Christina Cimenti

Thrombin Generation in Severely Obese Children

Since changes of hemostatic parameters in obese patients may contribute to the development of cardiovascular disease [1], we investigated whether there is a relationship between obesity in childhood and alterations in thrombin generation (TG). In a recently published pilot study we have shown that severely overweight children after a significantly prolonged lag phase and a significantly prolonged time to peak, generate significantly higher amounts of thrombin in comparison to age matched, normal weight, healthy controls. Furthermore, the ETP was significantly higher in obese children compared to controls [2].

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

Summary.  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.

Low endogenous thrombin potential in trained subjects

INTRODUCTION A paradox seems to exist: exercising leads to clotting activation in conventional clotting tests, but exercising persons have a low risk of thrombosis. In this study we tried to evaluate the effect of exercise performance status on in vitro plasma thrombin generation, which represents an overall function test of hemostasis. MATERIALS AND METHODS We compared 56 trained subjects to 98 healthy age matched sedentary volunteers. Blood samples were analyzed for thrombin generation using calibrated automated thrombography. Microparticles were quantified using ELISA. Additionally prothrombin fragments 1 + 2, thrombin-antithrombin complex, tissue factor pathway inhibitor, antithrombin and prothrombin were measured. The group of the trained subjects performed an incremental cycle-ergometer exercise test after taking the blood sample. RESULTS A significantly lower endogenous thrombin potential was observed in the group of the trained subjects compared to the sedentary individuals (p = 0.007). Microparticles (ELISA) were significantly lower in the trained subjects compared to the sedentary subjects (p = 0.001). Prothrombin fragments 1 + 2 (p < 0.001) and thrombin-antithrombin complex (p = 0.01) were significant higher in the trained subjects and antithrombin (p = 0.02) as well as prothrombin (p < 0.0001) were significantly lower in this group, whereas tissue factor pathway inhibitor values did not show significant differences. Both maximal and submaximal power output was significantly negatively related to endogenous thrombin potential (r = -0.43, r = -0.45) and thrombin peak (r = -0.44, r = -0.42). CONCLUSIONS Trained subjects have a lower endogenous thrombin potential than sedentary subjects possibly explaining the lower incidence of thrombosis in this group despite a higher acute clotting activation during strenuous exercise.

Thrombin generation before and after multicomponent blood collection

BACKGROUND: Apheresis technology has made tremendous progress up to the development of automated blood component collection, which offers increased efficiency in donor blood use, but the concern about the contact of donor blood with artificial surfaces remains. Activation of the hemostatic system is a major issue in this context and is controversial. The aim of this study was to estimate the effect of apheresis on continuous thrombin generation (TG), representing a new tool to examine the overall function of the plasmatic clotting system.

Regular smoking is not associated with increased thrombin generation in young adults

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Only minor changes in thrombin generation of children and adolescents with type 1 diabetes mellitus - A case-control study.

BACKGROUND Micro- and macrovascular diseases are frequent complications in patients with diabetes. Hypercoagulability may contribute to microvascular alterations. OBJECTIVE In this study, we investigated whether type 1 diabetes in children is associated with a hypercoagulable state by performing a global function test of coagulation - the thrombin generation assay. SUBJECTS 75 patients with type 1 diabetes aged between 2 and 19years were compared to an age-matched healthy control group. Diabetes patients were divided into high-dose and low-dose insulin cohorts with a cut-off at 0.8Ukg-1d-1. METHODS Measurements were performed with platelet poor plasma using Calibrated Automated Thrombography and 1 pM or 5 pM tissue factor. Additionally, we quantified prothrombin fragments F1+2, thrombin-antithrombin complex, prothrombin, tissue factor pathway inhibitor, and antithrombin. RESULTS Patients with type 1 diabetes exhibited a significantly shorter of lag time as well as decreased thrombin peak and endogenous thrombin potential compared to control subjects with 5 pM but not with 1 pM tissue factor. In high-dose insulin patients peak thrombin generation was higher and time to peak shorter than in low-dose patients. Thrombin-antithrombin complex was decreased in patients with type 1 diabetes, whereas prothrombin fragments F1+2 was comparable in both groups. Thrombin generation parameters did not correlate with parameters of metabolic control and the duration of diabetes. CONCLUSIONS Taken together, we found only minor changes of thrombin generation in children and adolescents with type 1 diabetes which - in contrast to type 2 diabetes - do not argue for a hypercoagulable state.

Monocytes Enhance rVIIa Induced Thrombin Generation in Absence of Platelets and Microparticles

Recombinant activated factor VII (rVIIa) was developed for the treatment of bleeding episodes in patients suffering from hemophilia with inhibitors. Its use is also established in non hemophilia patients with acquired antibodies against factor VIII [1, 2, 3]. In factor deficiency where no specific factor concentrates are available, such as V, VII and XI, use of rVIIa is also a therapeutic option. In clinical studies a beneficial effect of rVIIa in therapy of intra cerebral hemorrhage is reported [4]. There are studies in progress that rVIIa might be beneficial in trauma therapy [5, 6]. First studies in liver failure are also promising [7, 8]. Good results are reported in case studies with rVIIa as an alternative to platelet transfusion in preventing or controlling bleeding, including surgical bleeding, in patients with Glanzmann Thrombasthenia [9]. Results are not so consistent in thrombocytopenia [10, 12, 31, 32].

Thrombin Generation is Age-Dependent in Children as well as in Adults

Children have prolonged routine coagulation parameters like PT and aPTT, mainly due to lower concentrations of procoagulant clotting factors, but also inhibitory factors are reduced in children. These differences are pronounced in the early life years, but the raise to adult values lasts for all the time of childhood and adolescence [1]. Existing data of thrombin generation in childhood concentrate on the neonatal period. It was found that newborns are able to generate only 30–50 % of adult amounts of thrombin [2]. This was due to the use of high amounts of tissue factor (TF) – similar to the routine coagulation assay PT – to initialize coagulation. It has been shown that, using TF in lower quantities, the assay reflects probably the physiological conditions better [3]. We have shown that neonatal plasma can generate thrombin nearly at adult values when low amounts of tissue factor (<10 pmol) are applied, suggesting – in accordance to the clinical observation of excellent hemostasis in newborns – that the low levels of inhibitors compensate for low procoagulant protein levels [4, 5]. With the calibrated automated thrombography (CAT) an instrument was developed, that allows simultaneous analysis of several samples, contrary to the time consuming subsampling method. Importance of thrombin generation measurement lies in the key enzyme role of thrombin, so this method may become a new tool better reflecting overall hemostasis than global tests such as PT and aPTT or specific factor assays. Effects of different proand anticoagulants have been shown to be detected with thrombin generation measurement [6]. Parameters of thrombin generation measured by CAT show an intraindividual stability for healthy adults, but a wide interindividual scatter [7]. To our knowledge no data have been published about the age dependency of thrombin generation measured with CAT.

Effects of melagatran on activated partial thromboplastin time and on ecarin clotting time in cord versus adult plasma.

Melagatran is the active form of the oral direct thrombin inhibitor ximelagatran. Melagatran does not require antithrombin as a cofactor. Its administration is therefore of special interest in neonatal patients, whose plasma is relatively deficient in antithrombin. We investigated the effects of increasing amounts of melagatran (0.05–1 μmol/l) on the activated partial thromboplastin time (APTT) and ecarin clotting time (ECT) in cord versus adult plasma. Both the APTT and ECT were dose-dependently prolonged in the presence of increasing amounts of melagatran. Furthermore, the ECT revealed a higher susceptibility of cord plasma to addition of melagatran than adult plasma. Whereas similar amounts of melagatran were required in cord and adult plasma samples to double the APTT (IC50, 0.47 vs 0.46 μmol/l), significantly less melagatran was required in cord versus adult plasma to double the ECT (IC50, 0.26 vs 0.56 μmol/l). Based on APTT measurements, similar plasma levels of melagatran might be required in neonates and in adults to treat thromboembolic complications. The APTT, however, is relatively insensitive to plasma melagatran concentrations. When the sensitive indicator ECT is used, results suggest that lower amounts of melagatran might be required in neonates than in adults. This has to be scrutinized in future clinical studies.