1.0 Introduction 1.0 Coagulation Cascade Following injury to a blood vessel, the haemostatic response is initiated. Platelets and the coagulation cascade work synergistically to form a platelet and fibrin rich clot (Chambers, 2003). The clot seals the damaged area and prevents excessive blood loss. The coagulation cascade is defined as a series of enzymatic reactions in which inactive zymogens are converted to activated clotting factors, ultimately leading to the generation of thrombin. There are 2 distinct coagulation pathways, the Tissue Factor (TF) pathway (previously referred to as the extrinsic pathway) and the contact activation pathway (previously referred to as the intrinsic pathway). TF is also known as thromboplastin or FIII, and it initiates the coagulation cascade (Mackman, 2009). It is also referred to as CD142, a protein commonly found in white blood cells and subendothelial tissues to initiate the process of thrombin formation, starting from zymogen prothrombin activation (Versteeg et al., 2011). The damage to the blood vessels will expose the cells containing TF to the bloodstream, leading to the TF to bind to FVII and calcium forming a bridge between TF and FVII (Dahlback, 2005). The TF/FVII complex formed initiates an extracellular cascade that results in a number of serine protease activations. The TF/FVII is autocleaved and activated to TF/FVIIa, which then binds to TF. The resulting complex, FVIIa-TF, activates FIX and FX to FIXa and FXa respectively. FXa in the presence of FVa converts prothrombin (FII) to thrombin (FIIa), which then converts fibrinogen to fibrin. Fibrin is then deposited and the platelets are activated to form a clot. The fibrin clot is stabilized by crosslinking through the activation of FXIII to FXIIIa ((Dahlback, 2005). The contact activation pathway, also known as plasma kallikrein-kinin system is comprised of three serine proteases. These include the plasma prekallikrein (PK), coagulation factors FXII (Hageman factor) and FXI, and the high molecular weight kininogen (HK), which is a nonenzymatic cofactor (Wu, 2015). Fibrin is usually formed when blood is exposed to injured surfaces or when blood is exposed to blood activation surfaces. When the blood is exposed to blood activation surface in the cardiovascular cells, the contact system, namely, PK, HK and FXII, is activated and this further stimulates the intrinsic pathway to activate FXI (Wu, 2015). This activation initiates a series of proinflammatory and procoagulation reactions through the kallikrein-kinin system and intrinsic pathway of coagulation respectively. HK and factor XII readily bind to polyanions while FXI and PK will bind to the surface through the formation of a complex with HK. The surface binding will induce a change in FXII shape, thus resulting in limited contact factor activity. The activated ?¤FXII will cleave PK and FXI to FXIa and PK. The formed PK will activate FXII and a further cleavage of ?¤FXIIa will generate ??FXIIa which will have an active domain of the protease. ??FXIIa will cease to bind to the surface and cleave FXI and kallikrein will cleave HK to bradykinin (BK) (Maas, Oschatz & Renne, 2011). The activation of FXII leads to the activation of plasminogen by kallikrein and FXIIa. Fibrin formation is triggered by FXIIa through FXI in the contact activation pathway. These two pathways merge in the final common pathway which lead to thrombin generation by activation of FXII l to the activation of FIX and XI, converge at FX activation. FXa converts prothrombin to thrombin with the presence of FVa. Thrombin then converts fibrinogen to fibrin, which is a clot as shown in Figure 1. . Figure 1:TF pathway, contact pathway and common pathway (Gailani & Renne, 2007) Thrombin is a multifunctional serine protease. The protease activates the endothelial cells and a host of other critical responses in the circulatory systems apart from being involved in platelet aggregation in haemostasis. Thrombin has a procoagulant activity (converts fibrinogen to fibrin, activates FV, FVIII. As an anticoagulant, thrombin activates Protein C and platelets (via PAR-1/PAR-4), induces endothelial cell activation (via PAR-1), and influences fibrinolysis (activates TAFI) (Coughlin, 2000). The TF pathway is tightly controlled by a number of anticoagulant proteins such as the tissue factor pathway inhibitor (TFPI), protein C pathway (thrombomodulin or TM and activated protein C or APC) and anti-thrombin (Coughlin, 2000). TFPI acts to limit the initiation coagulation via directly inhibiting the unbound FXa and interacting with the complex formed from TF, FVIIa and FXa. The affinity of TFPI to bind with FXa is increased by protein S, and both exist in platelets in considerably high levels (Versteeg et al., 2011). Protein C controls the activities of FVIIIa and FVa, the key pro-coagulation factors in coagulation. The activation of protein C to APC is regulated by the thrombin which is bound to thrombomodulin. APC-mediated cleavage of both FVIIIa and FVa involves FV and protein S. Protein C also regulates the activation of prothrombin and FX, which consequently inhibits thrombin (Dahlback , 2005). In normal conditions, the anticoagulation pathway prevails over procoagulation pathway to maintain the normal blood flow in the body. Anticoagulation pathway works through antithrombin, which is a serine protease that inhibits the procoagulation enzymes (Dahlback, 2005). PT measures, also known as the international normalized ratio (INR) test which is the time it takes for a clot to form and thus used to diagnose bleeding complications. PT can also be used to demonstrate the working mechanism of anticoagulant therapies such as warfarin (Kuruvilla & Gurk-Turner, 2001). TT refers to a coagulation assay used to detect the duration it takes for clotting to occur in a plasma sample which has been pretreated with excess thrombin and an anticoagulant (Flanders, Crist & George, 2003). The assay checks for the abnormalities in the activation of fibrinogen to fibrin. TT is largely used in the evaluation of plasma samples that have prolonged APTT and PT values. APTT assay also measures the activity of factors involved in contact and the common pathways of coagulation. Apart from checking the abnormalities in coagulation, APTT can also be used to monitor patients following heparin administration (Platt, 2008). Thrombin generation test is used to diagnose thrombosis and can be invaluable in the management of haemorrhage (Lance, 2015). 1.1 Pregnancy Beyond, its critical role in haemostasis, the coagulation cascade has been shown to play an important role in many physiological and pathological processes, such as pregnancy and pregnancy related disorders like preeclampsia (Include reference to support statement) Pregnancy in humans begins from the point of fertilization of the female gametes which takes place in the Fallopian tube of the female reproductive system after the introduction of sperms by the male. After the sperm has been deposited near the entrance of the cervix they move up the cervical canal through the uterus and into the fallopian tube. After ovulation, the egg is moved to the fallopian tube in a location that it can be found by the sperms. The first sperm to penetrate the egg is the one responsible for the fertilization of the egg. After fertilization, the fertilized egg then moves to the uterus for implantation. It is after implantation that the process of placentation starts where there is a formation of the maternal portion by decidua basalis and the fetal portion by the chorion. The uteroplacental circulatory system then develops to provide the development of the entire hemochorial placenta which allows the movement of nutrients and waste products between the maternal and the fetal blood. The entire pregnancy period takes about 40 weeks divided into first, second and third trimesters. When the baby is fully developed and ready for delivery, hormones are produced which initiate the contraction and expansion of the muscles in the lower abdomen to ease the natural delivery of the baby (Jukic et al., 2013). The process of placenta development starts from morula movement into the uterus to result in the formation of a blastocyst. The blastocyst cells are the ones responsible for the development of the outer membrane of the placenta, known as the chorion and the trophoblastic layer. The inner cells of the blastocyst form the inner cell of the placenta known as the amnion. The development of the blood vessels of the developing zygote are the ones which provide nutrients and the materials responsible for the growth and development of the placenta which is the organ that connects both the fetus and the uterine wall of the mother for nutrient uptake and waste material exchange. This type of placentation in humans is known as hemochorial placentation and exhibits the unique feature of the chorion having a direct contact with the maternal blood (Davenport, 2016). The mother and the fetus do not have a direct contact in terms of blood. The direct contact of the maternal blood with the chorion of the fetus results inefficient transfer of nutrients and exchange of waste products. A figure showing the various parts of the placenta is as shown below. The boundary of the fetal blood and the maternal blood has also been shown to give an idea of how the materials are exchanged between the two bloods. Figure 2: The human placenta with the details about blood contact (Davenport, 2016). 1.2 The Role of Coagulation in Embryology During embryo development, it has been observed that some if not all of the coagulation factors vary in concentration as the embryo develops. During the early development stages of the embryo at around 29 to about 31 weeks of the gestation period, proteins of the contact system and the proteins which depend on vitamin K tend to reduce in concentration and a good example is factor FIX which becomes lower as compared to a factor such as FX and FVII (Include reference to support statement) . In even more early weeks of gestation typically 19 to 28 weeks of the gestation period, factors FIX and the antigen levels appear lower as compared to the factors such as FX and FVII. All these studies indicate that there is a possibility of factor FVII having a critical role in the coagulation in various stages of the embryonic development (Thornton and Douglas, 2010). Deficiency in specific coagulation factors result into different complications during embryonic development in relation to coagulation in the embryo. An example is the deficiency in tissues factor (TF) which leads to lethality during early stages of development as it is responsible for both the maintenance and development of vascular integrity. Deficiency in factor FX also has the same effect in the embryonic development as TF during the later stages of embryonic development. The only factor which has not shown embryonic lethality during the experiments with an embryo is factor FVII during the times of its deficiency (Kashif and Isermann, 2013). In the knockout (KO) mice model, there has been a intensive study with respect to the coagulation factors most of which are also found in human beings. Lack or very low levels of some of these coagulation factors in the KO mice models indicate the same symptoms of either bleeding or poor development as also observed in the development stages of the embryo. In the KO mice model, mice which have no or very little fibrinogen possess similar characteristics as the expectant mothers with very low values of fibrinogen as they cannot maintain pregnancy without proper clinical intervention. Mouse models have proven that some form of clot formation is important when it comes to the maintenance of pregnancy (Sharma, 2010). 1.3 Coagulation during Pregnancy During a normal pregnancy in humans, there are changes in coagulation through the entire gestation period. This is evident from the tests and studies which have already been carried out on various expectant mothers (Include reference for studies carried out). From the studies, hyper coagulation is a state developed in expectant mothers for the main reason of preventing too much blood loss during delivery. The consequence of this state of hyper coagulation is that the development of a state known as venous thromboembolism (VTE) which occurs mostly during the third trimester of pregnancy. According to James, 2009, the risk of acquiring VTE increases with pregnancy due to the reduced venous outflow and venous capacitance and this risk is about four to five fold for pregnant mothers as opposed to non-pregnant individuals. According to Kevane et al., 2014 there are many risk factors involves in an increase in pregnancy-associated VTE events such as obesity, infection, Caesarean section (CS), advanced maternal age, bleeding, diabetes, family history of VTE and thrombophilia, previous VTE and preeclampsia During pregnancy, factors such as VII, VIII, IX, X, XII, Plasminogen and D-Dimer always increase in levels of up to 1000% while fibrinogen levels tend to rise up to 200% above normal levels, factors such as XI and protein S reduce in levels while factors such as Factor II, AT-III and protein C remain unchanged. These changes in the levels of factors are as a result of the changing levels of the hormones during the various phases of the pregnancy. The coagulation disorders in expectant mothers can be tested through the use of Prothrombin time (PT) and Partial thromboplastin time (PPT) with the former majorly dealing with the extrinsic system while the latter dealing with the intrinsic system. A PT test is used to detect the presence of fibrinogen, prothrombin factors X, VII and V. An 11-15 second time indicates a normal presence of the listed clotting factors with a prolonged time indicating a lack of the clotting factors. Blood clots during pregnancy are always tested through the use of D-Dimer test which involves the test carried out on the substance that is generated after the breakdown of a blood clot. This is carried out on patients who are showing signs of venous thromboembolism, which can either be detected through tests or through physical symptoms shown by the expectant mother (James, 2009) 1.4 Preeclampsia Preeclampsia can be defined as a disorder of pregnancy that is associated with high amount of protein content in the urine and high level of blood pressure (Dacaj et al., 2016). It complicates up to 7% of the pregnancies (Include reference to support this statement). Globally, Preeclampsia is a leading foundation for maternal mortality. In the USA and Ireland, it is accountable for the deaths of the inborn and the expectant mothers. It does increase the danger of the outcome of the baby and to the mother. If the disorder is left untreated, the consequence may be a seizure which is also known as eclampsia (Include reference to support this statement). Preeclampsia is characterized by hypertension (readings of systolic blood pressure ? 140 mmHg and/or diastolic BP ? 90 mmHg) and proteinuria (with a urine dipstick of ? 1+ or ? 300 mg per 24 hours) (Bender & Ryan, 2013). However, the clinical presentation severity is very valuable thus making the outcomes to be always favorable when the mild preeclampsia starts to develop at around week 36 (Need to clarify this statement/ Include reference to support this statement). There is the risk of the increased rate of perinatal, maternal outcome when there is an earlier development of severe preeclampsia; this is usually before 24 weeks gestation (Include reference to support this statement). The Preeclampsia risk factors include; prior hypertension, obesity, diabetes mellitus and older age (Include reference to support this statement). The disorder is highly frequent in women whose first pregnancies are twins. Among the other factors, one major underlying factor involves abnormal or poor blood vessel formation in the placenta. Most of the Preeclampsia cases are diagnosed before the delivery of the baby. Other risk factors of Preeclampsia include women with chronic diseases of the kidney and those with preeclampsia previously. The disorder is more common in the primigravida women or ladies and its risk does increase with the increase in the pregnancy intervals. The risk is higher in women of above 40 years of age. Other risk factors may include women whose families have a history of the disorder and those that suffer from antiphospholipid syndrome (Jeyabalan, 2013). The pathophysiology of preeclampsia is characterized by a preclinical (asymptomatic) and clinical (symptomatic) stage. In a normal pregnancy, foetal extravillous cytotrophoblast invade the uterine wall and the uterine spiral arteries during placentation. During invasion of the spiral arteries, trophoblastic remodel the spiral arteries, turning the low capacity spiral arteries into high capacity blood vessels that can support sufficient blood flow to the placenta (Include reference to support this statement). In preeclampsia, this remodeling does not occur and a pathophysiology is intricate, with many stages entailing maladaptation in both the maternal and placenta physiology and clinical phases that are characterized by maternal hypertension syndrome and proteinuria (Include reference to support this statement). Hypoxic placenta that starts to release lots of inflammatory cytokines, trophoblastic debris, anti-angiogenic factors induce endothelial cell activation and damage. The increase in the hypoxic stress within trophoblasts could stimulate more release of sVEGFR-1 which could worsen the developing placenta outcome and the fetus. The activation of endothelial cell function and inflammatory state contributes to the maternal complications by the deposition of the trophoblastic debris in maternal circulation which then causes peripheral syndrome. The increase in the trophoblastic debris in the preeclampsia is a potential factor by its relationship with the micro particulate debris (Include references to support this paragraph). The immune adaptation and adjustments in the normal pregnancy are associated with the decrease in cytokines T helper one: interleukin 2, the transforming factor of growth beta (TGF ??) and the interferon gamma which are mainly involved in the cellular protection and the immune elimination of foetus. An increase in the cytokines T helper two: interleukins 5,4, 13 and 6 which are responsible for the mediation of the humoral immunity, will lead to the suppression of the cellular immunity thereby preventing the immune rejection or elimination of fetus (Include reference to support this statement). There is increased in the levels of inflammatory cytokines, TNF alpha, IL6 and inflammation marker and the CRP in the condition of severe preeclampsia. There may be the inducement of the release of a factor responsible for bioactive circulation; one of the factors may include the inflammatory cytokines which contributes to the mediation of the endothelial damage of the preeclampsia ((Include reference to support this statement). 1.5 Early Onset Vs Late Onset Preeclampsia. Preeclampsia is a very heterogeneous disorder with regard to presentation, time of onset, and severityand is defined as early onset and late onset preeclampsia. Both share a number of etiological characteristics, they differ in terms of their outcomes and risk factors (Aksnornphusitaphong & Phuong, 2013). Although Fang et al.,2009 failed to identify differences between early onset and late onset preeclampsia due to the sample size, later studies have shown major differences between the two categories of preeclampsia. According to Lisonkova &Joseph, 2013, the observed differences require the two forms of preeclampsia to be treated as independent entities. Early onset preeclampsia is often severe and poses high risks among patients, while late onset preeclampsia is rather mild with good perinatal and maternal outcomes (van der Merwe et al., 2010). Early-onset preeclampsia refers to the form of preeclampsia, which occurs prior to the 34th week of gestation. On the other hand, late onset preeclampsia develops after the 34th week of pregnancy. According to Aksornphusitaphong &Phupong ,2013, risk factors such as African race, a family history of chronic high blood pressure, and prior cases of hypertension differentiate the two categories of preeclampsia. Early cases of hypertension tend to predispose individuals to the early onset of preeclampsia compared to late onset preeclampsia, which has been associated with a family history of the condition. In addition to a history of preeclampsia in early pregnancies, early onset preeclampsia has also been shown to result due to the exposure to secondary smoking, obesity, and lower socioeconomic status (Aksornphusitaphong & Phupong,2013). Maternal age, family history of preeclampsia and BMI have been documented to be aetiological factors resulting in late onset preeclampsia (Valensise et al., 2008). Chronic hypertension has been marked as a predictor of early onset preeclampsia due to its association with vascular diseases and end-organ damage. Contrary, late onset preeclampsia is associated with family history because of the genes implicated in chronic hypertension (Aksornphusitaphong & Phupong, 2013). Remarkable differences between early onset and late onset preeclampsia have been observed in a number of studies. However, Stubert and a team of researchers have concluded that the frequency of predictors of preeclampsia, such as thrombophilia does not change between the two forms (Stubert et al., 2014). 1.6 Coagulation in Preeclampsia. Preeclampsia has been constantly recognized as one of the risk factors of pregnancy-associated venous thromboembolism (VTE). (Egan, Kevane, & Ni Ainle, 2015). As an idiopathic disorder affecting a number of systems of the body during the gestation period, preeclampsia can result in haematological complications among pregnant women (Jahromi & Rafiee, 2009). Notable hematological abnormalities associated with preeclampsia include a decline in plasma clotting factors and thrombocytopenia, which result in VTE during gestation. VTE, which comprises of pulmonary embolism (PE) and deep vein thrombosis (DVT), has remained to be the leading cause of morbidity and death among the affected women ((Include reference to support this statement). Preeclampsia has been identified as one of the novel risk factors of VTE and pregnant women are 5-times at risk of VTE compared to non-pregnant women. In recent years, the impact of preeclampsia on VTE risks at different gestation states has been documented. Although the existing evidence associate preeclampsia with increased risks of VTE, the molecular mechanisms involved in this observation are yet to be deciphered (Egan, Kevane & Ni Ainle, 2015). However, Egan, Kevane and Ni Ainle (2015) research demonstrates that coagulation changes among women with preeclampsia and due to this, their risks for VTE also increases. For instance, preeclamptic women exhibit elevated levels of plasma D-dimer and systemic activation of coagulation. The aetiology of preeclampsia-associated thrombosis is thought to be due to platelet activation, endothelial dysfunction, coagulation activation, and inflammation. These factors, which are unique to preeclampsia could contribute to VTE (Egan, Kevane &Ni Ainle, 2015) Coagulation can be altered both in vivo and in vitro through a number of mechanisms. Among preeclampsia women with DIC (Review sentence it is not clear) doctors recommend a blood transfusion, administration of clotting factors or transfusion of platelets to correct the problem in vivo. This is because serious postpartum bleeding and coagulation complications often develop among women with severe preeclampsia, which lead to increased morbidities (von Schmidt auf Altenstadt et al., 2013). Coagulation can also be altered in vitro, usually in laboratories to study the effect coagulants on coagulation factors (Main point of sentence is not clear). In order to identify predictors for preeclampsia onset and progress, it is important to check the changes in coagulation parameters such as clotting times, D-dimer generation, thrombin generation and change in the levels of coagulation factor in vitro (Han et al., 2014). In normal conditions, prothrombin time (PT), thrombin time (TT), activated partial thromboplastic time (APTT) and platelet count decreases in late pregnancy, and the level of fibrinogen as well as mean platelet volume (MPV) increases. This observation is contrary to what happens during early pregnancy. In women with preeclampsia, the D-dimer, TT, APTT and MPV increases after they reach their third trimester (Han et al., 2014). Disorders such as thrombophilia increase the risks of preeclampsia. For instance, in a study carried out by Mello and a team of other researchers revealed that thrombophilia augmented the risks of preeclampsia among pregnant women (Mello et al., 2005). 1.7 Review of the Methods used in this Study 1.7.1 In vitro Coagulation Studies 220.127.116.11 Thrombin Generation Testing Using CAT The generation of thrombin has been made possible by assays commonly referred to as Thrombin Generation (TG) tests (Add reference for this method). Unlike clotting assays that evaluates the time needed for a clot to be formed, TG assays evaluates the ability of a sample to produce thrombin over time. Clotting assays are mainly characterized by the coagulation initiation phase. During this phase, traces of thrombin that are needed for the formation of clot are made. TG assays involves both the propagation and termination phase (Hemker et al., 2003 / Castoldi & Rosing, 2011). The formation of thrombin can be best depicted using thrombin TG curve. The curve shows the various phases of thrombin formation. Each phase has a unique characteristic that is different from the other (Figure 3). The lag time is the same as the clotting time. It is characterized by the first formation of thrombin traces. The peak phase indicates a point in the assay where maximum amount of thrombin is formed. The period required to reach the maximal amount of thrombin is referred to as time to peak (ttpeak). The area that falls below the thrombin curve is known as the endogenous thrombin potential (ETP). The area is a representation of the catalytic work performed by active thrombin (Castoldi & Rosing, 2011). Figure 3 : Indicates the parameters of TG curve. (Practical-Haemostasis.com). In this investigation this assay is used to measure: Tissue Factor (TF) and contact pathway dependent thrombin generation TF dependent thrombin generation in the presence of different concentrations of thrombomodulin. TF dependent thrombin generation in the presence of different concentrations of activated protein C. 1.7.2 In vivo Coagulation Studies 18.104.22.168 Sandwich ELISAs Specific targets will be measured by Sandwich ELISA by using two layers of antibodies (capture and detection antibody). In this method, monoclonal or polyclonal antibodies can be used as the capture and detection antibodies. Monoclonal antibodies are used to recognize a single epitope. A polyclonal antibody is, in most cases used as the capture antibody to detect as much of the antigen as possible. The advantage of a sandwich ELISA over other forms of ELISA is that the sample does not have to be purified before analysis and very sensitive (up to 2 to 5 times more sensitive than direct or indirect ELISA) (Abcam). In this investigation, sandwich ELISAs method used to measured D-dimer and TAT levels in plasma. (include a diagram to represent your sandwich ELISA here).