TRANSIENT LIPOPOLYSACCHARIDE-INDUCED CYTOKINE RESPONSES IN THE MATERNAL AND FETAL GUINEA PIG

The aim of this study was to further investigate the role of pro-inflammatory cytokines in the pathogenesis of fetal cererbral white matter injury associated with chorioamnionitis by charaterizing the time course of the cytokine response in the pregnant guinea pig following a maternal inflammatory insult. Chorioamnionitis increases the risk for fetal brain injury. In the guinea pig, a threshold maternal inflammatory response must be reached for significant fetal brain injury to occur. However, a previous study demonstrated that, by seven days after an acute maternal inflammatory insult, cytokine levels in both maternal and fetal compartments are not different from controls. The purpose of this study, therefore, was to test the hypothesis that a significant cytokine response occurs within the first seven days following an acute maternal inflammatory response. Pregnant guinea pigs (n=34) were injected intraperitoneally with 100µg/kg lipopolysaccharide (LPS) at 70% gestation and euthanized at 24 hours, 48 hours or 5 days following endotoxin exposure. Control animals were euthanized at 70% gestation without exposure. Concentrations of interleukin-6, interleukin 1-β and tumour necrosis factor-α (IL-6, IL-1β, TNF-α) were quantified in the maternal serum and amniotic fluid by enzyme-linked immunosorbent assay. IL-6 and IL-1β concentrations were elevated in the maternal serum at 24 hours and returned to control levels by five days. In the amniotic fluid, IL-6 peaked at 48 hours and IL-1β at 24 hours. TNF-α levels were not significantly increased. A single maternal LPS injection produces transient increases in cytokine concentrations in the maternal serum and amniotic fluid. This further implicates the cytokines as potential mediators of fetal white matter damage. Although this response might not be sufficient to produce the brain injury itself, it may initiate harmful pro-inflammatory cytokine cascades, which could even continue to harm the fetus following delivery. A human diagnostic protocol was developed to assess the use of serial serum biomarkers, including IL-6 and TNF-α, in the prediction of histological chorioamnionitis. Preliminary analysis of the pilot study suggests that certain biomarkers might be worthy of further investigation in a larger-scale study.

COVALENT LIPOPROTEIN(A) ASSEMBLY: Characterization of Oxidase Activity Responsible for Catalyzing Covalent Lipoprotein(a) Assembly

Lipoprotein(a) (Lp(a)) has been identified as an emerging risk factor for the development of vascular diseases. The Lp(a) particle is assembled in a 2-step process upon secretion of the LDL and apo(a) components from hepatocytes. Work done by the Koschinsky group has identified an oxidase-like activity present in the conditioned medium (CM) harvested from human hepatoma (HepG2), as well as HEK 293 (human endothelian kidney) cells that catalyzes the rate of covalent Lp(a) formation. We have taken a candidate enzyme approach to identifying this oxidase activity. Specifically, we have proposed that the QSOX (Quiescin/sulfhydryl oxidase) is responsible for catalysis of covalent Lp(a) assembly. An oxidase activity assay developed by Dr. Thorpe (University of Delaware) was used to detect QSOX1 in CM harvested from cultured cell lines that catalyze covalent Lp(a) assembly. In addition, the QSOX1 transcript was identified in each cell line and quantified with the use of Real-Time RT-PCR. Quantitative assays of covalent Lp(a) assembly were performed to study some characteristics of the unkwown oxidase activity. First, conditioned medium was dialyzed through a 5 kDa cutoff, as this has previously been shown to reduce the aforementioned oxidase activity. Purified QSOX was then added back to the reaction and the rate of catalysis was observed. The addition of QSOX appeared to enhance the rate of covalent Lp(a) assembly in a dose-dependent manner. Additional covalent Lp(a) assembly assays were performed where various chemicals were added to determine whether Lp(a) assembly was affected. The addition of EDTA did not affect covalent assembly, suggesting that the oxidase activity may not be metallo-dependent. Moreover, dose-dependent addition of Calcium, DTT, Copper and glutathione to dialyzed medium also did not affect the rate of Lp(a) assembly. Taken together, these studies will aid in identifying the nature of the oxidase activity that catalyzes covalent Lp(a) assembly. This will provide us with valuable information on how Lp(a) particles are assembled, and may lead to the development of drugs inhibiting Lp(a) formation.

A Quantitative and Mechanistic Assessment of Activated Thrombin-Activatable Fibrinolysis Inhibitor and its Role in Pathological Bleeding and Thrombosis

The coagulation and fibrinolytic systems are linked by the thrombin-thrombomodulin complex which regulates each system through activation of protein C and TAFI, respectively. We have used novel assays and techniques to study the enzymology and biochemistry of TAFI and TAFIa, to measure TAFI activation in hemophilia A and protein C deficiency and to determine if enhancing TAFI activation can improve hemostasis in hemophilic plasma and whole blood. We show that TAFIa not TAFI attenuates fibrinolysis in vitro and this is supported by a relatively high catalytic efficiency (16.41μM-1s-1) of plasminogen binding site removal from fibrin degradation products (FDPs) by TAFIa. Since the catalytic efficiency of TAFIa in removing these sites is ~60-fold higher than that for inflammatory mediators such as bradykinin it is likely that FDPs are a physiological substrate of TAFIa. The high catalytic efficiency is primarily a result of a low Km which can be explained by a novel mechanism where TAFIa forms a binary complex with plasminogen and is recruited to the surface of FDPs. The low Km also suggests that TAFIa would effectively cleave lysines from FDPs during the early stages of fibrinolysis (i.e. at low concentrations of FDPs). Since individuals with hemophilia suffer from premature fibrinolysis as a result of insufficient TAFI activation we quantified TAFI activation in whole blood from hemophilic subjects. Both the rate of activation and the area under the TAFI activation time course (termed TAFIa potential) was determined to be reduced in hemophilia A and the TAFIa potential was significantly and inversely correlated with the clinical bleeding iii phenotype. Using a novel therapeutic strategy, we used soluble thrombomodulin to increase TAFI activation which improved the clot lysis time in factor VIII deficient human plasma and hemophilic dog plasma as well as hemophilic dog blood. Finally, we briefly show in a biochemical case study that TAFI activation is enhanced in protein C deficiency and when afflicted individuals are placed on Warfarin anticoagulant therapy, TAFI activation is reduced. Since TAFIa stabilizes blood clots, this suggests that reducing TAFI activation or inhibiting TAFIa may help restore blood flow in vessels with pathological thrombosis.

Analysis of parameters of haemostasis in pre-eclamptic, normotensive pregnant and non-pregnant women.

Pregnancy is characterized by a state of heightened coagulation, which is exacerbated in pathological conditions such as pre-eclampsia (PET). PET is further associated with abnormal maternal inflammation and increased circulating microparticles (MP); however, a mechanistic link between these pathological features has never been established. It is proposed in this thesis that abnormal maternal inflammation is causally linked to pro-coagulant trophoblast MP shedding via a mechanism mediated by the pro-inflammatory cytokine tumour necrosis factor alpha (TNF), thereby contributing to maternal coagulopathies associated with PET. Using thromboelastography (TEG) and standard laboratory tests, haemostatic function was evaluated in PET and normotensive subjects at delivery and post-partum. Furthermore, the effects of the menstrual cycle and oral contraceptive (OC) use on haemostatic function were assessed in non-pregnant subjects in order to understand their influence on post-partum haemostasis. Plasma TNF and pro-coagulant MP levels were evaluated in the pregnant subjects. Using chorionic villi explants from human term placentas, MPs were quantified after TNF administration. The pro-coagulant potential of placental MPs was evaluated by TEG by spiking whole-blood with medium containing MPs from chorionic villi. TEG identified increased whole-blood coagulability in PET subjects at delivery, demonstrating its increased sensitivity over standard laboratory tests at identifying haemostatic alterations associated with PET. Haemostatic alterations were normalized by six weeks post-partum. TEG also identified cyclic haemostatic variations associated with OC use. Chorionic villi treated with TNF (1 ng/ml) shed significantly more MPs than untreated placentas. MPs from chorionic villi increased the coagulability of whole-blood. Together, results provide evidence supporting the concept that abnormal maternal inflammation is causally linked to the development of maternal coagulopathies in pregnancy complications. Moreover, TEG may be superior to standard laboratory tests in evaluating haemostasis in pregnant and non-pregnant subjects.