Thrombin generation before and after multicomponent blood collection.

The development of apheresis technology has increased efficiency in donor blood use by collecting specific blood components in several combinations. The question of donor safety raised by the contact of donor blood with foreign, only in part biocompatible surfaces remains. The aim of this study was to estimate the effect of multicomponent blood collection on thrombin generation performing an overall function test of coagulation. DONORS, METHODS: 26 blood donors were included. Per apheresis two units of platelets and one unit of RBCs were collected by two cell separators (Amicus and Trima Accel). Each donor underwent the procedure on both apheresis systems. Samples were collected before, immediately after, and 48 hours after apheresis. Thrombin generation was measured by means of calibrated automated thrombography (CAT). RESULTS: CAT-data changed only slightly and no significant changes were seen before, immediately after, and 48 hours after apheresis. The parameters did not differ significantly between the two different apheresis devices. CONCLUSION: No change in parameters of continuous thrombin generation occurred, suggesting that apheresis did not lead to severe alterations in the haemostatic system.

Ghrelin does not regulate the GH response to insulin-induced hypoglycaemia in children but could be involved in the regulation of cortisol secretion.

OBJECTIVE: Ghrelin activates the growth hormone secretagogue receptor GHS-R. It strongly stimulates GH secretion and has a role in energy homeostasis. The relationship between plasma ghrelin and cortisol levels during insulin-induced hypoglycaemia in prepubertal and pubertal children has not yet been investigated. The aim of the present study was to establish whether insulin-induced hypoglycaemia stimulates ghrelin secretion and whether changes in ghrelin concentrations are related to changes in GH and cortisol in children. DESIGN AND PATIENTS: We studied a group of 20 children and adolescents (five girls, 15 boys, mean age 10.8 +/- 3.7 years) undergoing insulin tolerance tests (ITTs) for clinical investigation of GH deficiency. MEASUREMENTS: Stimulation tests were performed to investigate the relationship between ghrelin, GH, cortisol and glucose levels according to age and pubertal stage by determining the ghrelin profiles during insulin-induced hypoglycaemia (at 0, 60 and 120 min). RESULTS: Ghrelin was significantly and inversely related to body weight, height, body mass index (BMI) and age of children (P < 0.05). Significant changes in ghrelin levels (P = 0.00013) were found after the insulin bolus, with a decline at 60 min and an increase to baseline values at 120 min. Changes in cortisol levels were negatively correlated with changes in ghrelin at 60 min (r = -0.59, P = 0.004) and at 120 min (r = -0.605, P = 0.003). CONCLUSIONS: This study shows that ghrelin might not regulate the GH response to insulin-induced hypoglycaemia in prepubertal and pubertal children. A role for ghrelin in the regulation of cortisol secretion can be hypothesized concerning the negative correlation between changes in ghrelin and cortisol. Furthermore, the results imply that ghrelin secretion is age dependent and is a function of growth.

Thrombin generation in factor VIII-depleted neonatal plasma: nearly normal because of physiologically low antithrombin and tissue factor pathway inhibitor.

BACKGROUND: Bleeding in hemophilic neonates has a low incidence. A possible explanation for this could be the peculiarities of the neonatal hemostatic system, especially low levels of the inhibitors tissue factor pathway inhibitor (TFPI) and antithrombin (AT). OBJECTIVE: We investigated the influence of an elevation of these inhibitors to adult levels on the thrombin generation (TG) in normal neonatal plasma and factor (F) VIII-depleted neonatal plasma by means of incubation with anti-FVIII-antibodies. PATIENTS/METHODS: TG was measured after activation with low amounts of tissue factor (TF) by using Calibrated Automated Thrombography. RESULTS: TG in FVIII-depleted neonatal plasma was nearly as high as in normal neonatal plasma. TG decreased after elevation of AT in both neonatal plasmas. After elevation of TFPI TG decreased much more in FVIII-depleted neonatal plasma than in normal neonatal plasma. After elevation of both inhibitors their synergistic effect led to a stronger decrease of TG in FVIII-depleted neonatal plasma. TG measured in plasma of one hemophilic newborn showed the same pattern as in FVIII-depleted neonatal plasma. CONCLUSION: Our observation provides a biochemical basis for the rare bleeding in hemophilic neonates and shows the important role of the natural inhibitors in the hemostatic system of hemophilic patients.

Elevated thrombin-forming capacity of tissue factor-activated cord compared with adult plasma.

Clinically observed excellent hemostasis in neonates despite low levels of clotting factors is not completely understood so far. Therefore, we investigated whether physiological low levels of the inhibitor protein C (PC) facilitate thrombin formation in tissue factor (TF)-activated plasma samples. PC was activated by endogenously generated thrombin after addition of soluble thrombomodulin (TM). The capability of activated PC (APC) to suppress thrombin formation was significantly more pronounced in adult than in cord plasma. Addition of 4 nm of TM decreased the thrombin potential (TP) in cord plasma by 10%, and in adult plasma by 52% in the presence of 5 pm TF. We demonstrate that this low anticoagulant action of PC is attributable to the low levels of tissue factor pathway inhibitor (TFPI) and antithrombin (AT) physiologically present in cord plasma. Addition of 4 nm TM decreased the TP by 58% in cord plasma adjusted to contain TFPI and AT at adult levels in the presence of 5 pm TF. Thus, the combined low anticoagulant action of the three inhibitors APC, TFPI, and AT in cord plasma allows enhanced thrombin formation associated with shorter clotting times compared with adult plasma when low amounts of TF are applied to initiate clot formation. Although our laboratory experiments do not allow definite conclusions for various clinical situations, our data might contribute to explain excellent hemostasis in neonates despite low levels of procoagulants.

Low tissue factor pathway inhibitor (TFPI) together with low antithrombin allows sufficient thrombin generation in neonates.

Neonates have an excellent hemostasis despite, in comparison to adults, markedly decreased and delayed ability to generate thrombin. Only 30-50% of peak adult thrombin activity can be produced in neonatal plasma by means of conventional in vitro assays. We show that in contrast to conventional activation, activation with small amounts of lipidated tissue factor (<10 pmol L(-1)) results in shorter clotting times and faster activated factor X- and thrombin generation in neonates compared with adults due to the concomitant action of low tissue factor pathway inhibitor and antithrombin. The concentrations of both inhibitors in cord plasma are approximately 50% of the respective adult values. After addition of 2.5 pmol L(-1) lipidated tissue factor, cord plasma clotted approximately 90 s earlier than adult plasma and the amount of free thrombin generated was approximately 90% of adult value (291 +/- 14 vs. 329 +/- 16 nmol L(-1) min(-1), P < 0.01). Our results might help to explain the clinically observed excellent hemostasis of neonates despite low levels of procoagulant factors.

Respective roles of factors II, VII, IX, and X in the procoagulant activity of FEIBA.

Activated prothrombin complex concentrates (APCCs) are effective in the therapy of bleeding episodes in hemophilic patients with inhibitors. We investigated the respective roles of factor II, factor VII, factor IX, and factor X in the procoagulant activity of the APCC FEIBA. Factor II, factor VII, factor IX, and factor X were reduced in platelet-poor plasma, and the thrombin potential (TP) was determined using a chromogenic substrate in the absence or presence of FEIBA. Reduction of factor II resulted in a significant decrease of the TP without influencing the lag phase until the onset of thrombin generation. The reduction of factor VII showed no effect on the TP, but resulted in a prolongation of the lag phase. Changes of factor IX or factor X concentrations showed neither an effect on the TP nor on lag phases. Our study demonstrates that thrombin generation in the presence of FEIBA mainly depends on prothrombin.

[Selected blood coagulation problems in newborn infants]. / Ausgesuchte Gerinnungsprobleme beim Neugeborenen.

The paper deals with specific features of the neonatal coagulation system. The plasma concentration of the majority of coagulation factors is lower in neonates than in adults, whereby near-adult values are achieved for most components by 6 months of life. Bleeding episodes in haemophilic newborns are rare but may be of a profound nature, thus a proper communication between all involved physicians may aid in the management of such patients. On the other hand thromboembolic episodes might complicate neonatal intensive care, in particular in patients with an indwelling central line. Also inherited thrombophilia might contribute to the development of neonatal thrombosis. Prophylaxis and therapy of vitamin K deficiency bleeding are discussed.

Changes in serum interleukin-6 concentrations in obese children and adolescents during a weight reduction program.

OBJECTIVE: To investigate the effect of short term energy restriction combined with physical activity on serum concentrations of Interleukin-6 (IL-6) in obese children and adolescents. DESIGN: Longitudinal intervention study of 3.8-5 MJ daily with exercise. SUBJECTS: Forty-nine white obese children and adolescents (31 girls, age 11.9+/-1.8 y; 18 boys, age 11.6+/-1.7 y). MEASUREMENTS: Indexes of obesity, IL-6, leptin, estradiol, systolic and diastolic blood pressure, heart rate at baseline and after 3 weeks. RESULTS: All determined parameters decreased significantly during the 3 week program (IL-6: 3.9+/-4.7 vs 2.0+/-2.2 pg/ml; P<0.05). Body mass index (BMI) fat mass, percentage fat mass (indexes of obesity), and leptin were not related to IL-6 before the program. In contrast, IL-6 concentrations correlated significantly with indexes of obesity and leptin after weight loss. IL-6 concentrations did not correlate with estradiol, systolic and diastolic blood pressure, and heart rate. Changes in IL-6 concentrations correlated significantly with changes in BMI (r=0.25, P<0.05). CONCLUSION: An improved body composition induced by restriction of energy intake and increase in physical activity is associated with more favorable serum concentrations of IL-6 in obese children and adolescents.

The effects of changes in body mass and subcutaneous fat on the improvement in metabolic risk factors in obese children after short-term weight loss.

The pattern of subcutaneous fat (SAT) is related to metabolic risk factors in obese children. Because weight loss improves the risk-factor profile, we sought to determine whether changes in SAT or SAT-pattern contribute to the improvement in the risk-factor profile after 3 weeks of a low-calorie diet and physical activities. In 22 obese boys (mean age, 11.9 years) and 40 obese girls (mean age, 12 years), fat mass (by means of impedance) and fat distribution (waist and hip circumference) were assessed. The thickness of 15 different subcutaneous adipose tissue layers (SAT-layers) was measured using a Lipometer (Moeller Messtechnik, Graz, Austria). SAT and SAT-pattern (arm-SAT, trunk-SAT, leg-SAT) were calculated. Blood samples were taken for the determination of insulin, glucose, triglycerides, and cholesterol. After 3 weeks, fat mass, waist and hip circumference, SAT, arm-SAT, trunk-SAT (all P <.0001), and leg-SAT (P <.01) were reduced. Besides glucose, metabolic parameters were lowered (all P <.001) but changes in metabolic parameter were interrelated in boys and girls. Age- and sex-adjusted regression revealed that changes in body mass contributed to the variability in changes of insulin (adjusted R(2) =.15, P =.0015). For the change in triglycerides, changes in cholesterol together with subtle alterations in glucose and changes in leg-SAT were found to be the main determinants (adjusted R(2) =.587, P <.0001). The results indicate that the change in the atherogenic and metabolic risk factor profile is largely independent from the concomitant loss in SAT. The reduction in body mass explained only a small part of the variability in changes of insulin, but leg-SAT might participate in the lowering of triglycerides, especially in boys. The contribution of SAT-pattern to the risk factor profile is an issue that needs further investigation.

Determinants of homocysteine during weight reduction in obese children and adolescents.

Plasma homocysteine levels have been shown to be associated with indexes of obesity and insulin resistance in obese children and adolescents. We, therefore, investigated the contribution of changes in body composition, markers of insulin resistance, folate, and vitamin B(12) to changes in homocysteine during a weight reduction program in obese children and adolescents. Thirty-seven obese white girls (mean SD; age, 12 +/- 1.8 years, body mass index [BMI], 26.9 +/- 5.25) and 19 obese white boys (age, 11.9 +/- 1.7 years; BMI, 26.2 +/- 5.2) were investigated for body composition, fasting total plasma homocysteine (tHcy), insulin, C-peptide, folate, and vitamin B(12) before and after a 3-week weight reduction program including physical activities. During weight reduction BMI, fat mass (FM), percentage fat mass, insulin, and C-peptide decreased significantly, whereas homocysteine and vitamin B(12) showed a significant increase. Folate and lean body mass (LBM) remained unchanged. tHcy concentration before weight reduction was a function of age, folate, and C-peptide, whereas tHcy concentration after weight reduction was a function of folate and baseline LBM. Changes in tHcy during weight reduction correlated significantly with baseline LBM and were related inversely to changes in LBM during weight reduction. Children who increased LBM showed lower increases in tHcy compared with children who lost LBM. In multiple linear regression analysis, only baseline LBM contributed independently and significantly to changes in tHcy. Our study suggests that LBM has a significant impact on tHcy metabolism during weight reduction.

Effects of weight loss on leptin, sex hormones, and measures of adiposity in obese children.

Adipose tissue influences steroid conversion by paracrine and autocrine mechanisms. Leptin is secreted by adipocytes and influenced by sex hormones and adiposity. Short-term weight loss in the treatment of childhood obesity reduces leptin and adipose tissue. We therefore asked, Do alterations in sex hormones occur owing to weight loss? and can these alterations be explained by changes in fat mass or sc fat and are alterations in sex hormones directly related to the fall in leptin? Twenty obese boys and 40 obese girls were studied before and after 3 wk of low-calorie diet and physical activity. The weight loss program significantly lowered fat mass, abdominal fat distribution, sc fat (all p < 0.0001), leptin, insulin, and estradiol (all p < 0.0001) but not testosterone. Changes in leptin were related to changes in body mass and to changes in fat mass in boys. In girls, changes in leptin were related to changes in sc fatness and also to changes in insulin. In boys, the reduction in sc fat was positively correlated to changes in testosterone (r = 0.54; p < 0.01) and inversely related to the fall in estradiol (r = -0.41; p < 0.05). In girls, changes in testosterone (r = 0.33; p < 0.05) and in estradiol (r = 0.40; p < 0.01) were related to changes in insulin. Stepwise regression showed that initial leptin was the best determinant for the fall in leptin (adjusted R2 = 0.87; p < 0.0001). The results show that alterations in sex hormones are related to changes in certain fat depots in boys whereas in girls changes in insulin might participate in changes in sex hormones. A greater fall in leptin owing to short-term weight loss is not associated with greater alterations in sex hormones and initial leptin is the best determinant to explain the variability in changes in leptin. The possibility of sex differences in changes in sex hormones secondary to the reduction in fatness warrants further study.

Subcutaneous adipose tissue layers as a stable correlate of leptin in response to short term energy restriction in obese girls.

AIMS: We studied the relationship of subcutaneous adipose tissue layers (SAT-layers) measured at 15 specified body sites with leptin before and after a weight loss program for three weeks. SUBJECTS AND METHODS: In 70 obese girls, SAT-layers were measured by means of the optical device, lipometer. Fat mass (FM) was estimated by means of bioelectrical impedance. RESULTS: At the beginning of the study, all estimates of adiposity, insulin, and SAT-layers from the upper body (from 1-neck to 6-lateral chest) were correlated to leptin at a P-value of<0.0001. Percentage FM together with SAT-layer 4-upper back and insulin explained 75% of the variation in leptin (P<0.0001). After three weeks, estimates of adiposity and leptin were reduced (all P<0.0001). Most SAT-layers were reduced, but SAT-layers 8-lower abdomen and 9-lower back were significantly increased. Changes in leptin were best explained by initial leptin, but percentage change (Delta) in insulin, Delta SAT-layer 1-neck, and Delta SAT-layer 3-biceps contributed to the Delta leptin (adj. r(2)=0.47, P<0.0001). In the weight-reduced state, circulating leptin was best explained by three SAT-layers and insulin (adj. r(2)=0.67, P<0.0001). DISCUSSION: The results suggest that Delta changes in leptin are attributable to changes in the endocrine state and subcutaneous fat, and SAT-layers may serve as a stable correlate of leptin in the weight-reduced state.

Interrelationship between estimates of adiposity and body fat distribution with metabolic and hemostatic parameters in obese children.

Adiposity in childhood is often associated with metabolic abnormalities and accompanied by a dysregulation of the coagulation and fibrinolytic systems. We studied the interrelationship of metabolic and hemostatic parameters and explored their relationship with measures of adiposity and fat distribution in obese children. In 34 obese boys (mean age, 11.7 years) and 57 obese girls (12.1 years), blood samples were determined for insulin, glucose, triglycerides, fibrinogen, plasminogen activator inhibitor-1 (PAI-1), and tissue-type plasminogen activator-antigen (tPA-Ag). Body composition was assessed by means of impedance. Waist (Wc) and hip circumference were measured. The thickness of subcutaneous adipose tissue-layers (SAT-layers) was measured at 15 different body sites (from 1-neck to 15-calf) by means of the optical device, Lipometer. Overall subcutaneous fatness (SAT) was calculated and SAT-distribution was estimated by means of factor analysis. Significant correlations were found between different measures of adiposity and Wc with metabolic parameters. Fibrinogen was mainly associated with upper body subcutaneous fatness (factor 1) in boys. In girls, hemostatic parameters were associated with nearly all measures of adiposity and also with factor 1 and SAT. Regression analysis showed that factor 1 together with PAI-1 (both P <.0001) contribute to fibrinogen (adjusted [adj], R(2) =.30). PAI-1 together with trigylcerides (both P <.0001) and age (P <.04) were main determinants for tPA-Ag (adj, R(2) =.41). tPA-Ag (P <.0001) together with glucose (P <.001, negative slope), fibrinogen (P <.001, negative slope), and percentage fat mass (%FM) (P <.01) contributed to PAI-1 (adj, R(2) =.54). These results favor the concept of an interrelationship between metabolic and hemostatic parameters resulting from increased adiposity, perhaps influenced by pubertal development of children. Although upper body subcutaneous fatness was found to be a main correlate of metabolic and hemostatic parameters, it remains to be investigated whether this type of subcutaneous fat distribution is involved in the expression of metabolic and hemostatic risk factors and participates in the dysregulation of the hemostatic system in the state of childhood obesity.

Alpha-2-macroglobulin inhibits the anticoagulant action of activated protein C in cord and adult plasma.

Healthy newborns have a very low risk of thrombosis. It has been suggested that this is partly due to the anticoagulant effect of alpha-2-macroglobulin (a2-M). This broad-spectrum protease binding glycoprotein is physiologically elevated in newborns over adult values and has been shown to complex generated alpha-thrombin. In our present study, we point out that a2-M also acts as a procoagulant by inhibiting activated protein C (APC). In all experiments performed in cord and adult plasma the anticoagulant action of APC was diminished in a dose-dependent manner when a2-M levels were successively elevated, reflected in increased thrombin potential (TP), and enhanced at low a2-M levels, reflected in decreased TP.

Tumor- and treatment-related side effects after multimodal therapy of childhood intracranial germ cell tumors.

UNLABELLED: Multimodality treatment approaches have dramatically improved the outcome of patients with intracranial germ cell tumors and are resulting in an increasing number of long-term survivors. The aim of the present study was to evaluate prospectively the development of side effects in children, adolescents and young adults after treatment for intracranial germ cell tumors. Nine patients with a median age of 14 y at diagnosis and a median follow-up of 7.25 y underwent a detailed long-term evaluation including physical and neuro-ophthalmologic examinations, routine laboratory and endocrine stimulation tests, neuropsychometric testing, audiometry and spirometry at repeated intervals. Endocrine deficiencies requiring hormone replacement therapy occurred in all patients. Neuro-ophthalmologic side effects were observed in 8 of the 9 patients, urinary electrolyte wasting in 4 of the 9, alopecia in 3 of the 9 and high-frequency hearing loss in 2 of the 9. Neuropsychologic examinations revealed pathologic results in all five tested patients. CONCLUSION: The present study indicates that former intracranial germ cell tumor patients suffer from remarkable long-term side effects, and that some of these late effects can develop or worsen months or years after cessation of oncologic therapy. Since life quality is an important parameter of cancer survival, careful follow-up of long-term survivors is mandatory, aimed at counteracting side effects as early as possible and therefore at minimizing long-term morbidity, which may considerably compromise quality of life.

Effects of short-term energy restriction and physical training on haemostatic risk factors for coronary heart disease in obese children and adolescents.

OBJECTIVE: To study the changes of haemostatic risk factors for coronary heart disease during a weight reduction programme in obese children and adolescents. DESIGN: A short-term longitudinal study. SUBJECTS: Thirty-seven obese white girls (age, 12+/-1.8 y; body mass index (BMI), 26.9+/-5.25) and 19 obese white boys (age, 11.9+/-1.7 y; BMI, 26.2+/-5.2). MEASUREMENTS: Fibrinogen, factor VII coagulant activity, von Willebrand factor antigen, and soluble P-selectin were determined before and after a 3 week programme including energy restriction and physical activities. RESULTS: All determined haemostatic risk factors decreased significantly during the programme. Changes in risk factors were correlated to changes in body composition. Children and adolescents with the highest initial concentrations showed the greatest decreases. CONCLUSION: Energy restriction combined with physical activity improves the haemostatic risk profile in obese children and adolescents.

Effects of alpha(2)-macroglobulin and antithrombin on thrombin generation and inhibition in cord and adult plasma.

Thromboembolic complications rarely occur during infancy and childhood. It has been reported that increased capacity of cord plasma to inhibit thrombin due to elevated alpha(2)-macroglobulin (alpha(2)-M) levels may in part provide protection from thrombosis. In antithrombin (AT)-deficient plasma, alpha(2)-M exhibits anticoagulant action by complexing substantial amounts of generated free thrombin. It has been suggested that alpha(2)-M has the same impact on thrombin inhibition as AT, the most important thrombin inhibitor in adult plasma. The aim of our study was to examine this assumption by determining time-courses of free thrombin generation and prothrombin activation. Additionally, the amount of thrombin complexed to alpha(2)-M was assessed by comparing the heights of the end-level of amidolytic activity curves (AACs) after extrinsic activation of platelet poor plasma in the presence of different concentrations of AT or alpha(2)-M. Increasing the AT content by 30% resulted in significantly suppressed generation of free thrombin and prothrombin fragment 1+2 (F1+2) in cord and adult plasma. In contrast, increasing the alpha(2)-M content in plasma containing physiologic amounts of AT by the same percentage had no effect on free thrombin generation and on F1+2 generation in both cord and adult plasma. In addition, the effect of AT supplementation on the end-level of the AACs was significantly higher compared to the effect of alpha(2)-M supplementation. Since alpha(2)-M, in contrast to AT, had no effect on free thrombin generation and prothrombin activation, our study suggests that the action between alpha(2)-M and thrombin might not be fast enough to prevent thrombin from its feedback activation in both cord and adult plasma and, therefore, in cord and adult plasma containing physiological amounts of AT alterations of the alpha(2)-M content had no effect on thrombin generation and inhibition.