Masters Degrees (Haematology and Cell Biology)
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Item Open Access A yeast-based assay for detection of mutations in the human p16 gene(University of the Free State, 1999-05) Botha, Chantal; Pretorius, G. H. J.Cyclin-dependent kinases (CDKs) are crucial regulators of the cell cyele. CDKs themselves are subject to control by both cyelins and CDK inhibitors. Among the inhibitors, p 16 is very prominent, since it has been found to be mutated or lost in a variety óf tumours, We are interested in mutations involved in the progression of leukemia from the chronic to the acute phase. The p 16 gene has been implicated in this progression, therefore we needed an assay for p 16 status that could be applied to screen patients in chronic phase regularly. Traditional mutation screening makes use of physical methods such as Single Stranded Conformational Polymorphism (SSCP) analysis. These methods are generally labour intensive and are not always informative. If tests for the actual function for the gene products could be devised, it could be used to screen tumour samples for the status of these genes. We have decided to develop a yeast-based test that would directly assay for activity rather than just nucleotide changes. The assay is based on the yeast two-hybrid system, where protein-protein contact is reflected in colony colour. We have designed a primer set to amplify the p16 reading frame by RT-PCR from small amounts of leukocyte mRNA. This cDNA is then cotransformed with a gapped plasmid containing terminal p 16 overlaps, allowing homologous recombination to splice the reading frame into the plasmid. The host strain also contains a CDK4- expressing plasmid and if the amplified p16 can still bind to CDK4, the colonies would turn blue. We have successfully constructed and tested the system and found it to be very sensitive, being able to assay p 16 from as little as 300 microliters of whole blood.Item Open Access Co-expression and functional assay of human Rb and E2F1 proteins in yeast(University of the Free State, 1999-09) Wollenschlaeger, Alex; Pretorius, G. H. JThe mammalian cell cycle is composed of a myriad interactions occurring in a defined sequence dictated by the flow of entropy. The decision to study the cell cycle requires entry into a world where events take place, not because they want to, but because they have to. Cells do not 'decide' to perform certain actions, but are driven by the laws of nature, the same laws that are responsible for the existence of the universe. Study of this maelstrom of reactions is truly analogous to opening Pandora's proverbial box. A peek inside and all the inner workings of the cell start to spill out. Unfortunately, this is where things start to get complicated. Serendipitously, simpler alternatives are available. The yeast cell cycle is remarkably similar to that of higher animals. The Rb and E2F 1 proteins are integral components of the mammalian cell cycle, and as such, they are found in aberrant forms in numerous malignancies. This necessitates an adequate means for the determination of the cellular status of these proteins, as prognosis and diagnosis of several neoplastic disorders are dependentthereon. This study aimed to develop a yeast-based strategy for functional analysis of the human tumour suppressor protein Rb. This goal was impeded by one factor; the yeast cell cycle is too similar to that of humans. Several yeast strains, derived from W303-lA, were constructed that each contain a reporter gene -the lacZ gene of E. coli- regulated by E2F recognition elements introduced within the upstream CyC1 promoter. The theory was that in the absence of Rb, ectopically expressed human E2F 1 protein would be able to bind the RE and activate transcription of the reporter gene, ultimately resulting in a readily observable product. In the presence of functional Rb, E2Fl would be bound by the tumour suppressor protein, and thus be incapable of activating the reporter gene. This would provide an assay of Rb status, based not on tedious sequencing analysis of the genetic material, but on the actual functional activity of the protein. When dealing with Mother Nature, though, we are oftentimes reminded that she has thought of everything. S. cerevisiae contains an E2F-like activity, capable of binding the exact RE introduced into the reporter gene promoter. This was confirmed by experiments in this study, where the reporter gene was activated in the absence of ectopically expressed E2F1 protein. The endogenous yeast E2F-like protein is thus able to activate transcription of the reporter gene, negating the effect that would be observed by ectopic expression of human E2F 1, and thus, all Rb-expression was performed in the absence of eo-expressed human E2F 1. Since it is impossible to distinguish between the yeast and human E2F activities, it is impossible to create a functional assay for Rb activity in the W303-1Aderived chimeras constructed. As mentioned previously, it could be possible to overcome this problem by knocking out the yeast-borne E2F activity, but this approach is restricted by two barriers. Firstly, the yeast equivalent of the E2F-family is yet to be cloned. This problem can be approached with a transposon-based strategy. The endogenous E2F activity is capable of activating transcription of the lacZ reporter gene, and in so doing, provides a convenient assay for YE2F integrity. Through the use of a plasmid containing an inducible transposase it should be possible to disrupt the YE2F -encoding gene through integration of a transposon. This would be accompanied by an inability of the yeast to activate the reporter gene. The transposon, containing flanking genomic DNA, could then be retrieved and provide the basis for cloning the gene coding for YE2F. Since the reporter gene is specific for E2F-like binding, retrieval of non-specific factors should be negated. A second problem is that, since the E2F proteins play an important role in the progression of the cell cycle in higher animals, it is possible that the yeast would not survive knocking out its homologue. Relegation of YE2F to the role of bystander could possibly wreak havoc with the delicate mechanism that is the cell cycle. Still, it would be interesting to further pursue this idea. Ectopic expression of human Rb in the various strains used in this study provided some interesting results. Transformation of pAWl, followed by galactose-based induction of Rb expression resulted in observable differences in growth characteristics of certain strains. Those most affected were W-lf and W-1r, which each contain a single repeat of the E2F RE within the upstream promoter of the reporter gene, albeit in forward and reverse orientations, respectively. The effect was particularly evident in W-1r, where expression of Rb resulted in complete cessation of growth, probably due to binding of yeast E2F-like activity. These results could not, however, be reconciled with those obtained from cell cycle analysis with the aid of flow cytometry. These experiments did not show any significant effect of Rb expression on the cell cycle of the examined strains. This is possibly due to an insufficient period of observation, but this could, unfortunately, neither be confirmed nor dismissed. From the results obtained in this study it appears that construction of an apposite reporter system for the functional assay of Rb is perhaps more tricky than would be expected. The interference of endogenous proteins is a cause for concern in a development strategy such as this, and serves as a caveat for future studies in this field.Item Open Access Selection and characterization of a novel factor XI inhibiting peptide by using phage display technology(University of the Free State, 2002-11) Motloi, Nthabiseng Cecilia; Meiring, S. M.English: The role of factor XI in hemostasis can be seen as a combination of a procoagulant action (the formation of fibrin) and an antifibrinolytic action (the protection of fibrin). High levels of factor XI lead to a prolonged down regulation of fibrinolysis and therefore a risk of thrombosis (Meijers et ai, 2000). Under disease conditions associated with Disseminated Intravascular Coagulation (DIC), the continuous exposure to excess TF typically exhaust the available tissue factor pathway inhibitor (TFPI), leading to rampant thrombin generation by factor XI feedback and therefore also a risk of thrombosis (0sterud and Bjerlid, 2001). I selected possible .inhibitors of factor XI using phage display technology. I started the phage display selection by biopanning in immuno-tubes and eluted the factor XI binding phages non-specifically from the immuno-tube. I did four selection rounds, to enrich the factor XI binding phages. I found only two strong factor XI binding phage clones from a linear 12-mer phage library. Both phage clones bound dose dependently and with a high affinity to factor XI. Both clones also lengthened the partial thromboplastin time (aPTT) dose dependently. The amino acid sequences of the peptides displayed on these two clones indicate that both peptides contain three amino acid sequences of HMWK and thrombin. One clone also contains a three amino acid sequence of factor XII. None of them contains a three amino acid sequence of factor IX. I synthesize a linear peptide with the corresponding sequence as the peptide displayed on the clone that was prevented from binding to factor XI by both factor IX and thrombin. I characterized the peptide by studying its effect on the aPTT. This peptide lengthens the aPTT dose dependently. The lengthening in aPTT of our peptide however indicates that I have selected an inhibitor of the contact system factors of coagulation. In summary, this study shows that the phage display can be used to select novel factor XI inhibitors from random peptide libraries. With further studies, this peptide may be developed as an antithrombotic.Item Open Access DNA characterization of the FGA locus in the human genome(University of the Free State, 2002-11) Asfaw, Estifanos Kebede; De Kock, André; Pretorious, G. H. J.English: The human alpha fibrinogen (FGA) short tandem repeat locus is found in the long arm of chromosome 4. It is located in the third intron of the alpha fibrinogen gene. This complex highly polymorphic tetranucleotide repeat locus together with other STR DNA markers is extensively used in personal identification in medical and forensic sciences. STRs are also used to study genetic variation in distinct ethnic groups and in disease diagnosis. More than 80 alleles have been reported for this locus from various population frequency studies. A few sequence studies have also reported 11 sequence variants to date. The FGA locus was found to have high heterozygosity and power of discrimination. The aim of this study was to characterise the sequence of microvariant and off-ladder complete and microvariant alleles of the FGA locus that were observed during routine paternity analyses. The characterization of the sequence of as many as possible of alleles observed in our study population would also be attempted. A total of 62 DNA specimens were selected and sequence characterized either for one or both alleles. The DNA specimens were 52 from Negroid, 5 mixed ancestry, 4 Caucasian and 1 SAN origin. The PCR reaction was used to amplify the selected alleles. The band of interest was cut from the gel and purified in consecutive PCR and purification steps till separate single bands were obtained. The purified single bands were sequenced using a BigDye terminator ready reaction kit in both forward and reverse reactions separately. These products were precipitated with ethanol acetate and subjected to capillary electrophoresis on an ABIPrism 310 Genetic Analyser using POP6 polymer. The results were analysed using the "Sequence Analysis Software version 2.1". The data obtained were checked, printed and compared with STR analysis results and FGA sequence reports. From the selected 62 specimens a total of 76 complete and microvariant alleles, the size of which ranged between 16.1 (224bp) to 44.2 (337bp) were found. These represent 27 different alleles (13 complete and 14 interalleles). In this study 2 novel (previously undescribed) alleles (40.2 and 41.2) were found. Three sequence variants (26, 28 and 43.2) with two variants each were observed. Two alleles 43.2 and 44.2 that had reported sequence variants were found to have different sequence structures from the published sequences. Forty-nine of the 76 sequenced alleles were within ladder and the remaining 29 were off-ladder. Only 8.41% (4/49) of the within ladder alleles had been wrongly assigned allelic numbers with routine STR analysis. The difference between the routine assignment and the sequencing of these alleles was only 1 or 2 bp. In contrast, all of the 29 off-ladder alleles were wrongly assigned. In this instance the difference was 2 or more base pairs. Although this study was conducted on conveniently selected DNA samples, it had significant results. Three sequence variants, 2 newalleles and, 1 allele, which had been reported, but sequence had not been described was found. Additionally, two other alleles with reported sequences were found, but their sequence structure differed from the published sequences. The samples in this investigation were not representative of the population groups that are found in the Free State province and we suggest further population-based studies of STR loci that are commonly used in paternity and forensic investigation. The information obtained from such studies will disclose the frequency of sequence variant alleles.Item Open Access Investigating the genetic profile of the E-cadherin gene in squamous carcinoma of the esophagus(University of the Free State, 2003-06) Mabina, Boitumelo Desiree; Pretorius, G. H. J.; Van der Merwe, N. C.Item Open Access Construction of cDNA libraries, and the selection and expression of proteins and peptides involved in haemostasis(University of the Free State, 2004-06) De Bruin, Karen; Meiring, S. M.; Deckmyn, H.English: The need to find new manners in which to combat cardiovascular disease and associated thrombotic complications, remains a high priority in industrialised countries. Even in third-world countries the implications and associated risks of these diseases are being felt more and more. The advent of the biotechnology era and employment of recombinant DNA techniques has brought about exponential advances in understanding the complex mechanisms of haemostasis, and is employed to find new ways to combat pathological thrombotic complications. The challenge is to harness the many tools and techniques produced by the ongoing biotechnology explosion, and apply them to elucidate questions still unanswered and explore areas still unknown. In this study it was illustrated that modern molecular biology techniques can be applied in many areas of thrombosis and haemostasis research. The display of cDNA libraries on the surfaces of filamentous bacteriophages was used in the search for novel antithrombotic compounds from a haematophagous insect Hippobosca rufipes. Phages displaying the cDNA libraries were panned against human a-thrombin and selected according to their binding affinity and inhibition ability. To illustrate the use of a Escherichia coli expression system, a domain of a enzyme was cloned, expressed, and the recombinant peptide isolated and refolded. ADAMTS-13 was recently identified as an important role player in the realm of von Willebrand factor activity, including primary haemostasis and pathological disorders. The second carboxy-terminal CUB domain of ADAMTS-13 was amplified from full-length cDNA, cloned into a expression vector system, and expressed as insoluble inclusion bodies in the cytoplasm of E. coli, from where it was isolated and refolded. In this study, molecular techniques were used in different phases of research into the specific activity and interactions of a particular component of the haemostatic system. This illustrated the marriage of biotechnology with fundamental medical research in an era of interdisciplinary sciences.Item Open Access "Killer-cell immunoglobulin-like receptor haplotype diversity in three Free State population groups"(University of the Free State, 2006-11) Louw, Marius; De Kock, André; Louw, Vernon; Coetzee, MariusEnglish: In the foregoing project, an investigation was made into the relative KIR gene frequencies of three South African cohorts. Playing an important part in innate immunity, KIR fill a vital gap between viral onsets and cell mediated humeral immunity. Being able to sense when cells are abnormal, NK cells possess the ability to destroy cells which show altered HLA molecules during KIR/HLA interaction. Ethnic cohorts that were investigated included African black, mixed ancestry and the Caucasian populations. From these individuals DNA material was extracted using a “salting out” method before SSP-PCR genotyping. Seventeen primer pairs were used in the identification of individual KIR genes. PCR products were electrophoresed against a molecular weight marker in order to verify the correct fragment size. Products were viewed on a UV light where observations were noted, and indicated as present or absent. Data was recorded onto a spreadsheet indicating the absence or presence of each particular gene. Tabulated results were used in the construction of graphs as well as χ2 calculations. These graphs were used in the critical analysis of linkage disequilibrium as well as comparative analysis between the ethnic cohorts. Findings indicate that all framework genes are present in all cohorts. The Black African and mixed ancestry cohorts have not been genotyped for the KIR genes before. Investigation within non-framework genes revealed the identification of several new haplotypes, with the majority observed within the mixed ancestry cohort. Positive linkage disequilibrium was detected between 2DL2-2DS2 and 2DL5B-2DS5 for both the black African and Caucasian cohorts while 2DL1-2DL2 and 2DL5B- 2DS5 linkages were found in the mixed ancestry population. No negative linkages were observed for any of the three cohorts.Item Open Access Prevalence of helicobacter pylori and its relation to cytotoxin-associated gene a status in HIV positive and negative haematology patients(University of the Free State, 2007-10) Abbott, Tanya Claire; Louw, V. J.; Badenhorst, P. N.; Meiring, S. M.Abstract not availableItem Open Access Sequensing of exon 28 of Von Willebrand factor in five patients with type 2 Von Willebrand disease(University of the Free State, 2009-05) Mothabeng, Maliengoane Sylvia; Meiring, S. M.; De Kock, A.English: Von Willebrand disease (VWD) is a common bleeding disorder caused by either quantitative (type1 and 3) or qualitative (type 2) defects of von Willebrand factor (VWF). The diagnosis of VWD usually requires a panel of tests. Several analyses therefore are required to diagnose VWD. These tests are also subjected to pitfalls and it is important to take the pitfalls in to consideration when diagnosing VWD. Despite all these tests, the diagnosis and classification of VWD often remains a challenge. Identification of mutations that cause functional defects of VWF (type 2 VWD) is needed to improve the diagnosis of the disease. Mutations that cause functional abnormalities of VWF occur mostly in exon 28 of the VWF gene. Exon 28 primarily encodes the platelet GPIb and collagen binding domains of VWF (A1 domains) and the ADAMTS13 cleavage domain (A2 domains). Recently, studies in industrialised countries have been conducted on finding mutations on exon 28 but none have been done on South African populations. In this study we searched for mutations in exon 28 of the VWF gene in 5 patients with functional defects of VWF in order to set up the method for genetic analysis of VWD. We used two patients with type 2M, two with type 2B and one with type 2A VWD in this study. The whole exon 28 was analysed in four specific fragments, using PCR with primers that mismatch the pseudogene. The mutations were identified by automatic sequencing of the different fragments. The following polymorphisms were detected. A silent SNP 4641T/C in all five patients, the SNP 4141A/G in three patients, a silent SNP 3795G/A in one patient and a novel silent SNP 4923G/A in another patient. It is important to note that we found a novel SNP in an African patient with type 2B VWD, since no polymorphisms reported in exon 28 were from African populations. Several studies have proven the importance of mutational analysis is solving laboratory diagnosis paradox. The mutations found in the patients with type 2 VWD confirm the diagnosis and validates the importance of molecular diagnosis in VWD. With this study, we have successfully implemented a method to detect mutations in exon 28 of the VWF gene.Item Open Access The screening for single nucleotide polymorphisms of CYP3A4 in chronic myelogenous leukemia patients receiving imatinib(University of the Free State, 2009-05) Lamprecht, G. A.; Viljoen, C. D.English: Chronic myelogenous leukaemia (CML) is a malignant clonal disorder that results in the uncontrolled production of white blood cells. This disease is a result of a reciprocal translocation of the long arms of chromosome 9 and 22 resulting in a shortened chromosome 22, harbouring the BCR-ABL fusion gene, known as the Philadelphia chromosome. The BCR-ABL oncogene encodes for a constitutively activated tyrosine kinase that interferes with normal cell differentiation and apoptosis. CML can be effectively treated with tyrosine kinase inhibitors, such as imatinib mesylate (GleevecÒ). However, some CML patients experience adverse drugs reactions (ADRs) to imatinib and cannot be treated at the recommended dose. There is a concern that lowering the dose of imatinib to reduce the side effects can result in the development of resistant cancer cells, and thus a cessation in treatment is rather recommended. Imatinib is metabolized by the cytochrome P450 enzyme, CYP3A4. However, if the ADRs were a result of decreased metabolic effect of CYP3A4, it would be possible to reduce the dose of imatinib without effecting efficacy. It is hypothesised that single nucleotide polymorphisms (SNPs) can alter the catalytic activity of the CYP3A4 enzyme. Thus a decrease in metabolic rate can result in ADRs due an increased exposure to the drug. Therefore, the aim of this study was to determine whether SNPs in the CYP3A4 gene are associated with ADRs from imatinib treatment. In this study, the DNA sequence of the CYP3A4 gene from 25 CML patients treated with imatinib were compared to a reference DNA sequence obtained from Genbank. The SNPs identified during this study was statistically analysed, and their association with the presence of ADRs was determined using the online statistics package, SNPator. A total of six SNPs were detected, I193I, T15871G, CYP3A4*1G, C23187T, I369V and G73239A. Of these, I369V and G73239A are novel and not described previously in literature. It was found that I369V resulted in an amino acid change, involving a substitution of isoleucine with valine. The remaining SNPs identified in this study were located in intron regions, with the exception of I193I which is a synonymous SNP. There is little information available on the frequency of SNPs located in introns, since these SNPs are generally regarded to have no impact on the expression or activity of a protein. However, in this study an SNP located in intron 10 was significantly associated with the presence ADRs. Current hypothesises suggest that intron SNPs could affect the expression levels of a protein by influencing the splicing efficiency of mRNA and subsequently translation efficacy. Future research needs to elaborate on the role of CYP3A4*1G on CYP3A4 expression as well as on the prevalence of other alleles identified in this study in South African populations.Item Open Access The application of real-time quantitative PCR in the diagnostics of chronic myeloid leukaemia(University of the Free State, 2009-05) Van Deventer, Jacob Jacobus; Viljoen, C. D.; Louw, V. J.English: CML is a cancer of the white blood cells and it effects on average one individual in every 100,000. Since it was first described in 1845 by John Hughes Bennett and the subsequent discovery of the Philadelphia chromosome by Nowell and Hungerford in 1960, this hematopoietic malignancy has received much attention in terms of scientific study. Elucidating the pathogenic pathway has lead to the development of targeted therapy. In 2001 imatinib mesylate was introduced as first line therapy for CML. The success of imatinb was illustrated during the IRIS trial by Real-time quantification of BCR-ABL mRNA. BCR-ABL expression levels are correlated to disease stage and progression. BCR-ABL mRNA quantification is therefore the most accurate and sensitive prognostic marker to monitor CML patients. Hence, Real-time PCR for BCRABL has been introduced in many international laboratories to allow for accurate and reliable monitoring to improve and manage patient treatment. Standardization became problematic due to the ease of method development and robustness for Real-time quantification of BCR-ABL mRNA by different laboratories. As a result a plethora of methods for Real-time quantification of BCR-ABL mRNA have been published. This is especially problematic for laboratories with limited means undertaking to develop and implement such a method. Since there are no standardized guidelines, in-house development is required. Furthermore, availability of commercial copy number standards for control and target genes makes it difficult to implement any one method from the literature especially since there is criticism for the genes where standards are commercially available. From a thorough analysis of the literature, problem areas considering RNA extraction, the choice of priming for cDNA synthesis, primers and probes for Real-time PCR as well as a specific control gene together with copy number standards and reference material were clearly defined. Based on this information, best laboratory practice regarding common methodology from literature was established. Only recently through an initiative known as Europe Against Cancer (EAC) has there been a concerted effort to facilitate regional standardization of Real-time quantification of BCR-ABL mRNA. During this study a modified EAC method for Real-time quantification of BCRABL mRNA was developed and validated with the emphasis to improve reproducibility. Instead of ABL or BCR, GUS was used as control gene based on recommendations from literature. Based on statistical analysis it was concluded that the modifications did not bias the percentage BCR-ABL result. It cannot be emphasised enough that standardization for Real-time monitoring of BCR-ABL is most crucial as it will ultimately facilitate molecular laboratories to develop this diagnostic with much greater ease. In order for standardization to be realized, copy number standards as well as reference material for quality control purposes needs to become more readily available. In addition to that, specific guidelines for assay criteria such as appropriate Ct values and analysis of data must also be developed. By streamlining Real-time quantification of BCR-ABL the treatment and monitoring of CML patients can be improved on a global scale.Item Open Access The evaluation of tirofiban hydrochloride in a high shear rate arterial thrombosis model in baboons(University of the Free State, 2009-11) Janse van Rensburg, Walter James; Meiring, S. M.; Roodt, J. P.English: Background: Acute coronary syndrome (ACS) is a major cause of mortality and morbidity world-wide, and is responsible for roughly 2.5 million hospital admissions world-wide annually. ACS is commonly associated with platelet thrombus formation on disrupted atherosclerotic plaques, therefore effective and safe anti-platelet drugs are needed to help treat and prevent ACS. The current most popular anti-platelet drugs are associated with increased bleeding risk and reduced efficacy, thus drugs with a wider therapeutic window (more efficacy with less bleeding) need to be developed. Tirofiban hydrochloride is a small, short half-life molecule that inhibits platelet aggregation by antagonising the glycoprotein IIb/IIIa receptor on platelets preventing fibrinogen and von Willebrand factor to cross-link platelets, thereby inhibiting the final pathway of platelet aggregation. Tirofiban hydrochloride was believed to be a very promising drug due to its short half-life, as an antidote strategy is not needed to reverse adverse bleeding events, but it soon fell out of favour when it was found not to be as effective as for example abciximab in preventing ischaemic events. This was possibly due to the recommended dose being suboptimal. Methods and Results: We studied the efficacy of tirofiban hydrochloride to inhibit platelet thrombus formation on an injured and partially occluded artery by evaluating the effect of escalating doses on cyclic flow reduction (CFR) formation in a high shear arterial thrombosis model in baboons, and also evaluated its safety in two different bleeding models. We then compared our results to results found in the same model using clopidogrel. A significant effect on the number of CFRs was only observed after injection of three times (30 μg/kg bolus plus 0.45 μg/kg/min infusion) the therapeutic dose tirofiban, but it was a weak inhibitor at this dose. Only after injection of nine times (90 μg/kg bolus plus 1.35 μg/kg/min infusion) the recommended therapeutic dose, a strong complete inhibition was observed. A further dose of 27 times (270 μg/kg bolus plus 4.05 μg/kg/min infusion) the recommended therapeutic dose was given to evaluate the effect of an overdose on the bleeding tendency. A significant prolongation in bleeding time (3.05 minutes to 11.90 minutes) was observed after injection of nine times the therapeutic dose, an average 2.7 ± 2.44 fold increase in blood loss was also observed at this dose. A maximum increase in blood loss of an average of 3.4 ± 1.77 fold was seen after injection of 27 times the therapeutic dose. The efficacy of tirofiban hydrochloride was comparable to that of clopidogrel found in earlier studies, but the blood loss was much less when compared to the average 4.3 ± 2.6 fold increase with clopidogrel at 2.5 mg/kg and 8.0 ± 5.0 fold increase at 5 mg/kg. Conclusion: Tirofiban hydrochloride is an effective anti-platelet drug, but only offers adequate protection against arterial thrombosis at a dose between three and nine times the recommended therapeutic dose. However, it still remains safer in terms of bleeding than the most common anti-platelet drugs used today. We recommend that further in vivo studies be done to determine the optimal dose for tirofiban hydrochloride treatment, and that new clinical trials be done with higher dose tirofiban hydrochloride.Item Open Access Characterization of a human inhibitory antibody fragment against tissue factor(University of the Free State, 2011-11) Vermeulen, Jan-G.; Meiring, S. M.; Van Heerden, E.English: Tissue factor is a transmembrane glycoprotein that functions as the primary initiator of coagulation in response to mechanical or chemical damage. Due to its key position within the coagulation cascade it also plays an important role in the pathology of thrombosis and thrombotic complications associated with cardiovascular disease as well as in non-thrombotic disorders and diseases such as obesity, diabetes, cancer and HIV-AIDS. Recognising the potential in tissue factor inhibition as a novel approach to antithrombotic therapy, our laboratory utilized phage display technology in a previous study, in order to identify a 26 kDa single chain antibody fragment which functionally inhibits human tissue factor. In the current study, the tissue factor inhibitory single chain variable fragment (TFIscFv) was expressed by means of the pIT2 plasmid vector by Escherichia coli HB2151. This expression system was utilised in an up-scale setting in an attempt to improve the TFI-scFv yield. Although functional TFI-scFv was successfully purified from the culture media by means of Protein A affinity chromatography, the process was hampered by large sample volumes, low levels of expression as well as the high cost involved in Protein A purification. Due to an initial focus on improving TFI-scFv yield through the processing of larger sample volumes rather than the improvement of the expression system, immobilised nickel affinity chromatography was investigated as a more cost effective alternative to Protein A affinity chromatography. It was found that the original expression system was incompatible with immobilised nickel affinity chromatography as the protein was expressed into the culture media. The culture media contained nickel chelating elements that stripped the nickel from the column and consequently prevented TFIscFv purification. Subsequently the TFI-scFv gene was isolated, cloned into an over-expression system and modified to redirect the expression to the bacterial cytoplasm. Although TFI-scFv was successfully redirected to the bacterial cytoplasm and purified by means of nickel affinity chromatography, it was found that expression was hampered due to the presence of rare codons. The detrimental effect of rare codons on TFI scFv yield was addressed through the modification of the expression host by the coexpression of the pRARE plasmid as well as by the rare codon optimization of the TFI-scFv gene sequence for expression in E. coli. Although the co-expression of the pRARE plasmid only slightly improved TFI-scFv yield, a sufficient amount of TFIscFv was generated for functional testing. The modified TFI-scFv displayed a similar inhibition effect with reference to the original construct. The rare codon optimisation resulted in a substantial increase in TFI-scFv yield but consequently resulted in the loss of solubility and the production of inclusion bodies. Although the loss of TFIscFv solubility is unwanted, the high level of expression achieved provides an ideal platform for the further development and characterization TFI-scFv in animal thrombosis models.Item Open Access Mutational analysis of the Janus Kinase 2 gene in patients with polycythaemia vera, essential thrombocythaemia and primary myelofibrosis(University of the Free State, 2011-11) Goodyear, Quintin Clive; De Kock, A.; Viljoen, C. D.All the cells of blood arise from two lineages, the myeloid and the lymphoid lineage. The various cells of blood perform vital functions in the body. These cell counts are closely regulated by regulatory pathways. Mutations within genes that encode for the proteins involved in these pathways can occur. These mutations can cause uncontrolled proliferation of the cells. Myeloproliferative neoplasms are malignancies where there is an uncontrolled increase in the formation of the myeloid cells. The four classical neoplasms are polycythaemia vera, essential thrombocythaemia, primary myelofibrosis and chronic myeloid leukaemia. A mutation (V617F) in the tyrosine kinase, Janus kinase 2, has been found to be the cause of at least three of the classical MPNs. The mutation lies in the domain of the protein that controls its tyrosine kinase activity. The tyrosine kinase thus is constitutively active and causes proliferation of the myeloid cells. The V617F mutation lies in exon 14 and more recently several mutations have been described in the neighbouring exons encoding for the regulatory domain of the gene. Very few studies have been done on the other exons of the JAK 2 gene. In the study it was attempted to screen 15 MPN patients for mutations in the JAK 2 gene. Two different cell populations (lymphocytes and granulocytes) of each patient were screened. It was found that the cell purity was not sufficient in the study and better separation techniques are required for future studies. Only the granulocytes were used for the remainder of the study. High resolution melting curve analysis was used to screen the patients for mutations, however the data did not correlate with the sequencing results and it was decided to proceed with sequencing of all the samples. Seven of the 25 exons of the JAK 2 gene were successfully sequenced. The remaining exons could not be screened due to time constraints and complications such as multiple amplicon formation. Two previously reported single nucleotide polymorphisms were found in exons 11 and 15 in two patients. The clinical significance thereof is uncertain however, the patient whom had the SNP in exon 15 was negative for the V617F mutation and had a MPN. In exon 14 the V617F mutation was identified and the prevalence thereof correlates to that reported in literature. A novel SNP was found in exon 13 of a PV patient negative for the V617F mutation and the significance thereof is also uncertain. Additionally a novel inverse duplication consisting of at least of exon 13 was also identified. No mutations were identified in exons 10, 12, 16 and 17 of the JAK 2 gene. This was, to our knowledge, the first report in South Africa that found the prevalence of the V617F mutation in MPN patients correlating to the prevalence reported in literature. A novel SNP was identified in exon 13 and further studies are needed on the possible effect thereof. The previously reported SNPs found in exons 11 and 15 might be the cause of the formation of a MPN, however further research is needed. A novel duplication variant was also identified and this might be a possible splice variant. The study showed that the region between exons 10 and 15 in the JAK 2 gene is a mutational hotspot and further studies are needed to elucidate the effect thereof.Item Unknown The application of high-resolution melting curve analysis for the detection of mutations in the MCR-ABL kinase domain of patients with chronic myeloid leukaemia(University of the Free State, 2011-12) Wienand, Kirsty; Viljoen, C. D.; Louw, V. J.CML is a haematological malignancy that is characterized by the BCR-ABL fusion oncogene that encodes a constitutively active tyrosine kinase. The treatment of choice for CML is a tyrosine kinase inhibitor and molecular monitoring of patients forms an integral part of disease management. When the expected response to tyrosine kinase inhibitor is not achieved within internationally accepted time frames, acquired resistance to tyrosine kinase inhibitors is suspected. Acquired resistance to tyrosine kinase inhibitors is primarily due to mutations in the BCR-ABL kinase domain. Types of mutations include single base mutations, insertions, deletions as well as duplications. Characterization of these mutations is important for treatment, since the type and position of the mutation may have an effect on how the patient responds to treatment. Although several methods have been described for detecting mutations, DNA sequencing is mostly used. Sequencing is currently the only technique that can simultaneously detect single base mutations, insertions and deletions in the BCR-ABL kinase domain. However, sequencing is costly as some patient samples do not have mutations and the lack of response to treatment is due to non-compliance. Thus, a screening method to exclude samples without mutations would make mutational analysis more cost-effective. High resolution melting (HRM) is a relatively new technique that is being used to screen for mutations, prior to sequencing. HRM has recently been used to screen the region of BCR-ABL encoding for the kinase domain for single base mutations. However, it was unknown whether HRM could be used to identify insertions, deletions or duplications in the kinase domain. This study has shown that HRM can be used to screen for mutations including insertions, deletions and duplications the region of BCR-ABL encoding for the kinase domain, prior to sequencing. HRM was performed on 40 patient samples, 10 of which had confirmed mutations in BCR-ABL in the region of the kinase domain. Of the 10 samples with mutations, three had single base mutations, one with a previously described insertion, seven had novel deletion variants. Furthermore, HRM detected a tandem duplication of the kinase domain in two patient samples that was not previously been possible with sequencing. There was 100% congruency between the detection of mutations using HRM and sequencing results, indicating similar sensitivity. HRM proved successful to indicate the presence of deletion variants. However, the deletion variants were detected in the HRM region preceding the area affected by the deletion. It was confirmed that the detection of the deletion variants was due to the PCR extension of HRM 1 amplicon into the HRM area of the deletion. It has been suggested that the insertion, deletions and duplications detected in this study may result in acquired resistance to tyrosine kinase inhibitor. In conclusion, this was the first study to use high-resolution melting to detect insertions, deletions and duplications in the region of BCR-ABL encoding for the kinase domain, indicating the suitability of the assay for screening for mutations prior to sequencing.Item Unknown The effect of inflammatory cytokines and coagulation factors on Von Willebrand factor synthesis and cleavage(University of the Free State, 2012-02) Allers, Werner Ernst; Meiring, S. M.When injured, endothelial cells secrete inflammatory cytokines such as interleukin-6 (IL-6), interleukin-8 (IL-8), and tumour necrosis factor-α (TNF-α). These inflammatory cytokines stimulate the endothelial release of ultra large Von Willebrand factor (ULVWF) multimers that bind platelets to form thrombi in small vessels. The interaction between thrombosis and inflammation is not fully elucidated. A disintegrin-like metalloprotease with thrombospondin type I repeats-13 (ADAMTS-13) is freshly released from Weibel-Palade bodies of endothelial cells into the plasma and it cleaves the ultra large and hyperactive VWF multimers into smaller and less active forms. These VWF multimers mediate the initial adhesion of activated platelets, the first step in both inflammation and thrombosis. This process may be affected by the amount of ULVWF released and the processing capacity of ADAMTS-13. Little is known about the initial onset of HIV-associated TTP, a fatal thrombotic disease that is characterised by the absence of ADAMTS-13. The mechanisms underlying the initial onset and/or burst of TTP episodes still remain poorly understood. Interrelated components, such as coagulation factors and inflammatory cytokines, all contribute to the development of TTP, since increased levels of cytokines interleukin-6 and tumour necrosis factor and the coagulation factor, tissue factor is measured in these patients. Therefore, we hypothesised that certain inflammatory cytokines and coagulation factors released during inflammation may stimulate the release of VWF simultaneously while inhibiting the synthesis of ADAMTS-13, which results in an acquired deficiency of plasma ADAMTS-13 and ultimately in a TTP episode. Our aim was to examine the effects of inflammatory cytokines and coagulation factors such as tissue factor and thrombin as well as combinations thereof on the release and cleavage of ULVWF by cultured human umbilical vein endothelial cells (HUVECs). HUVECs were treated with cytokines, IL-6, IL-8, and TNF-α and coagulation factors, tissue factor and thrombin, and their combinations, for 24 hours under static conditions. The cells were then exposed to a shear stress of 2.5 dyne/cm2 to expose the VWF cleaving sites. The VWF, VWF propeptide and ADAMTS-13 secretion were measured by an ELISA technique. ADAMTS-13 content was measured using Western blot technology with densitometry. All treatments and their combinations, excluding IL-6, significantly stimulated the release of VWF and VWF propeptide from HUVECs. The VWF propeptide levels were constantly higher than the major VWF protein levels suggesting that the measurement of the VWF propeptide levels may be a better representation of the amount of VWF secreted from endothelial cells. Tissue factor alone and in combination with inflammatory cytokines increase the amount of VWF release from endothelial cells substantially. This correlates with the situation in thrombotic patients with inflammation where extremely high VWF levels are measured. Densitometric analysis of the Western blots indicated that lower levels of ADAMTS-13 secretion were found with all treatments. These results suggest that inflammatory cytokines such as IL-8 and TNF-α, coagulation factors such as thrombin and tissue factor, as well as combinations thereof, stimulate the release of ULVWF and inhibit the release of ADAMTS-13 in HUVECs, resulting in the accumulation of hyperreactive ULVWF in plasma and on the surface of endothelial cells to induce platelet aggregation and adhesion on the vascular endothelium. Our study may offer a logical explanation of how systemic inflammation and thrombosis might trigger the onset and/or burst of TTP in patients with HIV-associated TTP.Item Unknown Microparticles derived from stimulation of human umbilical endothelium(University of the Free State, 2013) Le Roux, Elzette; Meiring, S. M.; Coetzee, M. J.English: Endothelial microparticle reseach is currently a very novel and exciting topic in the field of haemosistasis and thrombosis. The role of microparticles in inflammatory and thrombotic disorders is however not fully understood. Dysfunction of endothelial cells is hypothesized to be a trigger of microparticle formation. In inflammatory disorders like sepsis and thrombotic disorders like atherosclerosis and thrombotic thrombocytopenic purpura, endothelial microparticle formation is altered and the numbers thereof may increase or decrease. It is not known if microparticles are the cause or the consequence of these disorders. To understand the role of endothelial microparticles in inflammation and thrombosis, the effect of inflammatory cytokines and coagulation stimuli was studied as well as combinations thereof on endothelial microparticle formation and on microparticle VWF and its regulating protease, ADAMTS-13 in HUVEC. In this study, the formation of microparticles in cultured human umbilical vein endothelial cells (HUVEC) was stimulated by different inflammatory agents: IL-6 (100 ng/ml), IL-8 (100 ng/ml) and TNF-α (100 ng/ml), coagulation stimuli: TF (2 U/ml) and thrombin (2 U/ml) and combinations thereof. The number of endothelial microparticles that formed was determined using flow cytometry. VWF and ADAMTS-13 levels of the microparticles were assessed by ELISAs and microparticle thrombin generation was measured by thrombin generation assays. VWF multimers were visualized by a Western Blot technique. IL-6 did not have any effect on HUVEC-derived microparticles due to the lack of the receptor for IL-6 on these cells. IL-8 only slightly increased effect on microparticle VWF and ADAMTS-13 levels. TNF-α had a significant effect on microparticle numbers and contributed to almost 80% of thrombin generated by the microparticles. It has however almost no effect on VWF levels. The coagulation stimulus TF, on the other hand, induced the highest increase in microparticle VWF levels and increased microparticle numbers impressively. Yet, it had no effect on the thrombin generation by the microparticles. TF in combination with TNF-α also induced an increase in microparticle VWF and a small decrease in ADAMTS-13 levels. So, TF may contribute to the increased VWF levels that are commonly found in TTP patients where inflammation and thrombosis occur. Interestingly, thrombin had a protective effect on the intact HUVEC by preventing microparticle formation. The combination stimuli of thrombin and inflammatory agents also had a protective effect on HUVEC. This highlighted the regulatory role of thrombin in intact endothelial cells and also the protection that it provides against thrombosis in extremely inflammatory environments. Endothelial microparticles can therefore be detrimental or beneficial, depending on the different stimuli and different environments. Inflammatory and coagulation stimuli may still pose a significant risk of clotting by altering microparticle quantity and content. This study contributes to understand the role that endothelial microparticles play in inflammation and thrombosis.Item Open Access Genotypic and expression analysis of CYP3A4 and CYP3A5 in patients with chronic myeloid leukaemia(University of the Free State, 2013-01) Thompson, Gaynor Gillian; Viljoen, C. D.Chronic myeloid leukaemia (CML) is a haematological malignancy characterised by the BCR-ABL fusion oncogene which encodes for a constitutively active tyrosine kinase. Imatinib mesylate is a tyrosine kinase inhibitor that has effectively been used in the treatment of CML. However, some individuals experience adverse drugs reactions (ADRs) to imatinib. One of the reasons for varied treatment response among individuals may be as a result of inter-individual differences in the metabolism of imatinib. Imatinib is metabolised by the drug metabolizing enzymes CYP3A4 and CYP3A5. Single nucleotide polymorphisms (SNPs) in CYP3A4 and CYP3A5 have been described, some of which have been associated with altered catalytic activity of these enzymes. SNPs in CYP3A4 and CYP3A5 may also impact the expression of these genes and result in a less favourable response to imatinib treatment. Patients with a decrease in catalytic activity of CYP3A4 and CYP3A5 may experience ADRs due to prolonged exposure to imatinib. On the other hand an increase in activity may lead to ineffective treatment as a result of increased clearance of the drug. Thus the aim of this study was to screen CYP3A4 and CYP3A5 for SNPs using high resolution melting curve analysis (HRM) and determine the impact of these SNPs on gene expression in CML patients treated with imatinib. A total of ten SNPs were detected in CYP3A4, of which two SNPs, namely A15619G and A15649T have not been previously described in literature. A15619G was a synonymous SNP while A15649T resulted in a change in amino acid from glutamine to a histidine. A total of four SNPs were detected in CYP3A5, of which one SNP namely, G7226A, had not previously been reported in literature and did not result in an amino acid change. Out of all the detected SNPs in CYP3A4, the G20338A SNP was statistically associated with the occurrence of ADRs but not with mRNA expression. The I369V SNP was statistically associated with increased CYP3A4 mRNA expression. None of the SNPs detected in CYP3A5 significantly affected mRNA expression. Expression of CYP3A4 and CYP3A5 was not dependent on ethnicity or gender, with the exception of CYP3A5 which showed a statistically significant difference between males and females. Currently a limited amount of literature exists regarding SNPs in CYP3A4 and CYP3A5 and CML treatment. Given the potential impact that SNPs can have on the CYP3A4 and CYP3A5 enzymes and therefore imatinib treatment, it is an important issue that needs to be investigated. Determining the potential impact of SNPs and differential gene expression of CYP3A4 and CYP3A5 is important as it may allow for more effective imatinib treatment.Item Open Access Development and application of a real-time PCR method to detect selected single nucleotide polymorphisms associated with hypertension in a black South African population(University of the Free State, 2014-08) Du Toit, Egardt; Viljoen, C. D.English: Hypertension is one of the leading causes of death and disability in the world. In 95% of individuals with hypertension, the condition arises from the interaction of multiple environmental factors with physiological systems. Environmental factors that have been found to increase blood pressure include obesity, aging, high salt and alcohol consumption, low potassium and calcium intake, stress and insulin resistance. Physiological systems that regulate blood pressure include the autonomic nervous system, the renal system, hormonal system and the cardiovascular system. Various genes in these systems including the β1-adrenergic receptor (ADRB1), α-adducin (ADD1), angiotensinogen (AGT), aldosterone synthase (CYP11B2), CYP3A5 and G protein-coupled receptor kinase 4 (GRK4) have been implicated in hypertensive blood pressure due to the presence of single nucleotide polymorphisms (SNPs). The occurrence of such SNPs in blood pressure regulatory systems is thought to result in altered gene expression or protein function. In South Africa, the prevalence of hypertension has been determined to be approximately 39.9% in males and 34.9% in females. The Assuring Health for all in the Free State (AHA-FS) study determined that the prevalence of hypertension was approximately 48.3% in the Mangaung population. The AHA-FS study also found that 37.6% and 51.2% of individuals in the study cohort were overweight or obese, respectively, and that high body mass could be an important risk factor for hypertension. The aim of this study was to determine whether genes in the sympathetic nervous system, renal system and hormonal systems could contribute to the high prevalence of hypertension in the Mangaung population. Previously identified SNPs associated with hypertension in ADRB1 (A145G and G1165C), ADD1 (G217T), AGT (G-217A, C521T and T704C), CYP11B2 (C-344T), CYP3A5 (A6986G) and GRK4 (G448T, C679T and C1711T) were genotyped in a cohort of the AHA-FS study, which comprised black individuals from Mangaung, Free State. Six of the 11 candidate SNPs did not appear to be associated with hypertension in the black population of Mangaung. These included G1165C (ADRB1), G-217A and T704C (AGT), G448T, C679T and C1711T (GRK4). None of the latter SNPs were associated with statistically significant elevations in either systolic or diastolic blood pressure. The association remained negative even after the cohort was stratified into underweight to normal weight and overweight to obese groups. The lack of association between these SNPs and hypertensive blood pressure in the black population group in Mangaung compared to other population groups could be attributed to population differences in environmental factors, ethnicity, cohort size and/or epistasis. Five SNPs were associated with hypertension in black individuals from Mangaung. The latter included SNPs in CYP3A5 (A6986G), ADRB1 (A145G), AGT (C521T), CYP11B2 (C-344T) and ADD1 (G217T). The hypertensive A allele of the A6986G SNP of CYP3A5 has been associated with systolic hypertension in homozygous individuals of the study. Similarly, an association between the A allele and hypertension in African-Americans and Swedish Caucasians has also been found. Thus, the A allele of the A6986G SNP seems to cause hypertension susceptibility in different populations, including the Mangaung population group. As for the A145G SNP of ADRB1, hypertensive systolic blood pressure was associated with individuals that were homozygous for the hypertensive A allele of the A145G SNP. A meta-analysis determined that individuals expressing the A allele of A145G had a 24% higher risk for developing hypertension. In the Mangaung population, hypertension risk associated with the A allele of A145G was especially increased in overweight to obese individuals. The presence of the hypertensive T allele of the C521T SNP of AGT was associated with hypertensive diastolic blood pressure in the Mangaung population. Similar results were found in Hutterite, Russian and Tartar population groups. Furthermore, in the Mangaung population the hypertension risk conferred by the T allele was significantly increased in overweight and obese individuals. This suggests that the T allele of C521T may be involved in particularly overweight and obesity related hypertension. The hypertensive T allele of the C-344T SNP of CYP11B2 was only associated with hypertensive systolic and diastolic blood pressure in the overweight to obese individuals of the Mangaung population. In another study conducted on black South African individuals the T allele was also associated with hypertension. The cohort of the latter study furthermore had an overweight to obese body mass index average. It therefore appears that the T allele of C-344T could primarily be a risk factor for overweight and obesity related hypertension. Interestingly the normotensive G allele of ADD1 was implicated in obesity related hypertension, instead of the hypertensive T allele. In contrast, another study on black South Africans found that the T allele was associated with hypertension. However, results from a previous study on African-Americans suggested that the T allele may be protective against hypertension. It has been proposed that other unidentified polymorphisms, which also could affect hypertension susceptibility, could be in linkage disequilibrium with the G217T SNP and that allelic variation of the other polymorphic loci could contribute to the inconsistent findings of association studies. Several individuals in the cohort from the AHA-FS study could not be genotyped for the candidate SNPs. Attempts to obtain conventional PCR product for individuals where genotyping failed did not prove entirely successful. In the cases where no PCR amplicon could be amplified even after attempts at assay optimization, it was concluded that primer mismatch, especially at the 3‟ end of the primer may be the likely cause. Where PCR amplicon was successfully amplified, sequencing proved difficult due to short amplicon size. However, partial sequence data revealed additional SNPs in some individuals in the probe binding region that would account for failed genotyping. A limitation of this study was that only selective SNPs in genes associated with hypertension were genotyped in the cohort. Furthermore, since the study did not investigate the role of potentially novel SNPs in candidate genes, it is possible that additional unidentified SNPs in these genes may also contribute to hypertension. Despite these limitations, this study is currently the most comprehensive of its kind on the Mangaung population. Future research could focus on additional genes as well as screening these genes for novel SNPs, especially in the genes where the SNPs investigated were not associated with hypertension in the Mangaung cohort. In conclusion, the A6986G SNP in CYP3A5 appears to be an independent risk factor for hypertension, whereas A145G in ADRB1, C521T in AGT, C-344T in CYP11B2 and G217T in ADD1 may be associated with hypertension related to overweight and obese body weights. To our knowledge, this is the most comprehensive study investigating a combination of several gene SNPs associated with hypertension in a black Mangaung population.Item Open Access Thrombogenecity of decellularized and re-endothelialized baboon arteries(University of the Free State, 2015-02) Khemisi, Mmakgabu Martha; Meiring, S. M.; Smit, F.English: The use of decellularized biological scaffolds for the reconstruction of small-diameter (< 6 mm) vascular grafts has been the focus of tissue engineering studies. These biological materials constructed through decellularization processes are thought to be ideal graft materials for replacement of diseased vessels. However thrombogenecity is a major cause of obstruction in these vascular constructs and result in early graft occlusion. Seeding of the decellularized vascular constructs with endothelial cells (ECs) is an attractive proposition as the endothelial layer incorporates many of the anti-thrombogenic properties of blood vessels. The aim of this study was to determine if we could successfully re-endothelialize decellularized baboon arteries, thereafter perfuse the newly engineered arteries with whole blood to investigate the possibility of thrombus formation. First, the histology of the decellularized baboon arteries were compared to normal arteries in order to assess the efficiency of the decellularization process. Collagen and elastin fibres were also stained to determine whether the remaining extracellular matrix scaffold was preserved after decellularization. Human umbilical vein endothelial cells (HUVECs) were then cultured under optimal conditions. The viability and proliferation rate of the cultured ECs were assessed using the MTT cell viability and proliferation assay. The cultured endothelial cells were then used to seed the luminal surfaces of decellularized baboon arteries. The confluent endothelial monolayer of the seeded decellularized arteries were then assessed using scanning electron microscopy (SEM) after 7 days. The seeded cells were then detached from the graft surfaces of small sections and tested for viability (metabolic activity and proliferation) using the MTT assay again. Afterwards, normal, decellularized and seeded decellularized arteries were perfused for 2 hours with baboon whole blood collected in 3.2% sodium citrate tubes. Thereafter, small artery sections were examined with SEM to determine whether thrombi was formed on the luminal surfaces of all arteries. Histology examinations showed that the decellularized arteries were not completely cell free. Nuclear and cellular remnants were still retained within the scaffold materials. Histology also revealed that the femoral arteries had retained more nuclear and cellular materials than the carotid and radial arteries which showed much less nuclear material remaining. However, the ECM of the decellularized arteries was preserved after the decllularization processes. Enough viable ECs were obtained in culture to seed three decellularized baboon arteries. After 7 days post seeding, a confluent endothelial monolayer was observed on the luminal surfaces of the decellularized scaffolds using SEM. The perfused normal artery and the seeded decellularized arteries showed no possible thrombus formation on their luminal surfaces. The decellularized arteries however showed wide-spread platelet adhesion and activation on the surface of the ECM. In conclusion, decellularization of arteries was not successfully and proved to be dependent on the thickness of the tissues used. However, the decellularization process produced morphologically preserved extracellular matrix. The endothelialization process was successful since the endothelialization of decellularized vascular grafts does prevent thrombus formation on artery surfaces after perfusion with whole blood, while a decellularized scaffold does promote thrombus formation.