[Truncated abstract] Every year thousands of individuals suffer from thrombotic related complications that in some cases can be fatal and every year millions of women take some form of hormonal contraceptive. In some cases, there is a cause and effect relationship between the two as users of the combined oral contraceptive pill have an increased risk of developing a thrombotic event. Increased circulating levels of oestrogen cause a prothrombotic shift in the coagulation cascade resulting from upregulation of several procoagulant proteins and a decrease of key anticoagulant proteins. One of the most oestrogen sensitive anticoagulants is Protein S (PS), a product of the PROS1 gene. PS acts as a cofactor to activated protein C (aPC) and the PS-aPC complex serves to downregulate clot formation by deactivating the tenase and prothrombinase complexes via proteolytic cleavage of activated factors VIII and V, respectively. As such, low PS levels are associated with an increased risk of developing thrombotic disorders such as pulmonary embolism, stroke or coronary thrombosis and deep vein thrombosis. During pregnancy when oestrogen levels increase, a steady decline in PS is evident in the early weeks of gestation and continues to decrease to below the normal range in the 2nd trimester, remaining there until post-partum. In addition, reduced free and total PS levels are observed in users of the combined oral contraceptive (COC) pill that contains an oestrogen and a progestin. Interestingly, users of 3rd generation COCs have significantly greater reductions of PS than do 2nd generation COC users. The difference between the two forms is the type of progestin, not the oestrogen, which is predominantly ethinyl oestradiol in the majority of commercially available preparations. At present, a mechanism to describe the relationship between oestrogen and/or progesterone associated with the observed in vivo changes in the levels of PS has not been identified. The aim of this thesis was to define the molecular mechanisms involved in the regulation of PS expression by oestrogen and progesterone. In this study, a Combined Single-stranded conformational analysis and Heteroduplex Analysis (CSHA) iv methodology was optimised for screening both PROS1 DNA and mRNA for the detection of mutations. '...' This may explain why users of 3rd generation COCs display a greater reduction in circulating PS levels compared to 2nd generation users. To investigate potential PS interactions with other proteins that could be hormonally regulated, a yeast-2-hybrid (Y-2-H) screen was performed using the PS molecule as a 'bait' against molecules derived from liver and bone marrow cDNA libraries. A clone that contained a portion of another haemostatic protein, Protein Z (PZ) was isolated and confirmed via sequencing. As no full length PZ clones were identified, a second Y-2-H screen was performed once again using the PS molecule as bait and the PZ molecule as the fish. Interaction between the two proteins was shown to be possible via the successful growth of colonies on triple knock out selective media and by positive β-galactosidase activity.
|Qualification||Doctor of Philosophy|
|Publication status||Unpublished - 2007|