Pregnancy induces a hypercoagulable state peaking at delivery and reverting postpartum. This prospective cohort study evaluated the longitudinal progression of endogenous thrombin potential in 102 high-risk pregnant women in relation to the development of preeclampsia or gestational diabetes mellitus. Samples were collected from gestational weeks 8-15, with follow-ups every 2-12 weeks, and thrombin generation was assessed using Calibrated Automated Thrombography. Eleven women developed preeclampsia, and 19 developed gestational diabetes mellitus. Endogenous thrombin potential values were significantly elevated in patients who developed preeclampsia (2220 ± 357 nM*min, p < 0.001) or gestational diabetes mellitus (2298 ± 377 nM*min, p < 0.001) compared to the rest of the cohort (1995 ± 337 nM*min), with high levels evident from the first trimester-well before clinical symptoms. Notably, preeclampsia patients on acetylsalicylic acid therapy did not show further increases in endogenous thrombin potential, and acetylsalicylic acid intake in gestational diabetes mellitus patients effectively moderated endogenous thrombin potential progression. These findings suggest that higher early-pregnancy endogenous thrombin potential reflects an underlying hemostatic imbalance associated with the subsequent development of preeclampsia and gestational diabetes mellitus. Furthermore, acetylsalicylic acid appears to exert effects beyond its anti-inflammatory properties by moderating endogenous thrombin potential, providing new insights into the early pathophysiology and therapeutic modulation of high-risk pregnancies.

With great interest, we read the work of Meyer et al, who used a mock neonatal extracorporeal membrane oxygenation (ECMO) circuit to investigate the impact of varying flow rates on platelet activation, leukocytes, extracellular vesicles, and thrombin generation in vitro.1 We appreciate the authors’ efforts toward refining treatment to prevent device-related thrombosis as a major cause of morbidity and mortality in this particular group of patients. Indeed, we have previously discovered massive clotting activation in term newborns under ECMO support. Prothrombin fragments 1 + 2, thrombin-antithrombin complex, and D-dimer were substantially increased after 1 hour with a progression that fits the pattern of consumptive coagulopathy.2 We very much appreciate the comments given by Guzzetta and Downey, who emphasize the developmental peculiarities in neonatal hemostasis that need to be taken into account for a tailored treatment with improved outcome.3 Because the authors mentioned part of our work, we would like to add further aspects of the neonatal hemostatic system that are pivotal under ECMO support. First, the neonatal hemostatic system can be easier unhinged by contact activation because it is balanced at a lower level than that of adults. As Guzzetta and Downey mentioned, thrombin generation is markedly lower in neonates than in adults. Nevertheless, healthy neonates exhibit neither bleeding tendencies nor easy bruising because lower levels of procoagulant factors are compensated by lower levels of tissue factor pathway inhibitor, antithrombin, and protein C and S.4, 5 In fact, we have shown that spatial clot growth from immobilized tissue factor is faster in neonatal plasma at the initiation phase and comparable to adult plasma in the stationary phase despite substantially lower intra-clot thrombin levels.6 Generation of minute amounts of thrombin at the clot front promoted by decreased inhibitor levels seems to be more relevant for clot-growth than the overall amount of thrombin generated within the clot. Lower levels of clotting factors as well as inhibitors in newborns result in a balanced but more fragile hemostatic system than that of adults and can, therefore, be easily shifted toward bleeding or thrombosis. Massive contact activation during ECMO support may produce more deleterious effects on the neonatal than on the adult hemostatic system because of a lower capability to counteract this storm of prothrombotic stimuli. Adult blood is added to the circuit during ECMO support but peculiarities of the neonatal fraction may still influence the optimum flow rate obtained with aforementioned in vitro experiments. Second, the development of acquired von Willebrand syndrome (AVWS) during ECMO-support has aggravating consequences on neonatal primary hemostasis. Neonates exhibit a well-functioning primary hemostasis despite in vitro platelet hypoaggregability. Guzzetta and Downey summarize multiple signal transduction impairments in neonatal platelets that contribute to decreased platelet aggregation with several agonists. Still, according to our findings, phosphatidylserine expression and microparticle generation are not different from that of adult platelets, resulting in equal support of thrombin generation.7, 8 However, the authors mention improved platelet adhesion due to higher activity of neonatal von Willebrand factor (VWF). Indeed, shear stress and ristocetin induced platelet binding of neonatal VWF is increased. This effect is equally observable with neonatal and adult platelets and thus not a result of variations in the platelet GPIb receptor. Improved platelet adhesion is caused by a greater proportion of high molecular weight multimers of VWF in neonatal plasma. This important compensation for the observable neonatal platelet hypoaggregability is substantially hampered under ECMO support. High molecular weight multimers are unfolded because of high shear stress in the extracorporeal unit and are exposed to cleavage by a disintegrin and metalloproteinase with the thrombospondin type 1 motif, member 13. The resulting constitutional loss of high molecular weight multimers leads to a functional defect of VWF, resulting in the clinical picture of AVWS. All patients under ECMO support develop severe AVWS within 1 hour and recover rapidly after weaning from the device.9 In a pediatric cohort including 11 newborns on ECMO/extracorporeal life support, we observed AVWS with 100% prevalence.10 We acknowledge that Meyer et al did not find changes in the VWF antigen with their experimental setup, but this functional defect of VWF can only be detected by specialized methods such as VWF collagen binding capacity assay or VWF multimer analysis. Because neonatal VWF high molecular weight multimers compensate for reduced platelet aggregation under neonatal physiological conditions, a constitutional loss thereof potentially results in a more severe disbalance compared with adults. The degree of VWF degradation is shear-stress-dependent, which correlates with the flow rate. Although contact activation during ECMO therapy increases thrombotic risk, AVWS increases bleeding tendencies. Both need to be considered when evaluating treatment strategies. We applaud Meyers et al for their well-performed study, as well as Guzzetta and Downey for jolting a debate, which, hopefully, will lead to refined treatment regimens for neonatal ECMO taking developmental hemostasis into account. The authors have no conflicts of interest. Axel Schlagenhauf wrote the first draft. Barbara Zieger and Wolfgang Muntean critically appraised the manuscript and approved the final version for submission.

Healthy neonates exhibit no bleeding tendencies, but exhibit longer partial thromboplastin times than adults. Lower clotting factor levels may be balanced by lower inhibitor levels, which is not reflected in routine coagulation assays, but could result in normal clot formation in vivo. The novel thrombodynamics assay simulates a damaged vessel with tissue factor immobilized to a surface. We hypothesized that intra-clot thrombin levels and spatial fibrin clot formation with this assay are comparable in neonates and adults. Coagulation was tested in plasma from venous neonatal blood (N = 12), cord blood (N = 30), and adult blood (N = 20) using thrombodynamics and calibrated automated thrombography. Neonates exhibited a higher initial rate of clot formation than adults (adult: 60.7 ± 3.9 µm/min; neonatal: 66.8 ± 3.9 µm/min; cord: 68.1 ± 3.3 µm/min; P < 0.001) and a comparable stationary rate of clot formation (adult: 35.8 ± 8.5 µm/min; neonatal: 37.0 ± 4.6 µm/min; cord: 36.0 ± 5.2 µm/min; P = 0.834). Intra-clot thrombin levels were lower in neonates (adult: 41.9 ± 11.2 AU/l; neonatal: 22.6 ± 10.2 AU/l; cord: 23.6 ± 9.7 AU/l; P < 0.001), but the longitudinal rate of thrombin propagation was comparable (adult: 27.2 ± 4.2 µm/min neonatal; 27.9 ± 2.9 µm/min; cord: 27.6 ± 3.4 µm/min; P = 0.862). Despite lower intra-clot thrombin levels, neonates exhibit normal spatial fibrin clot growth, which concurs with clinically well-functioning hemostasis in healthy neonates.

Healthy neonates exhibit a well-functioning haemostatic system despite peculiarities regarding composition of clotting factors and inhibitors as well as impaired platelet aggregation. Thrombocytopenia and severe bleeding events are feared in sick infants. Recombinant factor VIIa (rFVIIa) is a haemostatic agent used as a last resort in neonates with refractory bleedings. Aim of this study was to investigate in-vitro (i) changes in thrombin generation with different platelet counts, (ii) effects of rFVIIa under conditions of thrombocytopenia and (iii) potentially differing dose-response of rFVIIa in cord blood as a surrogate for neonatal blood compared to adult blood. Thrombin generation parameters were observed in cord blood plasma and adult plasma with various platelet counts, with or without addition of rFVIIa, respectively. Low platelet counts did not influence thrombin generation in cord blood in contrast to adult blood. RFVIIa primarily affected lag time throughout all platelet concentrations. Interestingly, peak height was reduced exclusively in cord blood plasma after addition of rFVIIa. No significant differences regarding dose-response were observed between cord blood and adult blood. In contrast to adult blood, thrombocytopenia in cord blood does not significantly influence thrombin generation. Even at very low platelet counts there is enough negatively charged surface to support rFVIIa action in plasma from cord blood and adult blood in-vitro.

The optimal mode of delivery for a pregnant hemophilia carrier is still a matter of debate. The aim of the study was to determine the incidence of intracranial hemorrhage and other major bleeds in neonates with moderate and severe hemophilia in relationship to mode of delivery and known family history. A total of 926 neonates, 786 with severe and 140 with moderate hemophilia were included in this PedNet multicenter study. Vaginal delivery was performed in 68.3% (n=633) and Cesarean section in 31.6% (n=293). Twenty intracranial hemorrhages (2.2%) and 44 other major bleeds (4.8%) occurred. Intracranial hemorrhages occurred in 2.4% of neonates following vaginal delivery compared to 1.7% after Cesarean section (P=not significant); other major bleeds occurred in 4.2% born by vaginal delivery and in 5.8% after Cesarean section (P=not significant). Further analysis of subgroups (n=813) identified vaginal delivery with instruments being a significant risk factor for both intracranial hemorrhages and major bleeds (Relative Risk: 4.78-7.39; P<0.01); no other significant differences were found between vaginal delivery without instruments, Cesarean section prior to and during labor. There was no significant difference in frequency for intracranial hemorrhages and major bleeds between a planned Cesarean section and a planned vaginal delivery. Children with a family history of hemophilia (n=466) were more likely to be born by Cesarean section (35.8% vs. 27.6%), but no difference in the rate of intracranial hemorrhages or major bleeds was found. In summary, vaginal delivery and Cesarean section carry similar risks of intracranial hemorrhages and major bleeds. The ‘PedNet Registry’ is registered at clinicaltrials.gov identifier: 02979119.

The Austrian Haemophilia Registry collects epidemiological data on patients with haemophilia, on treatment modalities and potential side effects. The Registry covers more than 85% of the assumed total number of haemophilia patients in Austria. This report summarizes data on 753 patients: 84.3% (635) have haemophilia A and 15.7% (118) have haemophilia B. Patients' median age is 34 years (range: 1-93 years). Of the total cohort, 39.0% (294) patients have severe haemophilia, 11.3% (85) moderate haemophilia, and 49.4% (372) mild haemophilia. Of the patients with severe haemophilia, 38.4% (113) have been infected with hepatitis C virus (HCV) and 12.6% (37) are human immunodeficiency virus (HIV) positive. Overall, 10.6% (67) of patients with haemophilia A and 1.7% (2) of those with haemophilia B have had an inhibitor in their history. Among patients with severe haemophilia, 68.4% (201) receive prophylaxis and 28.6% (84) receive on-demand therapy. There are 65.0% (191) patients with severe haemophilia who are treated with recombinant products. In conclusion, most patients with severe haemophilia receive prophylactic treatment. HCV and HIV infections are still important issues in the Austrian haemophilia population.

Background: Patients with inflammatory bowel diseases (IBD) exhibit chronic inflammation of the digestive tract associated with a prothrombotic shift in the plasmatic coagulation system as well as heightened platelet reactivity and preactivation. Recently, microbial and platelet-derived inorganic polyphosphate has been shown to inhibit tissue factor pathway inhibitor (TFPI), thus, influencing the hemostatic balance during inflammation. We hypothesized that polyphosphate plays a role in the pathophysiology of inflammatory bowel disease resulting in refractoriness to TFPI activity.

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.

IThe 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: CATdata 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. Schlüsselwörter Thrombingeneration, Multikomponentenspende, Hämostase

Thrombin generation was studied in paediatric patients with congenital heart disease (CHD) undergoing cardiac surgery using the calibrated automated thrombography (CAT) in terms of the lag time until the onset of thrombin formation, time to thrombin peak maximum (TTP), endogenous thrombin potential (ETP), and thrombin peak height. The suitability to determine the coagulation status of these patients was investigated. Patients, material, methods: CAT data of 40 patients with CHD (age range from newborn to 18 years) were compared to data using standard coagulation parameters such as prothrombin (FII), antithrombin (AT), tissue factor pathway inhibitor (TFPI), prothrombin fragment 1.2 (F 1.2), thrombin-antithrombin (TAT), activated partial thromboplastin time (aPTT), and prothrombin time (PT). Results: A significant positive correlation was seen between ETP and FII (p < 0.01; r = 0.369), as well as between peak height and F II (p < 0.01; r = 0.483). A significant negative correlation was seen between ETP and TFPI values (p < 0.05; r = –0.225) while no significant correlation was seen between peak height and TFPI. A significant negative correlation was seen between F 1.2 generation and ETP (p < 0.05; r = –0.254) and between F 1.2 generation and peak height (p < 0.05; r = –0.236). No correlation was seen between AT and ETP or peak. Conclusions: CAT is a good global test reflecting procoagulatory and inhibitory factors of the haemostatic system in paediatric patients with CHD. Schlüsselwörter Kalibrierte automatisierte Thrombographie, Thrombinentstehung, endogenes Thrombinpotenzial, Prothrombin, Herzoperation, angeborene Herzfehler

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.

Growth factor independent 1 (GFI1) and Growth Factor Independent 1B (GFI1B) are zinc finger transcriptional repressors that share about 90% homology on amino acid sequence and are expressed during hematopoiesis. While GFI1 is most important for granulocyte-monocyte lineage commitment, GFI1B is an essential master regulator of erythroid and megakaryocytic lineages. Mice lacking Gfi1b are embryonic lethal due to anemia and thrombocytopenia. In humans, alternative splicing leads to a shorter p32 isoform that lacks the first 2 of 6 zinc fingers. GFI1B germline mutations have been reported to cause autosomal-dominant macrothrombocytopenia with a grey-platelet syndrome phenotype, implying that the mutant protein acts in a dominant-negative manner.

This guideline which is endorsed by the Austrian Society of Haemophilia, the Austrian Society of Paediatrics, and the Austrian Society of Haematology & Medical Oncology is intended to give a clear and practical guidance for diagnosing and treating haemophilia in Austria. In the treatment of haemophilia there are few controlled interventional trials, and recommendations usually have a rather low level of evidence.The main basis for this paper are the new international guidelines by the World Federation of Hemophilia, published in 2013. These were adapted according to the local situation and experience.Covered topics are diagnostics, control visits, pharmacological treatment options, prophylaxis and treatment in children and adults, possible problems arising in haemophilia carriers and special aspects like home therapy, options for venous catheters, management of various traumas, bleedings and interventions, including dental procedures, and last not least inhibitors and their treatment.

Die vorliegende Leitlinie, die unter Patronanz der Österreichischen Hämophiliegesellschaft (ÖHG) sowie der Österreichischen Gesellschaft für Kinder- und Jugendheilkunde (ÖGKJ) und der Österreichischen Gesellschaft für Hämatologie und Onkologie (OeGHO) erstellt wurde, soll einen praxisnahen Leitfaden für die Diagnostik und Therapie von Hämophiliepatienten in Österreich darstellen. In der Hämophilietherapie gibt es wenige vergleichende Interventionsstudien, und die Empfehlungen haben einen meist niedrigen Evidenzgrad. Als wesentliche Grundlage dieser Leitlinie dienten neue, internationale Guidelines der „World Federation of Hemophilia“ aus dem Jahr 2013. Diese wurde den nationalen Gegebenheiten und Erfahrungen angepasst. Behandelt werden Themen wie Diagnostik, Verlaufskontrollen, medikamentöse Therapieoptionen, Prophylaxe und Therapie bei Kindern und Erwachsenen, weiters mögliche Probleme bei Konduktorinnen und spezielle Aspekte wie Heimtherapie, Optionen für venöse Zugänge, Umgang mit verschiedenen Formen von Traumata, Blutungen und Eingriffen einschließlich Zahnextraktionen und das Thema Hemmkörper und dessen Behandlung.

Objective:Celiac disease (CD) is a risk factor for venous thromboembolism (VTE) and stroke, but the mechanisms are unclear. Continuous measurement of thrombin generation in plasma is a feasible way to detect hypercoagulable changes. The aim of this pilot study was to investigate thrombin generation in pediatric patients with CD compared with pediatric controls.Methods:Plasma samples were collected from 19 pediatric patients with CD and 20 healthy controls. In each patient diagnosed as having CD, thrombin generation was determined twice by means of calibrated automated thrombography. The first measurement was undertaken when CD was diagnosed; the second measurement was undertaken after normalization of their IgA antitissue transglutaminase antibody (tTG-Ab) titers following a gluten-free diet. In the controls, measurement for TTG-Ab and thrombin generation was undertaken once during recruitment.Results:Patients with CD at diagnosis showed a significantly shorter lag time compared with controls (P<0.001) and a shorter time-to-peak compared with controls (P<0.02). These differences were no longer detectable after normalization of TTG-Ab values. The overall amount of generated thrombin, represented by the endogenous thrombin potential (ETP), showed no significant difference between the study groups.Conclusions:Our results show that alterations in coagulation can be found in untreated CD that may help to explain the described increased risk of stroke or VTE. A shorter lag time in patients with untreated CD indicates a more rapid onset of thrombin generation as a sign of hypercoagulability. ETP, the best predictive parameter for thromboembolic disease, however, was not altered.

Objectives: We aimed to determine how loss of response (LOR) to adalimumab (ADA) in juvenile idiopathic arthritis (JIA) may be related to anti-ADA antibodies (AAA). Method: AAA and ADA levels were measured in 23 consecutive patients with JIA responding significantly to treatment with ADA. Results: Six out of 23 (26%) patients developed AAA and had low ADA levels. Five out of six AAA-positive patients experienced LOR. In these patients use of concomitant methotrexate (MTX) was significantly lower. Conclusions: The occurrence of AAA is a frequent event associated with LOR. Monitoring of AAA and serum ADA levels should be considered in JIA patients under ADA therapy.