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To investigate changes in von Willebrand factor (VWF) concentration, function, and multimers during pediatric extracorporeal membrane oxygenation (ECMO) and determine whether routine monitoring of VWF during ECMO would be useful in predicting bleeding.Prospective observational study of pediatric ECMO patients from April 2017 to May 2019.The PICU in a large, tertiary referral pediatric ECMO center.Twenty-five neonates and children (18 yr) supported by venoarterial ECMO.None.Arterial blood samples were collected within 24 hours pre-ECMO, daily for the first 5 days of ECMO, every second day until decannulation, and 24 hours post-ECMO. The STA R Max analyzer was used to measure VWF antigen (VWF:Ag) and ristocetin cofactor (VWF:RCo) activity. VWF collagen binding (VWF:CB) was measured using an enzyme-linked immunosorbent assay. VWF multimers were measured using the semi-automated Hydragel 11 VWF Multimer assay. Corresponding clinical data for each patient was also recorded. A total of 25 venoarterial ECMO patients were recruited (median age, 73 d; interquartile range [IQR], 3 d to 1 yr). The median ECMO duration was 4 days (IQR, 3-8 d) and 15 patients had at least one major bleed during ECMO. The percentage of high molecular weight multimers (HMWM) decreased and intermediate molecular weight multimers increased while patients were on ECMO, irrespective of a bleeding status. VWF:Ag increased and the VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios decreased while patients were on ECMO compared with the baseline pre-ECMO samples and healthy children.Neonates and children on ECMO exhibited a loss of HMWM and lower VWF:CB/VWF:Ag and VWF:RCo/VWF:Ag ratios compared with healthy children, irrespective of major bleeding occurring. Therefore, monitoring VWF during ECMO would not be useful in predicting bleeding in these patients and changes to other hemostatic factors should be investigated to further understand bleeding during ECMO.

Objectives In adults, the elevation of cardiac troponin (cTn) above the 99th percentile upper reference limit defines myocardial injury. The use and interpretation of cTn in a paediatric population, however, is difficult given the 99th percentile for different assays is not well established. Using paediatric blood samples from healthy neonates, infants and children we derived continuous and partitioned 97.5th and 99th percentiles for the Ortho VITROS hs-TnI assay. Methods A total of 328 samples for infants, children and adolescents aged 0–17.8 years were obtained. Age partitioned reference limits were derived in accordance with CLSI EP28-A3C. Continuous reference limits were established as described previously by the HAPPI Kids Study team. Results hs-TnI as measured by the Ortho VITROS Assay is highly elevated above the adult 99th percentile at birth and declines to lower levels within the first 6 months of life. The 99th centile upper reference limit for ages 0–3 months was 72 ng/L (90% CI: 52–91) and 9 ng/L (90% CI: 5.2–17.4) for ages 3 months to 18 years. Continuous upper 99th centile reference limits were comparable. Conclusions Partitioned and continuous 99th percentiles for hs-TnI were derived for the new Ortho VITROS assay in healthy neonates and older children. This will assist clinicians to appropriately assess for the presence of myocardial injury in this population.

Proteomics, the simultaneous study of all proteins in a given cell, tissue or organism, is an innovative approach used to identify novel markers for diagnosis, prognosis and the pathophysiological mechanisms associated with diseases. Proteomic methodologies have been used in a variety of contexts such as investigating changes in protein abundance that may occur with disease presence, the response to therapeutic treatments as well as the impacts of age on the plasma proteome.Over the last decade, significant technological advancements in proteomic techniques have resulted in an increase in the use of proteomics in thrombosis and hemostasis research, particularly in order to identify relevant and novel clinical markers associated with bleeding and thrombosis. This mini-review explores the use of proteomics in the setting of thrombosis and hemostasis from 2010-2020, across five main domains (platelets, blood clot composition, stroke, venous thromboembolism, and therapeutics), as well as provides insights into key considerations for conducting proteomic studies.

Objectives Children with very short telomeres commonly develop bone marrow failure and other severe diseases. Identifying the individuals with short telomeres can improve outcome of bone marrow transplantation, with accurate diagnosis requiring the use of age-matched reference intervals (RIs). This study aimed to establish RIs for telomere length (TL) in children using three commonly used methods for TL measurement. Methods Healthy children aged 30 days to 18 years were recruited for assessment using age as a continuous variable. Venous blood samples were collected and leukocyte TL was measured using terminal restriction fragment (TRF) analysis, quantitative PCR (QPCR) and flow cytometry with fluorescence in situ hybridization (Flow-FISH). Fractional polynomial model and quantile regression were performed to generate continuous RIs. Factors that might contribute to variation in TL, such as gender, were also examined. Results A total of 212 samples were analyzed. Continuous RIs are presented as functions of age. TRF analysis and QPCR showed significant negative correlation between TL and age (r=−0.28 and r=−0.38, p Conclusions This study provides three options to assess TL in children by establishing method-specific continuous RIs. Choosing which method to use will depend on several factors such as amount and type of sample available and required sensitivity to age-related change.

Serum cystatin C (CysC) is a promising biomarker of kidney function, which has higher accuracy and sensitivity when compared with creatinine. To better utilize serum CysC in clinical practice, this study aimed to establish continuous paediatric reference intervals (RIs) for serum CysC.The study subjects consisted of healthy term neonates and children aged 30 days to 18 years. Venous blood samples were collected and serum CysC levels were measured using the immunoturbidimetric measurement principle. Fractional polynomial regression model and quantile regression was applied in the statistical analysis to generate continuous RIs.A total of 378 samples with equal numbers of males and females were analysed for serum CysC. No outliers were found in this analysis. The continuous RIs are presented as equations and graphical scatterplots.This study established continuous paediatric reference intervals (RIs) for serum CysC in healthy term neonates and children. The continuous RIs generated from this study show age-based dynamic changes as well as blood group and gender-specific differences for serum CysC. Graphical abstract.

Human blood plasma is a complex biological fluid containing soluble proteins, sugars, hormones, electrolytes, and dissolved gasses. As plasma interacts with a wide array of bodily systems, changes in protein expression, or the presence or absence of specific proteins are regularly used in the clinic as a molecular biomarker tool. A large body of literature exists detailing proteomic changes in pathologic contexts, however little research has been conducted on the quantitation of the plasma proteome in age-specific, healthy subjects, especially in pediatrics. In this study, we utilized SWATH-MS to identify and quantify proteins in the blood plasma of healthy neonates, infants under 1 year of age, children between 1-5 years, and adults. We identified more than 100 proteins that showed significant differential expression levels across these age groups, and we analyzed variation in protein expression across the age spectrum. The plasma proteomic profiles of neonates were strikingly dissimilar to the older children and adults. By extracting the SWATH data against a large human spectral library we increased protein identification more than 6-fold (940 proteins) and confirmed the concentrations of several of these using ELISA. The results of this study map the variation in expression of proteins and pathways often implicated in disease, and so have significant clinical implication.

Summary Background Antithrombin, a hemostatic protein and naturally occurring anticoagulant, is a major thrombin inhibitor. The capacity of antithrombin to inhibit thrombin is known to increase a 1000-fold whilst in the presence of unfractionated heparin. β-antithrombin is an isoform of antithrombin with a high affinity for unfractionated heparin. This study aimed to determine the differences in the anticoagulant activity of the β-antithrombin isoform in children compared with adults. Methods Plasma samples were obtained from 105 healthy individuals from the following age groups: neonates (day 1 and day 3), 28 days to 1 year, 1–5 years, 6–10 years, 11–16 years and adults. The method utilized to measure the activity of β-antithrombin in plasma is a modified version of the total antithrombin assay routinely used in diagnostic laboratories. The modified version of this assay allows for the specific quantification of the β-antithrombin glycoform anticoagulant activity alone, as the β-antithrombin molecule is activated under a high salt concentration, which in turn does not allow activation of other antithrombin isoforms. Conclusions This study demonstrated that there are no age-specific differences in the activity of β-antithrombin. However, considering that the total AT activity is significantly reduced in neonates, our results suggest that in this population β-antithrombin activity is a major contributor to the overall activity of AT.

SummaryIntroduction Developmental hemostasis recognizes the physiologic differences between the hemostatic system of neonates and children and that of adults. As compared with the knowledge of hemostatic system physiology in adults, our understanding in neonates and children remains inadequate. Routine clinical coagulation testing most commonly measures functional parameters of the hemostatic system. Very few studies have measured age-specific levels of hemostatic proteins. An understanding of the normal fluctuations in the levels of hemostatic proteins is vital in the prevention, diagnosis and treatment of hemostatic problems during infancy and childhood. This study was designed as the first comprehensive study of the age-specific changes in the levels of important hemostatic proteins in healthy neonates, children, and adults. Methods Plasma samples were obtained from 120 healthy individuals from the following age groups: neonates (day 1 and day 3), 28 days to 1 year, 1–5 years, 6–10 years, 11–16 years, and adults. Factor II, FV, FVII, FVIII, FIX, FX, FXI, FXII, FXIII, plasminogen, protein C and total and free protein S were quantified with commercially available ELISA kits. Results The levels of 10 proteins were significantly different between neonates and adults, and these differences persisted throughout childhood for most of these proteins. Conclusion The results of this study confirm that the levels of the majority of coagulation proteins vary significantly with age. Future studies should investigate how hemostatic protein level relates to functional changes with age.

Post-translational modifications (PTMs) such as glycosylation and phosphorylation play an important role on the function of haemostatic proteins and are critical in the setting of disease. Such secondary level changes to haemostatic proteins have wide ranging effects on their ability to interact with other proteins. This review aimed to summarize the knowledge of the common PTMs associated with haemostatic proteins and the implications of such modifications on protein function. Haemostatic proteins that represent the main focus for studies specific to PTMs are von Willebrand factor, tissue factor, factor VIII, antithrombin and fibrinogen. These proteins are susceptible to PTMs by glycosylation, phosphorylation, sulphation, citrullination and nitration, respectively, with a significant impact on their function. During synthesis, vWF must undergo extensive PTMs, with N-linked glycosylation being the most common. Increased phosphorylation of tissue factor results in increased affinity for platelets to the vessel endothelium. Citrullination of antithrombin leads to an increased anticoagulant function of this protein and therefore an anticoagulant state that inhibits clot formation. On the contrary, nitration of fibrinogen has been shown to result in a prothrombotic state, whilst sulphation is required for the normal function of Factor VIII. From this review, it is evident that PTMs of haemostatic proteins as a change in protein structure at a secondary level greatly influences the behaviour of the protein at a tertiary level.

Introduction Infants and children have a lower incidence of thrombosis compared with adults. Yet, the mechanism of blood clot formation and structure in infants and children, as the end product of coagulation, has not been studied. This study aimed to establish differences in the mechanism of thrombin generation, fibrin clot formation and response to thrombolysis in infants and children compared with adults. Materials and methods We studied thrombin generation, fibrin clot formation, structure and fibrinolysis in healthy infants, children and adults. Results Younger populations had a decreased potential to generate thrombin, at a slower velocity compared with adults, correlating positively with age. Clot formation at venous shear rate was decreased in infants and children compared with adults, with increased time for fibrin formation, decreased fibrin formation velocity, resulting in decreased tendency for fibrin formation in younger populations. These differences were less pronounced at arterial shear rate. Studies of the fibrin clot structure in paediatric age groups showed a significantly larger pore size compared with adults, suggestive of a clot that is less resistant to fibrinolysis. The presence of tissue plasminogen activator (tPA) resulted in a significant decrease in the pore size of infants and children, but not in adults. Conclusions This is the first study to suggest that the mechanism of blood clot formation and nanostructure, as well as response to thrombolytic therapy is different in infants and children compared with adults.

Major age-related diseases such as cardiovascular disease and cancer are the primary causes of morbidity and mortality in Australia and worldwide. In our recent study characterising differences in the plasma proteome between healthy children and adults, a large number of proteins differentially expressed with age were found to be of platelet origin. This study aimed to characterise differences in the resting platelet proteome and the platelet releasate of healthy children compared to healthy adults. This study represents the setup of a procedure for the proteomic analysis of platelets from children. Differentially expressed platelet proteins were identified using Two-dimensional Differential In-Gel Electrophoresis and mass spectrometry. Significant differences in the expression of nine proteins (1.1%) in the resting platelet proteome were observed in children compared to adults. Serotransferrin, fibrinogen alpha chain, glyceraldehyde-3 phosphate dehydrogenase, serum albumin, transgelin-2, calponin-2/LIM and SH3 domain protein 1 and human chorionic gonadotropin 2039797 were up - regulated , whereas thrombospondin-1 was down - regulated in children. Eleven proteins (1.5%) were differentially expressed in the platelet releasate of children compared to adults, where transferrin was also upregulated and TSP-1 was down regulated . Identified proteins are involved in processes including tissue and organ development, cell proliferation regulation and angiogenesis. Our results provide novel insights into platelet physiology as well as growth, development and ageing in healthy individuals. Biological significance The incidence of major diseases such as cardiovascular disease (CVD) and cancer increase with increasing age and are the major causes of morbidity and mortality both in Australia and worldwide. As the aged population continues to increase dramatically, so too will the financial strains associated with the long term care of the elderly population. Compared to adults, children have a significantly lower incidence of major diseases such as thromboembolic disease. This suggests that children have a protective mechanism against the development of disease. Therefore, studies focussing on the molecular changes of proteins, the machinery of the cell, between children and adults are the key to determining the underlying mechanisms responsible for the onset of major diseases. A well-defined example of how protein expression can change with age is that of the plasma proteome. Significant differences in the expression of numerous plasma proteins between healthy children and adults have been recently demonstrated. Interestingly, a large number of differentially expressed proteins were found to be of platelet origin. This finding forms the basis for the current study, presenting as strong evidence for the age-specific differences of the platelet proteome.

Introduction: Cardiopulmonary bypass (CPB) can be associated with deleterious clinical effects. However, the impact of CPB on inflammatory, immunological and other homeostatic pathways remains poorly understood. We investigated the impact of CPB on the plasma proteome in children undergoing tetralogy of Fallot repair. Methods: Blood samples were taken from 20 children prior to and at the end of CPB and 6h, 12h and 24h after CPB. Plasma was analysed by liquid chromatography-mass spectrometry (LC-MS) in a label-free, untargeted approach. Data were analysed using Genedata software to identify peptides that were differentially expressed (pResults: The proteins that were found to be differentially expressed were haptoglobin isoform 1 preproprotein, isoform 2 of semaphorin-6C, vitamin D-binding protein, inter-alpha-trypsin inhibitor, ceruloplasmin, apolipoprotein B100 and fibrinogen alpha. Conclusion: CPB alters the plasma proteome with differences most apparent at 6h and 12h post CPB. There was a return to baseline with no proteins differentially regulated by 24h.

The most widely used anticoagulants for prophylaxis and treatment of thrombosis are unfractionated heparin (UFH) and low molecular weight heparin (LMWH), with UFH remaining the most commonly used parenteral anticoagulant [1]. Despite the frequency of use, extensive experience and numerous studies with these drugs, the treatment failure rate and risk of clinical bleeding remains problematic. There is also significant variation in the dose of UFH required to achieve the therapeutic range, leading to frequent dose modifications. For adults, the inter-individual variation in drug effect for fixed dose of UFH and LMWH is 50% and 35%, respectively, as measured by the Anti-Xa assay [2]. This large variation in dose effect, combined with the risk of suband supra-anticoagulation is a major reason why these drugs need to be regularly monitored. New Oral Anticoagulants (NOACs) such as rivaroxaban and dabigatran have been developed and introduced into the adult clinical setting. Benefits of these NOACs are that they are synthetic, administered orally, specifically target haemostatic proteins, have a shorter half-life with a more predictable response compared to conventional anticoagulants, fixed dosing and no requirement for monitoring. Despite these benefits, NOACs are known to exhibit high inter-individual variability in dose response. Rivaroxaban demonstrates up to 40% inter-individual variability, while dabigatran reaches up to 81%, as measured by standard coagulation tests [3].

Protein S (PS) is a vitamin K-dependent plasma glycoprotein. Around 60-70% of PS in plasma is noncovalently bound to C4-binding protein (C4BP). Free PS functions as a cofactor that enhances the activity of activated protein C (APC) in the proteolytic degradation of activated factors V and VIII. PS also has a more recently described APC-independent ability to directly inhibit prothrombinase and tenase by direct binding of activated factors V, VIII, and X. Given that PS is one of the major naturally occurring inhibitors of coagulation, acquired or hereditary deficiencies of this protein result in excessive thrombin generation. As a vast array of mutations are responsible for hereditary PS deficiencies, screening for their presence by DNA testing would require sequencing each entire gene involving numerous exons. Moreover, the knowledge of the gene mutation does not offer any benefit in the treatment of thrombophilic families, so the routine molecular characterization is not indicative. These defects are detected by functional or immunological assays for free and total PS forms. Given that functional PS assays may detect some forms of PS deficiency that free PS immunoassays may miss, it is recommended to include them for initial testing along with immunoassays for free PS, although they should be used with caution. Functional PS assays are subject to multiple interference. For example in the presence of lupus anticoagulant (LA), only free PS immunoassays are recommended for initial testing. PS antigen assays are more popular with most laboratories.

Antithrombin (AT) is a heparin cofactor and a member of the serine protease inhibitor family (serpin). The mature AT molecule is composed of 432 amino acids and it is produced mainly in the liver. Initially, several different AT activities in plasma were reported, leading to the classification of antithrombin in a range from I to IV. It was subsequently shown that these various antithrombin activities were the function of one molecule, antithrombin III, whose name was reduced to antithrombin at the meeting of the International Society in Thrombosis and Haemostasis in 1993. AT is an important protease inhibitor of thrombin and factor Xa. However, AT is also able to inhibit factors IXa, XIa, XIIab, kallikrein, and plasmin. Given that AT is one of the major naturally occurring inhibitors of coagulation, acquired or hereditary deficiencies of this protein result in excessive thrombin generation. As a vast array of mutations are responsible for hereditary AT deficiencies, screening for their presence by DNA testing would require sequencing each entire gene involving numerous exons. Moreover, the knowledge of the gene mutation does not offer any benefit in the treatment of affected families, so the routine molecular characterization is not indicative. These defects are detected by functional or immunological assays. AT amidolytic assays are recommended for initial testing for AT deficiency. There is no need to routinely perform AT immunological assays. However, they are useful in order to distinguish type I from type II hereditary AT deficiency.

Protein C (PC) is a 62-kDa vitamin K-dependent plasma zymogen which, after activation to serine protease, plays an important role in the physiologic regulation of blood coagulation. Given that PC is one of the major naturally occurring inhibitors of coagulation, acquired or hereditary deficiencies of this protein result in excessive thrombin generation. As a vast array of mutations are responsible for hereditary PC deficiencies, screening for their presence by DNA testing would require sequencing each entire gene involving numerous exons. Moreover, the knowledge of the gene mutation does not offer any benefit in the treatment of thrombophilic families, so the routine molecular characterization is not indicative. These defects are detected by functional or immunological assays. Measurement of PC activity is essential to identify subjects with both type I and type II PC defects. There is no need to routinely perform PC immunological assays. However, they are useful in order to distinguish type I from type II PC hereditary deficiency.

Introduction Age specific differences in the function and concentration of haemostatic proteins are well established and are thought to contribute to the lower incidence of thromboembolism in infants and children compared to adults. We hypothesized that there are age-specific differences in the fibrin clot nanostructure and that these differences impact on fibrinolytic and anticoagulant treatment. Aim To determine the differences in fibrin clot nanostructure and response to tissue Plasminogen Activator (tPA) and Unfractionated Heparin (UFH) in infants compared to adults. Materials and Methods Four plasma samples from infants (0.86±0.16 years) and four samples from adults (32.04±15.80 years) were investigated. Thrombin generation (TG) was performed on paper discs in a microplate with 300mmol/L rhodamine substrate (P2Rho), 4mmol/L phospholipids, 5x10-9 mmol/L tissue factor and 16.7 mmol/L CaCl2; with and without additional 50ng/ml tPA, and 0.01IU/ml UFH. Fluorescence signal was recorded with a Fluoroskan Ascent fluorometer, with samples tested in triplicate. The resulting fibrin clots were fixed with 2.5% glutaraldehyde; washed with PBS and fixed with osmium tetroxide OsO4. The clots were dehydrated in ethanol and hexamethyldisilazane and were placed on carbon adhesive tabs. Each clot was coated with gold and studied using Scanning Electron Microscopy (SEM) on a Phenom Pro desktop SEM. The age-specific differences in fibrin fibre thickness and pore size were quantified using the Fibermetric software, with 36 representative images for infants and the same number for adults at each baseline, tPA and UFH, at magnification 10.000, counting 500 fibres per image. Results We observed significant differences in fibrin clot nanostructure between infants and adults (Fig 1). Results are expressed as median and range. Fibrin fibers were thicker in infants compared to adults (343.3nm, 61.2nm to 997.1nm vs 352.3nm, 26.1nm to 997.5nm, p In the presence of tPA in the adult sample, the fibrin fibers were thicker (352.3nm, 26.1nm to 997.5nm vs 355.8nm, 70.0nm to 999.6nm, p0.05) and pore size (0.057pm2, 0.0041pm2 to 19.97pm2 vs 0.050pm2, 0.0041pm2 to 19.34pm2, p>0.05). In the presence of UFH in adults fibrin fibers were thicker (352.3nm, 26.1nm to 997.5nm vs 339.0, 66.9nm to 996.4nm, p Discussion and Conclusion Our study suggests that the fibrin clots in infants are weaker compared to those of adults and is consistent with the lower TG in infants. This is further supported by published evidence that clots produced with low thrombin concentrations are composed of thick fibrin fibres, whereas those generated with high thrombin concentrations are composed of a tight network of thin fibres. The effect of both tPA and UFH on fibrin clots nanostructure in adults is consistent with published literature and is investigated in children for the first time. The fact that tPA did not have an effect on the fibrin clots of infants is not surprising considering that the concentration of plasminogen is 39%, tPA is 40% and TAFI function is 60% of the adult value; while PAI function is 50% higher compared to adults. UFH had an effect of the fibrin clot nanostructure in both infants and adults, with the effect on pore size being more pronounced in adults. While developmental haemostasis has been understood for many years, this is the first study to provide definitive proof that the end product of coagulation is a different clot structure in children. This further enhances our understanding of why thrombosis are less common in children, and has significant implications for treatment. Disclosures: No relevant conflicts of interest to declare.

Introduction Developmental Haemostasis, the concept describing maturation of the haemostatic system is based on established age specific differences in the quantity and function of haemostatic proteins. Interestingly, infants and children have a lower incidence of thromboembolic events than adults. Understanding the precise mechanism of thromboprotection in infants and children may therefore provide novel prevention and treatment strategies for adults. We hypothesized that a significant component of the thromboprotective mechanism is contributed to by the age-specific differences in thrombin generation. Aim This study was designed to investigate differences in thrombin generation in different age groups from infants to adults, to obtain more insight into age related differences in thromboembolic rates. Materials and Methods Plasma samples from 86 infants, children and adults were tested individually and the results were grouped according to age: 1 month to 1 year (N=13), 1-5 years (N=20), 6-10 years (N=19), 11-16 years (N=16) and adults (N=18). Thrombin generation (TG) was measured using Calibrated Automated Thrombinography (CAT) triggered with tissue factor (TF) at 5pM and 1pM final concentration. Parameters measured from the TG curve included: lag time, maximal thrombin concentration (peak), time to peak, as well as the area under the curve (endogenous thrombin potential, ETP). The raw data from TG was also used to calculate the overall, as well as alpha-2-macroglobulin (A2M) dependent decay constants. A2M function was measured with a thrombin inhibition assay developed in house. Results Both ETP and peak thrombin in infants and children was consistently significantly lower than in adults at both 1pM and 5pM tissue factor concentrations. The difference in ETP ranged from 27% to 35% (p Discussion and Conclusions This study confirms that TG in children (including adolescents) is lower than in adults and shows for the first time that this is predominantly caused by higher thrombin inhibition, largely due to an increase of A2M activity. Considering that the levels of A2M are increased by 50%, Antithrombin is decreased by approximately 37%, whilst the levels of Prothrombin, Fibrinogen and Heparin Cofactor II remain the same in infants and children compared to adults; therefore A2M is an important inhibitor of thrombin in early life and during childhood. Age-specific differences in thrombin generation as demonstrated by our study possibly play an important role in the thromboprotective mechanism functioning in infants and children. Disclosures: No relevant conflicts of interest to declare.