Alpha-linolenic acid and marine long-chain n-3 fatty acids differ only slightly in their effects on hemostatic factors in healthy subjects.

The effects of alpha-linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3), and docosahexaenoic acid (DHA, 22:6n-3) on hemostatic factors were compared. Healthy subjects (29 women and 17 men aged 20-44 y) received either linseed oil (average ALA intake: 5.9 g/d) or fish oil plus sunflower oil (average EPA + DHA intake: 5.2 g/d) for 4 wk. The supplemented amount of fat was 1.19 mg/kJ (1 g/200 kcal) calculated energy expenditure. Stability of habitual diets was monitored. Blood samples were collected at baseline, at the end of the experimental period, and after a 12-wk follow-up period. Different changes in the study groups were seen only in serum cholesterol and triacylglycerols, platelet fatty acid composition, and ADP-induced platelet aggregation. The treatments did not differ in their effects on collagen-induced platelet aggregation and thromboxane production, aggregation to the thromboxane A2 mimic I-BOP, urinary excretion of 11-dehydro-thromboxane B2 and beta-thromboglobulin, bleeding time, plasma fibrinogen concentration, antithrombin III activity, factor VII coagulant activity, or activity of plasminogen activator inhibitor 1. The results indicate that supplemented ALA from vegetable oil and EPA and DHA from a marine source have largely parallel effects on hemostatic factors.

Abnormal diurnal changes in in-vivo platelet activation in patients with atherosclerotic diseases.

We measured the urinary excretion of beta-thromboglobulin in timed urine samples collected by 2 groups of healthy volunteers, (group I, n = 20, mean age 34 years, group II, n = 15, mean age 64 years) and by patients (n = 40) with symptomatic atherosclerotic diseases. Older healthy subjects were found to excrete high amounts of BTG in comparison to young subjects (302.25 +/- 50.61 vs 219.65 +/- 59.31 ng/day, P less than 0.05). Higher (P less than 0.01) levels of urinary BTG were observed in patients with coronary (427.61 +/- 179.96 ng/day), cerebral (422.13 +/- 223.2 ng/day) and peripheral (454.16 +/- 269.05 ng/day) arterial diseases and in diabetic patients with diffuse vascular complications (613.71 +/- 253.07 ng/day). The diurnal variability of BTG excretion, measured as coefficient of variation (C.V. %) of the mean daily excretion rate, was higher (P less than 0.001) in atherosclerotic patients (70.59 +/- 26.57) as compared with the similar values observed in the control groups of young (32.05 +/- 14.54) and older subjects (26.38 +/- 8.4). Comparable diurnal variabilities of the creatinine excretion rate were observed in the control groups and in patients. These data indicated that in vivo platelet activation may occur in atherosclerotic patients with a distinctive high fluctuation rate.

Increased urinary beta-thromboglobulin excretion in diabetes assayed with a modified RIA kit-technique.

A modification of the commercial radioimmunoassay (RIA) kit adapting to the assay of beta-thromboglobulin (beta-TG) in the urine, is described. The urine was concentrated by dialysis against dry Sepharose G 200. 131I-albumin was used as marker. Experiments in which 125I-beta-TG and 131I-albumin were added simultaneously showed a parallel recovery of both markers in the final sample. The sensitivity of the RIA was enhanced by incubation overnight of the urine sample with the antiserum. Increased urinary beta TG values were found in 20 patients with diabetes mellitus as compared to 20 young healthy normals. Increased urinary beta-TG values correlated with decreased creatinine clearance, but the difference between diabetics and normals was still observed when patients with a creatinine clearance of less than 100 ml/min were excluded. The urine beta TG level was correlated with the presence of neovascularization within the diabetic group.

Urine beta-thromboglobulin concentration or beta-thromboglobulin/creatinine ratio in single voided urine samples cannot be reliably used to estimate quantitative beta-thromboglobulin excretion.

Different procedures are currently used in the urine beta-thromboglobulin (BTG) assay. We investigated the reliability of limited urine collections and of different expressions of urine BTG results (concentration, urine BTG/creatinine ratio) for the measurement of hourly or daily BTG excretion rates. BTG was measured by a sensitive RIA method in various urine collections of normal subjects (n.80) and patients (n.120) with miscellaneous diseases where an enhanced in-vivo platelet activation could be expected. The BTG concentration in a 6-hour urine collection appeared to change in relation to the urine flow rate (r = -0.53 in normals, r = 0.27 in patients, p less than 0.01) and urine osmolality (r = 0.46 in normals, r = 0.31 in patients, p less than 0.01). In both normals and patients not a very good correlation was observed between the urine BTG/creatinine ratio and the BTG excretion rate (r = 0.54 and r = 0.48; p less than 0.001, respectively). Variable coefficients of correlation (r = 0.83-0.34) were observed between the BTG excretion rate of single voidings of the morning, afternoon-evening and night and the daily BTG excretion both in normals and patients. Reliable measurements of the BTG in urine should be expressed as the hourly excretion rate in a given period of the day for limited urine collections or as the daily excretion for 24-hour urine collections.

Urinary beta-thromboglobulin correlates with impairment of renal function in patients with diabetic nephropathy.

Beta-thromboglobulin (beta TG) is a platelet-specific protein and since its concentration in plasma rises when platelets are activated, it has been used as an indicator of platelet involvement in vascular disease. Since platelets might be involved in the pathogenesis of diabetic microvascular disease we measured urinary beta TG in 20 insulin-dependent diabetics with nephropathy and compared the results with those from 20 normal subjects. Measurement of beta TG in urine was undertaken to avoid errors induced by blood sampling and to gain information over a prolonged period using a single assay. Measurements were made of beta TG, beta 2-microglobulin and total protein in urine collected for 24 h and creatinine and beta 2 microglobulin in plasma. Survival of indium-111-labeled platelets was measured in nine patients. Urinary beta TG was significantly (p less than 0.02) increased in the 20 patients compared with 20 normal volunteers (median value 1.3 vs 0.8 microgram/24 h). There was a strong correlation between urinary beta TG excretion and plasma creatinine concentration (r = 0.8, p less than 0.0001) and plasma beta 2-microglobulin concentration (r = 0.9, p less than 0.0001). Urinary beta TG concentration did not correlate with platelet survival. The results indicate that although urinary beta TG is significantly increased in patients with diabetic nephropathy its concentration in urine correlates with indicators of glomerular filtration rather than with a test of platelet activation.

Cigarette smoking increases thromboxane A2 formation without affecting platelet survival in young healthy females.

Smoking is a risk factor for the development of atherosclerotic cardiovascular disease, in men as well as in women. An increased urinary excretion of the thromboxane metabolite 2,3-dinorthromboxane B2 (Tx-M) has been observed in smokers of both genders, suggesting that cigarette smoking may facilitate cardiovascular disease via an action on the platelets. The present study addressed the hypothesis that the increased Tx-M excretion in female smokers reflects a true facilitation of platelet reactivity in vivo, rather than an increased destruction of the platelets. In healthy female volunteers (aged 20-46 years, 18 smokers and 17 non-smokers) platelet life-span and indices of platelet activity were determined, together with plasma levels of plasminogen activator inhibitor-1 (PAI-1), fibrinogen, peripheral blood cell counts and hematocrit. The urinary excretion of Tx-M was higher in smokers than in non-smokers (361 vs. 204 pg/mg creatinine, respectively, p < 0.05), while plasma and urinary beta-thromboglobulin, plasma platelet factor 4, platelet mean life-span and platelet production rate did not differ between the groups. PAI-1 activity, white blood cell count and hematocrit were higher in smokers than in non-smokers (p < 0.05). These data indicate that smoking facilitates platelet formation of thromboxane A2 without affecting platelet survival; i.e. it increases the activity of platelets without affecting their viability to a measurable extent. Such an increase in platelet activity, operating in parallel to a reduced fibrinolytic activity and a higher hematocrit and white blood cell count, may play an etiological role in smoking-induced cardiovascular disease in women.

A new assay for beta-thromboglobulin in urine.

Measurements of beta-thromboglobulin (beta TG) excretion in urine may be of value for "field" studies and due to problems with sampling artifacts for beta TG in plasma. Previous studies have used a radioimmunoassay designed for plasma without characterizing the "beta TG" immunoreactivity in urine. We describe modifications of the assay which increase its sensitivity and a sample work-up procedure using Sephadex G-25M columns separating high molecular weight (HMW) components (presumably intact beta TG) from low molecular weight (LMW) immunoreactivity (i.e. beta TG fragments and/or non-specific interferences). The sensitivity of the assay (with 2.5 ml sample) is less than 12 pg/ml HMW beta TG. Inter- and intraassay coefficients of variation were 7-10%. Only 33 (range 5-75)% of beta TG immunoreactivity in urine represented HMW beta TG. LMW immunoreactivity may be related to salt and other non-specific influences in the sample. Recoveries of beta TG were quite variable (9-100%) in unextracted urines, but high and reproducible (80 +/- 2%) in the HMW fraction. Thus, nonspecific interferences with beta TG measurements in certain urines are overcome by the separation step. Using Sephadex fractionation beta TG immunoreactivities in night urines (n = 15) were: 20 +/- 3 pg/ml in the HMW fraction, 70 +/- 8 pg/ml in the LMW fraction, and 85 +/- 10 pg/ml by direct assay. HMW beta TG increased in daytime samples (to 30 +/- 5 pg/ml; p less than 0.01), but no diurnal variation was seen in the LMW fraction or with the direct assay. Thus, selective analysis of HMW beta TG in urine circumvents problems with nonspecific immunoreactivity and apparent interferences with measurements of intact beta TG. The present more selective assay for HMW immunoreactivity increases the possibility of detecting physiological changes in beta TG release in vivo by urinary measurements.

Immediate effects of heparin and LMW heparin on some platelet and endothelial derived factors.

Heparin and a low molecular heparin fragment, injected intravenously in volunteers, increased the plasma concentrations of platelet factor 4, but did not induce platelet activation as judged from excretion of 2,3-dinor-TxB2 (a major thromboxane A2 metabolite) and beta-thromboglobulin (btg) in urine and from btg levels in plasma. Heparin prolonged, within the normal range, the bleeding time in all six subjects. Platelet aggregation in platelet rich plasma was potentiated by both heparins, but platelet number, mean platelet volume and platelet distribution width were not affected. No evidence for endothelial release of prostacyclin was obtained as judged from urinary excretion of 2,3-dinor-6-keto-PGF1 alpha (a major prostacyclin metabolite), and plasma concentrations of tissue plasminogen activator, its inhibitor (PAI-1) and the von Willebrand-factor were unchanged.

A sensitive enzyme immunoassay system for the measurement of beta-thromboglobulin in plasma and urine.

A sensitive enzyme immunoassay system for the measurement of beta-thromboglobulin (beta-TG) in plasma and urine was developed using purified antibodies to beta-TG linked to beta-D-galactosidase from Escherichia coli as label. The assay system consisted of polystyrene beads with immobilized antibody F(ab')2 fragments and the antibody Fab' fragments labeled with beta-D-galactosidase. The assay had a sensitivity of 10 pg/assay tube. beta-TG levels in plasma and urine could be determined with 1-2.5 microliters and 100 microliters samples, respectively. The results obtained with the enzyme immunoassay correlated well with those of a radioimmunoassay (r = 0.946, slope = 0.846, y-intercept = 4.71 ng/ml; n = 60). Plasma beta-TG levels in healthy subjects determined with the present assay system were 38.4 +/- 12.2 ng/ml (+/- 1 SD). Levels were markedly increased in 19 out of 22 patients with myeloproliferative syndrome. Urinary beta-TG levels in healthy subjects were estimated to be 0.17 +/- 0.05 ng/ml (+/- 1 SD). Urine levels were increased in 16 out of the 19 patients with myeloproliferative syndrome whose plasma levels were high. These findings indicate that urinary beta-TG levels may be reflection of the beta-TG in plasma and can be an indicator of platelet activation.

Intermittent pneumatic compression in chronic venous insufficiency favorably affects fibrinolytic potential and platelet activation.

Nineteen patients with symptoms of chronic venous insufficiency (CVI) were treated with 13-week cycles of intermittent pneumatic compression (IPC) during 2 h sessions twice weekly, with most treatments at home. At study completion, quantitative subjective scores for total symptomatology were improved in 16/19 patients (84%). Enhancement of fibrinolytic potential in vivo was detected in 86% of observations on specimens from CVI patients over 2 h of IPC, with accelerated euglobulin clot lysis times (ELT) noted within 15 min of initiating compression. The enhanced fibrinolytic potential was attributed to increased urokinase plasminogen activator (u-PA), probably released from perturbed endothelial cells by IPC. Significant decreases in total t-PA antigen (mass concentration) but not t-PA activity, were produced by IPC in CVI patients only (P = 0.0001), with greater effects noted in the non-anticoagulated versus the anticoagulated cohort. Plasminogen activator inhibitor type 1 (PAI-1) levels rose rapidly after IPC only in the controls and non-anticoagulated CVI patients. PAI-1 decreased in those receiving anticoagulation. No platelet perturbation was detected during IPC by measuring levels of beta-thromboglobulin or the thromboxane A2 metabolite, 11-dehydrothromboxane B2; however, significant (P < 0.003) decreases in plasma prostacyclin (PGI2) levels (measured as the stable 6-ketoprostaglandin F-1-alpha-metabolite) were observed after 15 min of IPC in non-anticoagulated CVI patients only. There was no evidence of increased thrombin generation by IPC, determined by urinary excretion of fibrinopeptide A and prothrombin fragment 1. Concurrent anticoagulation appears to mediate more favorable biochemical alterations in CVI, although subjective improvement did not correlate with anticoagulation. The mechanism(s) by which these physiologic changes compliment the mechanical effects of IPC remain to be elucidated and will require adequately controlled and powered studies.

[Plasma and urine beta-thromboglobulin determination in the detection of thrombocyte hyperactivation in diabetic nephropathies]. / A plasma és a vizelet béta-thromboglobulin meghatározás a diabeteses nephropathiát kísérö thrombocyta hyperaktiváció kimutatásában.

Serum creatinine, immunoreactive serum and urine beta-2-microglobulin, plasma and urine thromboglobulin, plasma thromboxane-B2 levels and daily protein excretion were determinated in 61 insulin treated diabetic patients, comparing the different patient groups (complication free, nephropathy without azotaemia and nephropathy with azotaemia) with the control subjects. In the groups of all diabetic patients plasma and urine beta-thromboglobulin and plasma thromboxane-B2 levels were higher that in the controls. There was a positive significant correlation between urine beta-thromboglobulin and beta-2-microglobulin in the group without complication, and between the plasma beta thromboglobulin and beta-2-microglobulin, and plasma beta thromboglobulin and thromboxane levels in the diabetic group with azotaemia. In contradiction to some previous assumptions, the increased level of plasma beta-thromboglobulin reflects a real platelet hyperactivation also in patients with diabetic nephropathy. At the same time urine beta-thromboglobulin also increases. Determination of urine beta-thromboglobulin is more simple with less possibility of methodological error.