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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.

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.