Harald Haidl

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].

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

affect platelet dense granules. Semin Thromb Hemost 2004; 30: 537–47. 10 Ren Q, Wimmer C, Chicka M, Ye S, Ren Y, Hughson F, Whiteheart S. Munc13-4 is a limiting factor in the pathway required for platelet granule release and hemostasis. Blood 2010; 116: 869–77. 11 Savage JS, Williams CM, Konopatskaya O, Hers I, Harper MT, Poole AW. Munc13-4 Is Critical for Thrombosis through Regulating Release of Adp from Platelets. J Thromb Haemost 2013; 11: 771–5. 12 Braun A, Varga-Szabo D, Kleinschnitz C, Pleines I, Bender M, Austinat M, Bosl M, Stoll G, Nieswandt B. Orai1 (CRACM1) is the platelet SOC channel and essential for pathological thrombus formation. Blood 2009; 113: 2056–63. 13 Gachet C. P2 receptors, platelet function and pharmacological implications. Thromb Haemost 2008; 99: 466–72. 14 Stoll G, Kleinschnitz C, Nieswandt B. Molecular mechanisms of thrombus formation in ischemic stroke: novel insights and targets for treatment. Blood 2008; 112: 3555–62. 15 Hankey GJ, Eikelboom JW. Antithrombotic drugs for patients with ischaemic stroke and transient ischaemic attack to prevent recurrent major vascular events. Lancet Neurol 2010; 9: 273– 84. 16 Koch H, Hofmann K, Brose N. Definition of Munc13-homology-domains and characterization of a novel ubiquitously expressed Munc13 isoform. Biochem J 2000; 349: 247–53. 17 Gruner S, Prostredna M, Schulte V, Krieg T, Eckes B, Brakebusch C, Nieswandt B. Multiple integrin-ligand interactions synergize in shear-resistant platelet adhesion at sites of arterial injury in vivo. Blood 2003; 102: 4021–7. 18 Massberg S, Brand K, Gr€ uner S, Page S, M€ uller E, M€ uller I, Bergmeier W, Richter T, Lorenz M, Konrad I, Nieswandt B, Gawaz M. A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation. J Exp Med 2002; 196: 887– 96. 19 Denis C, Methia N, Frenette PS, Rayburn H, Ullman-Cullere M, Hynes RO, Wagner DD. A mouse model of severe von Willebrand disease: defects in hemostasis and thrombosis. Proc Natl Acad Sci USA 1998; 95: 9524–9.

Recessive grey platelet-like syndrome with unaffected erythropoiesis in the absence of the splice isoform GFI1B-p37

Gene expression during hematopoietic lineage commitment is tightly regulated by a complex network of transcriptional regulators. Growth Factor Independent 1 (GFI1) and GFI1B are recruiters of histone deacetylases, histone methyltransferases, and key transcriptional repressors during hematopoiesis.[1

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.

Non-enzymatic quantification of polyphosphate levels in platelet lysates and releasates.

Inorganic polyphosphate has been shown to be shed upon platelet activation inducing prothrombotic stimuli on the coagulation system. Several methods have been published to detect and quantify polyphosphate in various cells and tissues, but evaluation of platelet content has only been achieved by indirect detection of orthophosphate after enzymatic digestion, thus, relying heavily on specificity of an exopolyphosphatase that is not commercially available. We present a non-enzymatic method for quantification of platelet-derived polyphosphate featuring optimized extraction on silica spin-columns, followed by specific fluorescence detection using DAPI. This allowed us to quantify polyphosphate in platelet lysates, but also in releasates of TRAP-activated platelets for the first time. Extraction of exogenous polyphosphate from buffer and sample matrices resulted in quantitative yields while removing matrix effects observed with direct fluorescence detection. Treatment of eluted fractions with phosphatase completely abrogated polyphosphate-specific fluorescence arguing for no additional compounds influencing the fluorescence detection. This was confirmed by no change in fluorescence intensity in samples previously treated with DNase and RNase. Taken together, we developed a robust and easily standardizable method to quantify polyphosphate in platelet lysates and releasates that will facilitate polyphosphate related investigations of platelet physiology and coagulation.

Prostaglandin E2 levels and platelet function are different in cord blood compared to adults

Neonatal platelets support primary haemostasis and thrombin generation as well as adult platelets, despite observable hypoaggregability in vitro. High prostaglandin E2 levels at accouchement could account for inhibited platelet function via the EP4 receptor. We set out to determine prostaglandin E2 plasma levels in cord blood of healthy neonates and evaluate the impact of prostaglandin E2 on platelet function in adult and cord blood samples. Prostaglandin E2 plasma levels were measured in cord blood and venous adult blood using GC-MS. Impact of prostaglandin E2 on platelet aggregation was measured by spiking cord blood and adult samples. Contributions of EP3 and EP4 receptors were evaluated using respective antagonists. Intracellular cAMP concentrations were measured using a commercial ELISA-kit. Prostaglandin E2 plasma levels were substantially higher in cord blood than in adult samples. Spiking with prostaglandin E2 resulted in a slight but consistent reduction of platelet aggregation in adult blood, but response to PGE2 was blunted in cord blood samples. Aggregation response of spiked adult samples was still higher than with non-spiked cord blood samples. Blockage of EP4 receptors resulted in improved platelet aggregation in adult platelets upon prostaglandin E2 spiking, while aggregation in cord blood samples remained unaltered. Intracellular cAMP concentrations after preincubation with prostaglandin E2 were only increased in adult samples. In conclusion, very high prostaglandin E2 concentrations in cord blood affect platelet function. This effect may partially explain neonatal platelet hypoaggregability. Peak levels of prostaglandin E2 can potentially protect against birth stress-induced platelet activation.

Polyphosphate in Neonates: Less Shedding from Platelets and Divergent Prothrombotic Capacity Due to Lower TFPI Levels

Background: The neonatal hemostatic system exhibits a fragile balance featuring lower levels of clotting factors as well as inhibitors. Neonatal platelets show in-vitro hypoaggregability, but neonates exhibit well-functioning primary and secondary hemostasis despite this impairment. Recently, polyphosphate shed by activated platelets has been shown to induce a prothrombotic shift on the plasmatic coagulation system of adults. The impact of platelet derived polyphosphate might differ in neonates due to aforementioned peculiarities. Aims: We aimed to comparatively determine polyphosphate content and release from adult and neonatal platelets and to determine its impact on thrombin generation in plasma from adult and cord blood. Methods: Polyphosphate was extracted from adult and neonatal platelet lysates and releasates using silica spin-columns and quantified with a DAPI based fluorescence assay. The impact of exogenous polyphosphate in various concentrations (208–0.026 μg/ml) on thrombin generation was evaluated in plasma from adult and cord blood as well as in adult plasma with reduced tissue factor pathway inhibitor (TFPI) levels using calibrated automated thrombography. Results: Polyphosphate content was comparable in both groups, but the fraction of released polyphosphate upon stimulation with thrombin receptor activating peptide was lower in neonatal samples (adult: 84.1 ± 12.9%; cord: 58.8 ± 11.2%). Relative impact of polyphosphate on lag time of thrombin generation was higher in adult samples compared to samples from cord blood (adult: 41.0% [IQR: 35.2–71.8%] of vehicle; cord: 73.4% [IQR: 70.2–91.4%] of vehicle). However, in samples from cord blood, lower concentrations of polyphosphate were required to obtain maximal impact on thrombin generation (adult: 26 μg/ml; cord: 0.814 μg/ml). PolyP affected thrombin generation in adult plasma similarly to cord plasma, when the TFPI concentration was reduced to neonatal levels. Conclusion: Differences in the impact of polyphosphate on adult and neonatal coagulation are largely caused by differences in TFPI levels. Lower polyphosphate release from neonatal platelets, but lower optimum concentration to drive neonatal plasmatic hemostasis emphasizes the well-matched, but fragile interplay between platelets and coagulation in newborns. A potential developmental mismatch should be considered when transfusing adult platelets into neonates.

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.