Paediatrics (RCH) - Theses
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ItemCharacterisation of the age-specific differences in platelet physiology and function( 2014)Cardiovascular disease represents a major healthcare burden and is also the major cause of death in Australia. Thrombotic and haemostatic problems involving platelets is an integral part of the disease progression not only in cardiovascular disease but also in other high burden diseases such as cancer and diabetes. The incidence of thrombosis is rare in children but the morbidity associated with it is clinically significant. Children are apparently protected from spontaneous thrombosis. This ‘thrombo-protection’ is consistent with clinical observations where children do not develop the same degree of thrombotic complications as seen in adults when exposed to similar thrombogenic stimuli such as prolonged immobilisation after surgery. Developmental haemostasis is important as it informs the physiological changes of the haemostatic system with age and potentially identifies the pathophysiological changes. Platelets are crucial protective cellular elements of haemostasis and are also known to be immune mediators in pathologic thrombosis. Despite this integral role of platelets, there is a paucity of information relating to normal platelet physiology and function in children. The research presented in this thesis aimed to characterise platelets in healthy children compared to healthy adults. Our study hypothesised that there are age-specific differences in the physiology and function of platelets in healthy children compared to healthy adults. These differences may play a role in the thrombo-protection observed in children. This hypothesis was tested by addressing the following specific aims: 1. To characterise molecular indices of circulating platelet activation and monocyte-platelet interaction in healthy children compared to healthy adults. 2. To characterise the platelet response to physiological stimulation in healthy children compared to healthy adults. 3. To characterise the proteome of platelet releasate and identify candidate proteins which are differentially expressed in healthy children as compared to healthy adults. A total of 58 healthy children (median 5.5 years, age range: 8 months to 14 years) and 56 healthy adults volunteers (median age 30.5 years, age range: 20 to 45.2 years) were recruited into this study. Citrated whole blood was collected and processed immediately for each of the experiments. The physiology and function of platelets were characterised using multi-colour flow cytometry and proteomics approaches. We reported the first evidence of elevated monocyte-platelet-aggregates (MPA) in healthy children compared to healthy adults. MPA formation in adults is associated with thrombo-inflammatory response and is linked to underlying thrombotic diseases. Platelets bound to monocytes in children did not show the elevation of P-selectin expression that is typically associated with MPA formation in adults. Elevated levels of P-selectin are associated with platelet activation. These results suggest that elevated circulating MPA in children are not a result of increased platelet activation and granule exocytosis. In addition, the expression of Mac-1 on circulating monocytes with bound platelets in children was significantly lower than in adults, where an increase in Mac-1 expression is associated with monocyte activation. Hence, this differential expression of Mac-1 in MPA implies a different interaction mechanism that is responsible for MPA observed in children and adults. MPA in healthy children are unlikely to play the same role in thrombo-inflammatory response as reported in adult studies. Platelet responsiveness to physiological agonist stimulation was tested and it was demonstrated that children showed hyper-responsiveness to submaximal dose of Adenosine Diphosphate (ADP) and thrombin receptor activating peptide (TRAP). These results implied that children may respond differently to antiplatelet treatment which inhibits the specific platelet activation pathways. Moreover, these results demonstrated a negative correlation of agonist-stimulated MPA formation in children correlated with age. This result highlights the need to establish appropriate age-specific reference range for MPA when this parameter is used in paediatric population, rather than extrapolation from the adult data. This is the first study of the platelet releasate proteome in healthy children. In this study, there were 11 proteins that were differentially expressed in healthy children compared to healthy adults. In children, the effects of decrease in TSP-1, reduced FXIII but increase in PAI-1 and reduction in fibrinogen gamma chain seem to converge on a common mechanism which supports thrombo-protection observed in paediatric population. In summary, this study demonstrated that platelets in healthy children are different from platelets in adults. There was a significant difference in cellular interactions as seen in elevated circulating MPA in healthy children compared to healthy adults. Platelets from children showed hyper-responsiveness to physiological stimuli. Furthermore, the platelet releasate proteome in healthy children showed a different spectrum of protein abundance compared to healthy adults. The hypothesis of this study, that there are age-specific differences in physiology and function of platelets in healthy children comparing to healthy adults is well-supported. This research provides new insights into developmental changes in platelet physiology that have clinical significance. Future study will continue to contribute to the understanding of the potential thrombo-protective mechanism observed in children.
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ItemThe impact of remote ischaemic preconditioning on the human plasma proteome( 2014)Remote Ischemic Preconditioning (RIPC) is an intervention involving intermittent periods of ischaemia and reperfusion of peripheral tissue to provide multi-organ protection, including the heart, from ischaemia-reperfusion (IR) injury. RIPC has been shown to improve clinical outcomes in a variety of settings. It appears that humoral factors that are released into the blood in response to the RIPC stimulus. Previous studies have demonstrated a global genomic response to RIPC in healthy adult volunteers. The focus of the studies described in this thesis was to examine the proteomic changes in plasma in response to RIPC in healthy adult volunteers and children undergoing repair of Tetralogy of Fallot (ToF) with cardiopulmonary bypass (CPB). The latter was in the setting of a randomised controlled trial (RCT). RIPC was induced by cyclic inflations of a standard blood pressure cuff applied to a limb, to a pressure exceeding systolic in order to completely interrupt blood flow. Complete interruption of blood flow was confirmed by pulse oximetry. The protocol involved four cycles of five minutes of ischaemia alternating with five minutes of reperfusion. In the preclinical study, the proteomic change in plasma originating from the limb subjected to the RIPC and the proteomic response to RIPC at both 15 min and 24 h post RIPC was studied. Forty eight proteins were found to be differentially expressed with up-regulation dominating during the cycles of reperfusion and down-regulation evident at 15 min and 24 h post RIPC stimulus. These results suggested that in response to brief episodes of ischaemia and reperfusion in an upper limb, proteomic changes in the plasma are induced. This may result in a protective state systemically, that significantly modifies both the early and late proteomic response to IR injury. We then extended this idea into the clinical setting to determine if a similar proteomic response could be detected in children with ToF undergoing repair with CPB. A double blind RCT was performed to investigate whether RIPC modifies clinical markers of IR injury and cardiopulmonary function after CPB in children undergoing ToF repair. Although there was no clinical evidence that RIPC promotes recovery of cardiac function, reduces post-operative complications or inotrope requirement in the first 24 h post CPB, the proteomic response to the RIPC was noticeable. We presented the results characterising the proteomic changes in response to CPB that are most apparent at 6 h and 12 h post CPB and return to baseline within 24 h. CPB induced significant changes to the global proteomic response in plasma of children undergoing cardiac surgery for repair of ToF. We further investigated the impact of the RIPC on modifying the global plasma proteomic response during the peri-operative period in plasma from children undergoing ToF repair with CPB. There were no differences between the control group and the RIPC group at baseline or at the end of CPB. At 6h post CPB, there were 48 peptides that were found to be differentially expressed which related to six proteins. There was a return to the baseline within 24 h of CPB. In conclusion, the findings of this thesis provided evidence for the first time that there were proteomic changes in human plasma in response to RIPC, supporting the hypothesis that humoral factors are released into the blood to render a protective state against IR injury. Together, findings from the preclinical study and the RCT indicated that the observed proteomic changes were consistent in healthy adults and children undergoing heart surgery.