Siegfried Gallistl

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

Summary.  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 ∼90 s earlier than adult plasma and the amount of free thrombin generated was ∼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.

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 significiantly 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.

Activation of the clotting system during extracorporeal membrane oxygenation in term newborn infants

OBJECTIVES To determine the degree of clotting activation that occurs with the usual anticoagulation regimen with systemic heparinization. METHODS To allow a standardized comparison of the patients, this study focused on the first 48 hours of extracorporeal membrane oxygenation (ECMO) in term newborn infants. The ECMO perfusion circuit consisted of a roller pump, silicone membrane lungs, and silicone rubber tubing. Coagulation was controlled routinely by measuring prothrombin time, fibrinogen, antithrombin III, and reptilase time. Platelet counts, activated clotting time, and heparin concentration were controlled regularly. The following specific activation markers of the clotting system were measured: prothrombin activation fragment 1 + 2(F1+2), thrombin-antithrombin III complexes, and D-dimer. Measurements were done before the start of ECMO, after 5 minutes, and at hours 1, 2, 3, 4, 6, 12, 24 and 48. RESULTS All seven term infants had excessively high levels of clotting activation markers within the first 2 hours of ECMO: F1+2, 11.6(+/- O.9) nmol/L (mean +/- SEM); thrombin-antithrombin, 920(+/- 2.2) microg/L; D-dimer, 15.522(+/- 3.689) ng/L. During the next 46 hours of ECMO, F1+2 and thrombin-antithrombin III complexes decreased from those high values, whereas D-dimer did not. The increase of activation markers was accompanied by low fibrinogen, low platelet counts. and prolongation of reptilase time. CONCLUSIONS These findings fit the pattern of consumptive coagulopathy during neonatal ECMO, especially in the first 24 hours.

Calibrated automated thrombin generation in normal uncomplicated pregnancy

Pregnancy is associated with substantial changes in the haemostatic system and a six-fold higher incidence of venous thromboembolism. Conventional global tests, such as prothrombin time and activated partial thromboplastin time, do not definitely detect this hypercoagulable condition. We investigated whether the changes in haemostatic system during pregnancy are reflected in the calibrated automated thrombography (CAT). Thrombin generation was measured in platelet-poor plasma (PPP) of 150 healthy pregnant women without any pregnancy associated diseases by means of CAT. In addition, prothrombin (FII), antithrombin (AT), protein S, protein C, tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor-1 (PAI-1), thrombin-antithrombin complex (TAT), and prothrombin fragments 1+2 (F1+2) were measured. Endogenous thrombin potential (ETP) and peak of thrombin generation increased significantly with gestational weeks, while lag time and time to peak remained unchanged. A significant increase of PAI-1, TFPI, F1+2 and TAT as well as a significant decrease of free protein S, protein S antigen, and protein S activity was observed. Levels of AT and protein C remained stable during pregnancy. Division of population in trimester of pregnancy and analysis of differences between the trimesters showed rather similar results. Our study shows that endogenous thrombin potential does increase with duration of normal uncomplicated pregnancy. Whether parameters of continuous thrombin generation will correlate with thrombembolic disease remains to be shown.

The role of inherited thrombotic disorders in the etiology of Legg-Calvé-Perthes disease.

Inherited thrombophilia has been suggested as a cause of Legg-Calvé-Perthes disease in up to 75% of patients. This prevalence seems high compared to the prevalence of inherited thrombophilia in other thrombotic disorders (average, 30%). Therefore, we investigated 44 patients with Legg-Calvé-Perthes disease for antithrombin-III, protein-C, and protein-S deficiency and activated protein C (APC) resistance. Three (6.8%) of our patients were positive for APC resistance, and one patient (2.2%) showed deficiency of protein-C activity. Our results suggest that inherited thrombophilia is not associated with avascular necrosis of the femoral head (Legg-Calvé-Perthes disease).

Effects of antithrombin and protein C on thrombin generation in newborn and adult plasma.

It has been suggested that antithrombin (AT) and protein C (PC) might be less important in newborn plasma than in adult plasma for the inhibition of clotting activation. To assess the importance of AT and PC for the regulation of thrombin generation in newborn as compared with adult plasma, thrombin generation was determined in the presence of different concentrations of AT and PC. In both, newborn and adult plasmas, reduction of AT and PC resulted in an increase of thrombin generation after intrinsic activation. On the other hand, supplementation of AT and PC decreased thrombin generation in a dose-dependent manner. Clotting times were influenced by different PC concentrations, whereas clotting times were almost not affected by different amounts of AT. Our study suggests that the significance of AT and PC in the regulation of thrombin generation is the same in newborn and adult plasma. Our experimental results also support the notion that administration of AT or protein C concentrates may be beneficial in some clinical situations leading to an increased clotting activation in newborns.

Cystatin C, an early indicator for incipient renal disease in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic disease requiring potential nephrotoxic therapy with nonsteroidal antiinflammatory drugs (NSAIDs) and disease modifying antirheumatic drugs (DMARDs). The rationale of our study was to examine the renal status of patients suffering from prolonged RA by means of plasma cystatin C, a new parameter of renal function. Fifty-six patients affected with RA for more than 5 years, and treated with NSAIDs for more than 50 months, were included in the study. Besides conventional markers of renal function (i.e. plasma creatinine, estimated glomerular filtration rate, creatinine clearance), we analysed plasma cystatin C by an automated, nephelometric immunoassay on a Behring nephelometer. Sixty percent of the RA patients exhibited elevated levels of plasma cystatin C, whereas only three out of 56 patients showed an elevated plasma creatinine, even though the creatinine clearance was decreased in 57% of these patients. Cystatin C exhibited a by far better correlation with creatinine clearance than plasma creatinine. In conclusion, patients with prolonged RA for more than 50 months, show a disturbed renal function despite normal plasma creatinine. Elevated cystatin C indicates such incipient renal disease, and is, not least because of a simple, well reproducible technique, more recommendable for screening purposes than tedious clearance determinations.

Alpha 2-macroglobulin enhances prothrombin activation and thrombin potential by inhibiting the anticoagulant protein C/protein S system in cord and adult plasma.

Protein S (PS) is a vitamin K-dependent plasma protein and serves as a cofactor for the anticoagulant activities of activated protein C (APC). We investigated the effects of different PS concentrations on prothrombin activation and thrombin generation in cord and adult plasma containing APC and different amounts of alpha 2-macroglobulin (a2-M). Prothrombin activation was assessed by monitoring the time-course of prothrombin fragment 1+2 (F1+2) generation. Thrombin generation curves were determined by means of a subsampling technique using the chromogenic substrate S-2238. We demonstrate a dose-dependent inhibition of the anticoagulant action of PS by a2-M: suppression of F1+2 and thrombin generation due to addition of PS was stronger in plasma containing low amounts of a2-M than in plasma with elevated a2-M levels. Since no complex formation between a2-M and PS was observed by means of SDS-PAGE, we attribute decreased anticoagulant action of PS at high a2-M levels to enhanced complex formation between APC and a2-M. Thereby, APC is subtracted from its cofactor PS, resulting in suppressed formation of the anticoagulant APC/PS complex. Thus, our data suggest that a2-M, besides its well-known anticoagulant effects, also acts as a procoagulant by suppressing the formation of the anticoagulant APC/PS complex. Our findings have implications particularly on thrombin generation and inhibition in cord plasma, since a2-M levels in newborns are elevated over adult values and the antithrombotic APC/PS pathway is up-regulated at birth. Therefore, elevated levels of a2-M might restrict the up-regulation of the APC/PS pathway.

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

Summary.  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.

Thrombophilic polymorphisms – factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T mutations – and preterm birth

SummaryAIM OF THE STUDY: To evaluate the influence of three common thrombophilic polymorphisms, factor V Leiden (FV), prothrombin G20210A (PT), and methylenetetrahydrofolate reductase (MTHFR) C677T mutations, on preterm birth of unknown cause. PATIENTS AND METHODS: A single-centre case-control study of women with preterm infants of ≤35 weeks of gestation, in whom obvious maternal, uterine, and fetal causes responsible for preterm birth were excluded (n = 35). The controls were 54 women with term infants hospitalised in the same ward. RESULTS: There were no significant differences between the groups of mothers in history of fetal loss, venous or familial thrombosis, or previous preterm birth. FV was found in 8.6% of the cases, PT in 5.7%, and MTHFR mutation (homozygous) in 4.8% compared with 5.4% (p = 0.292; OR, 1.594; CI 95%, 0.303–8.384), 7.4% (p = 0.379; OR, 0.758; CI 95%, 0.131–4.374), and 4.5% (p = 0.485; OR, 1.050; CI 95%, 0.090–12.276), respectively, in the controls. Differences in the three thrombophilic polymorphisms in the two groups of infants were also not significant. CONCLUSION: We could not demonstrate a distinct association between these thrombophilic polymorphisms and preterm birth.