ABSTRACT
BACKGROUND Sickle cell disease is a genetic disorder of haemoglobin with a world-wide distribution. Nigeria, the most populous African country has the highest cohort of sickle cell anaemia patients. Hence the need for more research studies on sickle cell disease in this part of the world. The clinical manifestation of sickle cell disease vary enormously ranging from asymptomatic to patients disabled by recurrent pain and chronic complications, the most devastating probably being occurrence of stroke in a patient with sickle cell disease. Sickle cell disease is considered a prethrombotic state; certain characteristics of sickle cell such as abnormal adhesivity and absence of membrane phospholipid asymmetry are involved in the thrombotic process. Most of the morbidity of sickle cell disease is related to the appearance of occlusion of the microvasculature resulting in widespread ischaemia and irreversible organ damage. Protein C is a Vitamin K dependent serine protease and naturally occuring anticoagulant that plays a role in the regulation of haemostasis by inactivating Factors Va and VIIIa in the coagulation cascades. Activated Protein C is a
down regulator of blood coagulation resulting in protection against thrombosis. It also has anti-inflammatory effects through its inhibition of cytokine generation and also exerts profibrinolytic properties that facilitate clot lysis.
OBJECTIVE To evaluate Protein C in Nigerian patients with sickle cell anaemia in steady state so as to assess their prothrombotic tendency compared with normal individuals. Sub-objectives Evaluation of Prothrombin time (PT), Activated partial thromboplastin time (APTT) and liver function tests (LFT) in sickle cell anaemia patients in steady state.
METHOD The study was carried out at the University College Hospital Ibadan. The study population comprised of forty sickle cell anaemia patients who are in steady state, asymptomatic for at least two weeks and forty healthy normal HbA control subjects, age and sex matched who satisfy the inclusion criteria as contained in the methodology. Protein C was assayed with Amax Destiny plus Coagulometer using clot based method.
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RESULTS There was a significant decrease in the Protein C in sickle cell anaemia patients in steady state (median value 62.8%) compared with HbA controls (median value 74.6%) (P=0.000). The HbS patients had significantly higher APTT (median value 48.5s) than the control subjects (median value 44s). (P=0.025). However, no significant difference was found between liver function tests of the HbS patients and the control subjects. (ALT: P=0.823, Albumin: P=0.117, Total Protein: P=0.371). Spearman rank correlation coefficient (rho) between protein C and other parameters of the patients are as follows; PT (rho=0.30, p=0.06). APTT (rho=0.21, p=0.19). ALT (rho=0.1, p=0.54) Albumin (rho=0.23, p=0.16) Total protein (rho 0.02, p=0.92) None of the parameters had significant relationship with protein C in HbS patients in steady state. (p>0.05 in all cases.)
CONCLUSION The low Protein C level observed in HbS patients in steady state may not be due to hepatic dysfunction as LFT in the subjects were found to be normal. Further studies should focus on comparing Protein C in steady state and in crisis in Nigerian sickle cell disease patients.
CHAPTER ONE
1.0 INTRODUCTION
Sickle cell haemoglobin was the first hemoglobin variant discovered. It was first described in the medical literature at the beginning of the twentieth century. Over 500 structural variants of haemoglobin have since been discovered. The earliest known medical paper with the original name of sickle cell disease was published in 1874 by Africanus Horton [1]. Herrick’s was the first to describe the elongated crescent shaped cells in 1910 and this led to a variety of in vitro experiments which attempted to explain this phenomenon [2]. Hydrophobic Valine is substituted for the normal more hydrophilic Glutamic acid at the sixth residue from the NH3 terminal of the Beta-globin chain in HbS. This substitution is due to a nucleotide mutation (GAG/GTG) in the sixth codon of the beta globin gene of the DNA in the long arm of chromosome 16. Sickle cell is a germ line mutation and hence it is passed down intact over generations [3]. The mutation can be found in five different haplo types (Senegal, Benin, Bantu, Arab India and Cameroon) leading to the conclusion that the mutation appeared independently five times in five different founders in human history
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[4]. However, their highest frequency occurs in tropical areas but the population migrations have ensured that they are encountered in most different countries [4]. It apparently arose repeatedly in region where there is malaria endemicity particularly in Africa and Middle East. It is interesting to note that a single copy of sickle cell gene helps the carrier to survive malaria infection [5]. The sickled configuration of red blood cells occurs when haemoglobin is deoxygenated. The loss of potassium and water associated with the sickling phenomenon provides inhospitable environment for the plasmodium falciparum parasite [5]. The vicious cycle of the erythrocyte membrane damage of sickle cell disease is well documented [5]. The sickling phenomenon results from the formation of deoxyhaemoglobin S and the transition from a sol to a gel is accompanied by a dramatic increase in blood viscosity. The consequent increase in haemoglobin concentration accelerates and potentiates the rate of deoxygenation of the erythrocytes at which further polymerization can occur. This marks the beginning of the numerous structural and functional abnormalities of sickle red blood cells [6, 7].
The clinical manifestations of sickle cell disease vary enormously ranging from asymptomatic subjects to patients disabled by recurrent pain and chronic complications. Virtually every organ system in the body is subject to vaso-occlusion. This accounts for the characteristic acute and chronic multisystem failure in the sickle cell disease patient. A case of neuro-ophthalmological sequelae of sickle cell disease reported by Adeuja A.O ,Osuntokun O and Essien EM in 1990[9] revealed multiple cerebral ,pontine and cerebellar infarct at autopsy of a 16yr old sickle cell anaemia patient who presented with impairement of vision. The whole blood viscosity is a function of both the number of erythrocytes, their deformability and of the levels of plasma protein [5]. The plasma proteins have a mediating effect with RBC-RBC adhesive interaction at lower sheer rates. This is mediated by large plasma proteins e.g. fibrinogen [7, 8]. As reported by Ballas and Mohandas [10], a wide number of interrelated factors influence the micro and macro rheology of the sickle blood. The plasma components, the unsickling-sickling red cell cycles, the cellular dehydration, the erythrocyte deformability and mechanical fragility, the white cell populations, the environment and the alterations of the Virchow triad on the
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haemostatic system [1, 8] are responsible for the adhesive interaction of sickle RBC with the vascular endothelium. Patients with Sickle cell anaemia run the risk of having decreased levels of natural coagulation inhibitors [11]. They are considered as having a prethrombotic state. Certain characteristics of sickle cells such as abnormal adhesivity and loss of lipid asymmetry contributes to the thrombotic process [12]. Hepatic dysfunction found in these patients also contributes to the low levels of natural coagulation inhibitors. Deficiencies of protein C and enhanced thrombin generation have been reported in patients with sickle cell disease [13]. Protein C activity was found to be lowered in HbS patients during painful crises [14]. Protein C, a serine protease and its cofactor protein S are both vitamin K dependent proteins with anticoagulant, anti-inflammatory and profibrinolytic properties [15]. They are the major inhibitors of factors Va and VIIIa. The first step in this process is the activation of protein C by thrombin molecules bound to thrombomodulin. Thrombomodulin is a 75-KD a transmembrane protein found on the luminal surface of endothelial cells. Activated protein C then forms a complex with protein S on the phospholipid membranes of platelets. This complex subsequently enzymatically degrades factors Va and VIIIa [15].
The expression of thrombomodulin is down regulated by tumor necrosis factor, endotoxin and interleukin 2 hence coagulation is thereby promoted by these mediators of inflammation. Of the total protein S in the circulation, approximately 60% is complexed with C4b-binding protein and thus has procoagulant activity unlike the unbound protein S that has anticoagulant and prothrombotic activities through its catalytic activity of protein c function. Hepatic synthesis of C4b- binding protein is increased by inflammatory mediators resulting in reduced free protein S levels [15]. All these predispose persons with with SCD to increased coagulation and reduced fibrinolysis. This study therefore is designed to evaluate protein C in Nigerian patients with sickle cell anaemia in steady state since low levels of natural anticoagulants may be a major factor in their prothrombotic tendency compared with normal HbA individuals. Sickle cell anaemia patient is said to be in steady state when he or she is asymptomatic or free of any acute illness for at least two weeks.
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OBJECTIVE AND SUB OBJECTIVES 1.1-Broad objective: Protein C assay in Nigerian patients with Sickle Cell Anemia in steady state so as to assess their prothrombotic tendency compared with normal HbA individuals and its contribution if any to morbidity and mortality. 1.1.1-The sub objectives PT and APTT determination in Nigerian patients with Sickle Cell Anaemia in steady state to assess the subjects’ global test of coagulation. This information will help to assess whether they are hypo-coagulable or hyper-coagulable. LFT in the selected subjects for the study to assess synthetic function of the Liver or rule out primary liver dysfunction since Protein C synthesis occurs in the Liver.
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