Chantal Attard

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.

First Report of Elevated Monocyte-Platelet Aggregates in Healthy Children

Platelets are subcellular fragments which circulate in blood and have well established roles in thrombosis and haemostasis in adults. Upon activation, platelets undergo granule exocytosis and express P-Selectin on the cell membrane which binds a ligand on monocytes, leading to monocyte-platelet aggregation. Elevated circulating monocyte-platelet aggregates in adults are linked to atherothrombosis, but have not been investigated in children where thrombosis is less common. This study aimed to measure monocyte-platelet aggregate formation in children using whole blood flow cytometry. Monocyte-platelet aggregates as well as activation and granule exocytosis of platelets were measured in healthy adults (n = 15, median age 28 years) and healthy children (n = 28, median age 7 years). Monocyte-platelet aggregates in healthy children were elevated compared to healthy adults (37.8±4.4% vs 15.5±1.9% respectively, p<0.01). However, this was not accompanied by any difference in platelet activation (PAC-1 binding 6.8±1.5% vs 6.3±2.0% respectively, p = ns) or granule exocytosis (P-selectin expression 4.4±0.5% vs 3.1±0.5% respectively, p = ns). Despite comparable numbers of platelets bound per monocyte (GPIb MFI 117.3±13.7 vs 130.9±28.6 respectively, p = ns), surface P-selectin expression per platelet-bound monocyte was lower in children compared to adults. We therefore provide the first data of elevated monocyte-platelet aggregates in healthy children.

The in vitro anticoagulant effect of rivaroxaban in children

INTRODUCTION Current anticoagulation therapy in children is less than ideal, requiring regular venous monitoring and dosing adjustments. Limitations associated with conventional anticoagulants have prompted the development of novel drugs that specifically target key proteins in the coagulation system. Rivaroxaban is the first oral, direct Factor Xa inhibitor available for the prevention of venous thromboembolism in adults. Its predictable pharmacokinetic profile, high oral bioavailability and once-daily dosing make rivaroxaban an optimal anticoagulant that warrants investigation in children. The aim of this study was to investigate the age-related anticoagulant effect of rivaroxaban in vitro. MATERIALS AND METHODS Age-specific plasma pools were created (i.e. 28 days-23 months, 2-6, 7-11, 12-16 years and adults) and spiked with increasing concentrations of rivaroxaban (0-500 ng/ml). Commercially available PT, APTT and anti-Factor Xa assays, as well as sub-sampling thrombin generation assays, were used to measure rivaroxaban effect. RESULTS The results of this study indicate that there are no significant differences in rivaroxaban effect across the age groups in vitro. CONCLUSION In vivo studies are required to confirm the consistency of dose-response across the paediatric age groups.

The in-vitro anticoagulant effect of rivaroxaban in neonates.

The use of anticoagulants in neonates is increasing because of the medical advances improving the long-term survival of very sick infants who are at risk of venous thromboembolism (VTE). Current anticoagulation therapy in neonates is less than ideal, because of the physiological differences compared to children and adults regarding the pathophysiology of thrombosis and pharmacology of the drug. Limitations associated with conventional anticoagulants have prompted the development of novel drugs that specifically target the key proteins in the coagulation system. Rivaroxaban is the first oral, direct Factor Xa inhibitor available for the prevention of VTE in adults. Its predictable pharmacokinetic profile, high oral bioavailability and once-daily dosing make rivaroxaban an optimal anticoagulant that warrants investigation in neonates. This study was designed to determine whether there are age-related differences in the pharmacodynamic effects of rivaroxaban in vitro amongst neonates. Neonatal and adult plasma pools were created and spiked with increasing concentrations of rivaroxaban (0–500 ng/ml). Commercially available prothrombin time (PT), activated partial thromboplastin time (aPTT) and anti-Factor Xa assays as well as a sub-sampling thrombin generation assay were used to measure the rivaroxaban effect. A dose-dependent response was observed for PT, aPTT and lag time in both the age groups. Rivaroxaban caused a significant increase in the clotting time for PT and aPTT as well as an increase in lag time (as measured by thrombin generation) in neonates when compared with adults. In-vivo studies are required to confirm the consistency of dose–response in neonates.

Epidemiology of venous thrombosis in children with cancer

There has been an extensive body of research focusing on the epidemiology of thrombosis in adult cancer populations; however, there is significantly less knowledge about thrombosis in paediatric cancer populations. Thrombosis is diagnosed with increasing frequency in children being treated for cancer, and there is an urgent need to increase our understanding of the epidemiology of thrombosis in this population. Currently, there are no guidelines for identification of high-risk groups, prophylaxis or management of thrombotic complications in paediatric cancer patients. We reviewed the available literature regarding the epidemiology, mechanisms, risk factors, prophylaxis and outcomes of thrombosis in children with cancer and identified areas that require further research. The reported incidence of symptomatic venous thromboembolism (VTE) in children with cancer ranges between 2.1% and 16%, while the incidence of asymptomatic events is approximately 40%. Approximately 30% of VTE in this population is associated with central venous lines (CVL). The most common location of VTE is upper and lower extremity deep venous thrombosis (43 to 50% of events, respectively), while 50% of events in ALL patients occur in the central nervous system. Key characteristics that increase the risk of thrombosis include the type of cancer, age of the patient, the presence of a CVL, presence of pulmonary/intra thoracic disease, as well as the type of chemotherapy. Outcomes for paediatric cancer patients with VTE include post-thrombotic syndrome, pulmonary embolism, recurrent thromboembolism, destruction of upper venous system and death. Prospective studies aimed at enabling risk stratification of patients are required to facilitate development of paediatric specific recommendations related to thromboprophylaxis in this population.

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.

Platelets from children are hyper-responsive to activation by thrombin receptor activator peptide and adenosine diphosphate compared to platelets from adults

Platelets are crucial subcellular elements of haemostasis at sites of vascular injury and are also known to be immune mediators in pathological thrombosis. Despite the integral role of platelets in many disease processes, there is very little information available on platelet function and response to agonists in healthy children. We recently reported important differences in the interaction of platelets with monocytes in the circulation, including increased formation of monocyte‐platelet aggregates (MPAs) without concomitant increase in P‐selectin expression. Our current study investigates parameters of platelet activation (PAC‐1 binding and P‐selectin expression) and MPA formation in response to a range of physiologically relevant platelet agonists in healthy children compared to healthy adults. All parameters were significantly higher in children in response to sub‐maximal concentrations of thrombin receptor activator peptide and adenosine diphosphate, reflecting an age‐specific difference in agonist‐stimulated platelet reactivity in children. The results of our study challenge the general assumption that platelet reactivity in children is similar to adults. This finding is fundamental to investigating the role of platelets in diseases of childhood and pathogenesis of adult‐based diseases that have their origins in childhood. Our findings underscore the need for age‐specific reference ranges for platelet function in children rather than extrapolation from adult data.

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.