Vasiliki Karlaftis

Developmental hemostasis: age-specific differences in the levels of hemostatic proteins

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.

Differences in the resting platelet proteome and platelet releasate between healthy children and adults

UNLABELLED 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.

Differences in the mechanism of blood clot formation and nanostructure in infants and children compared with adults

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.

Remote Ischemic Preconditioning (RIPC) Modifies the Plasma Proteome in Children Undergoing Repair of Tetralogy of Fallot: A Randomized Controlled Trial

Background Remote ischemic preconditioning (RIPC) has been applied in paediatric cardiac surgery. We have demonstrated that RIPC induces a proteomic response in plasma of healthy volunteers. We tested the hypothesis that RIPC modifies the proteomic response in children undergoing Tetralogy of Fallot (TOF) repair. Methods and Results Children (n=40) were randomized to RIPC and control groups. Blood was sampled at baseline, after cardiopulmonary bypass (CPB) and 6, 12 and 24h post-CPB. Plasma was analysed by liquid chromatography mass spectrometry (LC-MS) in an untargeted approach. Peptides demonstrating differential expression (p<0.01) were subjected to tandem LC-MS/MS and protein identification. Corresponding proteins were identified using the NCBI protein database. There was no difference in age (7.3±3.5vs6.8±3.6 months)(p=0.89), weight (7.7±1.8vs7.5±1.9 kg)(p=0.71), CPB time (104±7vs94±7 min)(p=0.98) or aortic cross-clamp time (83±22vs75±20 min)(p=0.36). No peptides were differentially expressed at baseline or immediately after CPB. There were 48 peptides with higher expression in the RIPC group 6h post-CPB. This was no longer evident at 12 or 24h, with one peptide down-regulated in the RIPC group. The proteins identified were: inter-alpha globulin inhibitor (42.0±11.8 vs 820.8±181.1, p=0.006), fibrinogen preproprotein (59.3±11.2 vs 1192.6±278.3, p=0.007), complement-C3 precursor (391.2±160.9 vs 5385.1±689.4, p=0.0005), complement C4B (151.5±17.8 vs 4587.8±799.2, p=0.003), apolipoprotein B100 (53.4±8.3 vs 1364.5±278.2, p=0.005) and urinary proteinase inhibitor (358.6±74.9 vs 5758.1±1343.1, p=0.009). These proteins are involved in metabolism, haemostasis, immunity and inflammation. Conclusions We provided the first comprehensive analysis of RIPC-induced proteomic changes in children undergoing surgery. The proteomic changes peak 6h post-CPB and return to baseline within 24h of surgery. Trial Registration ACTR.org.au ACTRN12610000496011

Beta (β)-antithrombin activity in children and adults: implications for heparin therapy in infants and children.

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.

Latent antithrombin levels in children and adults.

Antithrombin (AT), a key anticoagulation protein, has been shown to exist in various isoforms; native AT (NAT), cleaved AT, pre-latent AT (pre-LAT) and latent AT (LAT). Approximately 3% of the total AT circulating in adults exists in the latent conformation [1]. The conversion of NAT to LAT has been shown to be caused by an irreversible conformational change. This conversion is an essential function of AT which allows senescence to be maintained in the healthy individual [2]. LAT has been associated with the onset of severe and sudden thrombosis in seriously ill patients [2–6]. In addition, LAT has potent anti-angiogenic activity that is comparable to that of other naturally produced angiogenesis inhibitors such as endostatin [4,7,8]. The anti-angiogenic properties of LAT are particularly important in the setting of AT supplementation, especially in seriously ill paediatric patients that regularly receive this product [9]. AT concentrates are administered to seriously ill children in order to correct for decreased AT activity and potentiate the effect of unfractionated heparin therapy. We have recently shown that the use of AT concentrates at our paediatric tertiary centre has increased over the last ten years, particularly in infants [9]. AT concentrates are manufactured by purifying AT from pooled adult plasma and have been found to contain up to 40% LAT [10]. Our team has recently demonstrated that the specific brand of AT concentrate, Thrombotrol-VF, used at the Royal Childrens Hospital, Melbourne, contains up to 11% LAT, The administration of adult derived AT concentrates to seriously ill children could potentially increase the levels of the anti-angiogenic LAT form circulating in blood and thereby disturbing the normal physiological balance of AT isoforms in this vulnerable population. This study aimed to determine the levels of LAT in healthy infants, children and adults, in order to provide knowledge that could guide future clinical use of AT concentrates in the paediatric population. Citrated, platelet-poor plasma samples from children and adults were collected, processed, and stored using a previously established collection protocol [11–13]. All participants were healthy individuals, without previous thromboembolic events, family history of bleeding or thrombosis, and not subjected to any form of anticoagulant therapy. The paediatric cohort is representative of the general population as they were obtained from healthy children about to undergo minor elective day surgery (e.g. circumcision and tonsillectomy) at the Royal Childrens Hospital, Melbourne. Other than the need for elective surgery, these children were healthy and were not receiving any medications. This protocol was approved by the Royal Childrens Hospital, Melbourne, Ethics in Human Research Committee 20031. Individual plasma samples were pooled onto the following age groups: b1 year (mean age: 0.6 years), 1–5 year (mean age: 1.8 years), 6–10 year (mean age: 7.1 years), 11–16 year (mean age: 12.4 years) and adults (mean age: 30 years) with each age-specific plasma pool consisting of twenty individual donors.

Quantitative Age-specific Variability of Plasma Proteins in Healthy Neonates, Children and Adults

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.

The microparticle-specific procoagulant phospholipid activity changes with age.

Sir, Procoagulant microparticles (MPs) are small membrane vesicles shed from the plasma membrane surface and which express a variety of phospholipids and proteins specific to the cell they originate from [1]. MPs are released by any cell type into the vascular compartment during either cell activation or apoptosis [2]. Anionic phospholipids present in the microparticle membranes have procoagulant activity in vivo through their role as catalytic sites for factor Xa and thrombin formation [3]. Increased procoagulant phospholipid activity (PPA) of MPs is associated with many pathological states, including cancer, cardiovascular disease, infection and inflammatory diseases, as well as other clinical states associated with platelet activation. [4, 5]. Therefore, detection and measurement of MPs have a potential application in the study of different pathologies, as well as a prognostic role and could serve as a tool in monitoring effectiveness of therapy in the setting of major diseases. The procoagulant Phospholipid kit, STA-Procoag PPL (Asni eres-sur-Seine, Diagnostica Stago, France), determines the functional haemostatic effect of MPs. Specifically, the STA-Procoag PPL kit measures the clotting time of a plasma sample that is added to human phospholipid depleted plasma in the presence of excess exogenous factor Xa and calcium. In this assay system, the phospholipiddependent acceleration of prothrombin activation by the factor Xa/factor Va complex directly impacts the test result, recorded as clotting time. Therefore, shorter clotting times are associated with increased phospholipid content and are an indication of increased concentration of MPs. This study aimed to measure the age-specific differences in circulating MPs in healthy neonates, children and adults. All blood samples were collected in S-Monovette tubes (Sarstedt, N€ umbrecht, Germany), containing 1 volume of citrate per 9 volumes of blood. Samples were collected, processed and stored according to an established protocol [6]. Informed consent was obtained from the parents of the neonates and children and from the adult participants themselves. All participants were healthy individuals, were without previous thromboembolic events, had family history of bleeding or thrombosis and were not subjected to any form of anticoagulant therapy. For paediatric samples, the inclusion criteria were healthy children attending the hospital for minor day surgery and who did not have a family history of coagulation disorders (e.g. thrombosis) nor were they taking any medications. Adult samples were from healthy volunteers aged over 18 years who were not taking medication and did not have a family history of coagulation disorders. Family history was assessed via a brief interview with the parents of the children and the adult volunteers themselves. This study was approved by the Melbourne Royal Children’s Hospital Ethics in Human Research Committee, reference number 20031. Neonatal samples were collected from healthy term neonates from the postnatal wards at the Royal Women’s Hospital, Melbourne. Selection criteria included gestational age of more than 37 weeks, birth-weight over 2500 g, APGAR score of at least 7 at 5 min and absence of systemic abnormalities as well as no family history of coagulation disorders. One milligram of vitamin K was administered intramuscularly in the delivery room, as per normal clinical practice. Samples were collected on day one or day three postbirth, via clean venepuncture using the ‘broken needle technique’ from the dorsum of the hand. This study was approved by the Royal Women’s Hospital Research Ethics Committee (EHRC 02/08). Plasma samples were tested individually and grouped for analysis into the following age groups: Day 1 and Day 3 neonates, 1 month to 1 year, 1–5 year olds, 6–10 year olds, 11–16 year olds and adults based on seminal studies performed by Andrew et al. [7, 8]. Plasma samples were thawed at 37 °C for 10 min and assayed using the Procoagulant Phospholipid kit on the STA-R analyser (Diagnostica Stago) as per manufacturer’s instructions. Statistical analysis was performed using the statistical software package STATA (STATA Corporation, College

Cardiopulmonary bypass changes the plasma proteome in children undergoing tetralogy of Fallot repair

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 (p<0.01 above a false discovery rate). Proteins were identified from peptides that demonstrated differential expression. Results: 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.

Personalised anticoagulation approach to improve the prevention and treatment of thrombosis

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].