid precursor β-lipotrophin (β-LPH) in normal subjects and patients with ACTH-related disorders. DESIGN We produced monoclonal antibodies to β-EP and β-LPH for the development of two-site immunoradiometric assays (IRMAs) which specifically quantitate β-EP and β-LPH. PATIENTS Samples were obtained from patients with a range of ACTH-related disorders and compared with 18 normal subjects. Peptide levels were also compared in six patients with Cushing's syndrome undergoing bilateral inferior petrosal sinus sampling with corticotrophin releasing hormone administration. MEASUREMENTS In the β-EP IRMA, antibody 6B2, specific for β-EP 18–27, is radiolabeled and antibody 2E10, recognizing β-EP 1–5, is coupled to Sephacryl S-300 as solid phase. The IRMA is specific for β-EP (β-LPH cross-reacts <0.02%), has a detection limit of 14 ± 0 7 pmol/l (n = 7) and has a within-assay coefficient of variation of <10% between 4.9 and 1200 pmol/l. In the β-LPH IRMA, antibody 6B2, which recognizes an epitope common to β-LPH and β-EP, is radiolabeled and paired with solid-phase antibody 5C11 which recognizes β-LPH 39–56. The binding site of this antibody ensures that β-EP cannot be measured in the β-LPH assay. The detection limit is 0.8 ± 0.1 pmol/l (n= 9) and the within-assay coefficient of variation is <10% at concentrations 1.7–870 pmol/l. RESULTS In normal subjects, β-EP and β-LPH levels were < 1.4–1.7 pmol/l (< 5–6 ng/l) and 2.5–6.7 pmol/l (29–77 ng/l) at 0930 h and < 1.4–1.7 pmol/l (<5–6 ng/l) and 1.9–4.5 pmol/l (22–49 ng/l) at 1600 h, respectively. In patients with ACTH-related pathologies concentrations of β-EP and β-LPH paralleled those of ACTH. The ratio of β-LPH: β-EP in plasma varied between 3.2:1 and 38:1 in these patients demonstrating that β-LPH is the major circulating peptide derived from the C-terminal of pro-opiomelanocortin in man. However, in two patients undergoing bilateral inferior petrosal sampling with corticotophin releasing hormone for diagnosis of Cushing's disease β-EP concentrations increased rapidly during the first 5 minutes of the test, resulting in a sharp decrease in the β-LPH: β-EP ratio. These results suggest that β-EP is preferentially released in response to acute corticotrophin releasing hormone stimulation. CONCLUSIONS It is concluded that two-site IRMAs for β-EP and β-LPH provide an easy approach to study the dynamic changes in processing of β-LPH to β-EP.