Doctoral Degrees (Haematology and Cell Biology)
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Item Open Access Characterisation of the fibrinolytic system and the Von Willebrand factor-Adamts13 axis in the Chacma baboon(University of the Free State, 2021-11) Joubert, Jaco; Janse van Rensburg, W. J.; Meiring, S. M.Background: The Chacma baboon (Papio ursinus) model of acquired thrombotic thrombocytopenic purpura (aTTP) is ideally suited to investigate novel treatments with potential application in this lethal thrombotic disorder, which hinges on the dysfunction of the VWF–ADAMTS13 axis. One such modality is the thrombolytic drugs, which activate the fibrinolytic system. Our recently published pilot study demonstrated the thrombolytic drug streptokinase as ineffective in resolving aTTP in this model and highlighted the deficits in our knowledge of the Chacma baboon’s fibrinolytic system and VWFADAMTS13 axis. The present study aimed to characterise these components of the Chacma baboon’s haemostatic system to better understand the aTTP model, the effects of thrombolytics in this model, and the implications for other haemostatic disease models in this species. Materials and Methods: Forty baboons were tested using observational and experimental assays. The VWF–ADAMTS13 axis was investigated by determining ADAMTS13 antigen and activity levels, VWF:Ag, VWF:RCo, and VWF:CB levels and VWF multimer patterns. The fibrinolytic system was explored by measuring the concentrations of fibrinogen, plasminogen, tPA, PAI-1, PAP complexes, TAFI, and α2- antiplasmin, and by assessing its in vitro clot lysis ability in a modified clot lysis time assay.The plasminogen activation potentials of streptokinase and tPA were determined using concentration escalation experiments. The effect of tPA-induced plasmin activity on VWF multimer patterns when in the non-globular state was assessed in the presence of the anti-ADAMTS13 mAb 3H9. Thrombin generation, which can influence the aTTP model and its response to thrombolytics, was also evaluated, as well as the effects of sex and ABO blood group. Reference intervals and interindividual variation were calculated and compared with human values. Results and Discussion: ADAMTS13 activities were generally below (but still comparable to) human ranges. VWF:Ag and VWF:CB values tended towards the lower limit of the human reference interval, whereas VWF:RCo activities were higher. All VWF multimer patterns were essentially equivalent to normal human patterns. Fibrinogen concentrations were similar to human values, but tPA, PAP complex, PAI-1 and α2- antiplasmin concentrations all tended toward the lower human ranges. Meaningful results could not be generated for ADAMTS13 antigen, plasminogen, or TAFI, possibly due to structural differences with the human protein. Streptokinase resulted in minimal plasmin activity, but tPA led to concentration-dependent increases. All baboon samples exceeded 100% of human plasmin activity at baseline and had clot lysis times shorter than pooled normal human plasma when activated by tPA. In the absence of ADAMTS13 activity, tPA reduced the non-globular high molecular weight VWF multimers in both human and baboon samples. Baboons had greater overall endogenous thrombin potentials than humans, which was more prominent in females (p=0.0238). Except for lower fibrinogen concentrations (p=0.0134) in male baboons, and PAP complex concentrations which were higher (p=0.0188), no other sex-related differences were apparent. No baboons were typed as ABO group B or AB, and none were Rh(D) negative. Group O baboons had higher fibrinogen concentrations (p=0.0355), but all other parameters were unaffected. Fibrinogen and especially α2-antiplasmin were subject to considerable interindividual biological variation. Conclusion: The central thesis of this research is that the components of the Chacma baboon’s haemostatic system pertinent to the pathogenesis of aTTP and its treatment with thrombolytics are similar enough to their human counterparts to enable continued use of this species as a model of human haemostasis, provided quantitative results are interpreted within the context of the novel reference intervals, and the identified limitations and interspecies differences are considered. Finally, tPA should be explored further in the,Chacma baboon aTTP model in vivo to provide proof-of-concept for the use of thrombolytic drugs in the treatment of aTTP in humans.Item Open Access Haemostatic and thrombotic disorders: a journey from bench to bedside(University of the Free State, 2019-12) Meiring, Sarah Muriel; Brown, S. C.; Kotze, H. F.This compilation focuses on thrombotic and haemostatic disorders, illustrating my journey from basic research on thrombotic and haemostatic disorders to the differential diagnoses of these disorders. For purposes of clarity I shall divide it in three parts. The first part includes the testing of antithrombotic agents. My scientific career started with the testing of antithrombotic drugs in a baboon model of arterial thrombosis. These antithrombotic drugs were mostly targeted at platelets and, to a lesser extent, coagulation. For my PhD, I clarified the catabolism, pharmacokinetics and exctretion of recombinant hirudin, an anti-thrombin drug. The second part includes the development of cost-effective diagnostic tests, mostly for von Willebrand disease (VWD), the most common congenital bleeding disorder. This comprises the largest part of the thesis. I developed four anti-thrombotic peptides by using Phage Display technology that I mastered during my post-doctoral study at the University of Leuven in Belgium. I was also part of the group of researchers that developed many new thrombosis models in baboons. For my M.Med Sc study, I developed a flow chamber model to study in vitro endothelial function, which was subsequently used to test the thrombogenicity of tissue-engineered small vessels. This study was the first where endothelial cells were successfully seeded onto decellularised baboon arteries. This study was undertaken in collaboration with the Department of Cardiothoracic Surgery at the University of the Free State, Bloemfontein. The third part is a spin-off from my research on VWD. I established the only Special Haemostasis laboratory of the NHLS, situated at the University of the Free State in Bloemfontein, South Africa, by developing, validating and implementing four diagnostic assays. As a result the laboratory now functions as a reference centre for von Willebrand disease; the most prevalent, but underdiagnosed bleeding disorder in South Africa. The developed diagnostic assays resulted in nineteen peer-reviewed publications on the diagnosis of haemostatic and thrombotic disorders. As reference centre for VWD in South Africa, we published eight articles on its diagnosis, together with international leaders in the field. Additionally, we also published the first South African recommendations regarding the differential diagnoses of VWD. We published on other bleeding disorders and on a fatal thrombotic disorder, thrombotic thrombocytopenic purpura (TTP). Lastly, is it important to note that the research on VWD, haemophilia and TTP is ongoing.Item Open Access Telomeres and telomerase in cancer of esophagus(University of the Free State, 2001-11) Van den Heever, Wilhelmina Maria Jacoba; Pretorius, G. H. J.English: Squamous cell carcinoma of the esophagus is a cancer with a high incidence in South Africa. We have investigated the prognostic value of telomerase activity in tumors as well as in nearby normal tissue. Biopsies from 98 patients were analyzed using an adaptation of the TRAP assay. We found all tumor biopsies to have moderate to high telomerase activity, while one third of biopsies from normal mucosa were negative. The telomerase activity level of the tumors had no prognostic value (P=0.95) as determined by the log rank test. A P-value of 0.02 was found when the telomerase-negative and moderately positive normal biopsies were grouped together and compared to those with high activity. Our results show that telomerase activity of normal mucosa in the vicinity of the tumor can identify a population of patients with significantly worse prognosis, even in late stage patients. Telomerase has attracted intense interest as a possible target for cancer therapeutics. Previous attempts at inhibiting telomerase activity utilized antisense oligonucleotides targeted at the template region of the RNA subunit of the enzyme. Although it worked well in cell extracts, getting the oligonucleotides into intact cells are difficult. We attempted a peptide-based approach to overcome the transport problem. A phage-display selection strategy was designed to isolate peptides (7 and 12 amino acids) capable of binding to the RNA template with high affinity and in so doing preventing the enzyme from elongating existing telomeres. Both libraries showed no high affinity binding to the RNA. The most probable explanation is that such short peptides are incapable of binding sequence-specifically to nucleic acids. Many studies have indicated the importance of telomerase activity as an independent prognostic indicator in a variety of cancers, but recent results have shown that telomere length is actually a better indicator, as it shows the final result oftelomerase activity. We thus decided to develop a flow cytrometric method that would allow us to analyze telomere length in small tissue samples. After the initial technique had been established on lymphocytes, it was tested on the SNO cell line before we switched to solid tissue. The background of cellular debris and cell clusters was much worse than for lymphocytes, but differences in signal strength could be seen. From the results obtained it is clear that this method is not optimized yet. The results also indicate that at this stage of development, flow-FISH is inferior to telomerase activity as prognostic indicator. The best way to validate the current method is to analyze the same samples by Southern hybridization and flow-FISH, preferably well defined tumor and normal tissue.Item Open Access Recombinant production and evaluation of a multifunctional haemostatic fusion protein(University of the Free State, 1999-10) Van Zyl, Walda Brenda; Pretorius, G. H. J.; Kotzé, H. F.Platelets and coagulation both play a pivotal role in thrombosis, one of the major life-threatening diseases in our society. We have thus experienced a drastic increase in the development of potent and secure antithrombotic, antiplatelet and fibrinolytic agents during the past decade. Recently, much research has been devoted to the development of chimeric proteins, where haemostasis is simultaneously targeted at different levels. For the purpose of this study, such a chimera, named PLATSAK, was designed. A 29 amino acid antithrombotic and antiplatelet peptide, comprising three inhibitory regions, was linked to staphylokinase via a cleavable factor Xa recognition sequence. The overall activities of PLATSAK should include inhibition of thrombin, prevention of platelet aggregation and activation of fibrinolyis. The gene encoding PLATSAK was expressed in E. coli cells under controlled conditions. PLATSAK was produced as a strongly expressed protein of 18 kDa and was purified form native E. coli proteins using metal affinity chromatography. In vitro analysis of PLATSAK activity revealed strong inhibition of thrombin and potent fibrin degradation. However, no effect on platelet aggregation could be observed. Several attempts at producing more potent antiplatelet variants were unsuccessful. According to its in vitro activity, PLATSAK appeared to be a potent novel haemostatic agent and was prone to be evaluated in an in vivo system. The in vivo activity of PLATSAK was evaluated by assessing its effect on platelet deposition in a baboon model of arterial and venous thrombosis. Dacron vascular graft segments and expansion chambers, inserted as extensions into permanent femoral arteriovenous shunts, were used to simulate arterial and venous thrombosis, respectively. PLATSAK (3.68 mg/kg) was administered as a bolus. Platelet deposition onto the graft surface and in the expansion chamber was imaged in real time with a scintillation camera as the deposition of 111ln-labelled platelets. After two hours, platelet deposition in the graft segments and expansion chambers was inhibited by 50% and 85% respectively when compared to control studies. The aPTT was lengthened to >120 seconds. Interestingly, the level of FOP in plasma did not increase after administration of PLATSAK. These results demonstrate that PLATSAK effectively inhibited platelet deposition in both arterial- and venous-type thrombosis an animal model. This is in contrast to the lack of the antiplatelet activity of PLATSAK in vitro. This illustrates that in vitro platelet aggregation results can not be directly applied to an in vivo situation. In summary, the recombinant production of a multifunctional haemostatic fusion protein, PLATSAK, was successful. In vitro PLATSAK showed significant antithrombin and fibrinolytic activity, but trivial antiplatelet activity. In vivo studies revealed that PLATSAK is a potent antithrombin and also prevented platelet deposition on thrombogenic material. The strong immuun response of PLATSAK however needs to be investigated and a variant with a weak immunogenic nature needs to be produced.Item Open Access Phage display selection of peptide inhibitors of FVIIa and their functional characterisation(University of the Free State, 2002-06) Roets, Catharina Elizabeth; Meiring, S. M.English: The importance of FVlla and the FVlla/TF complex for the initiation of not only hemostasis but also thrombosis is now generally accepted. It was shown that the blockade of coagulation at the level of FVlla provided full anti thrombotic protection without abnormal bleeding (Harker et aI, 1996), therefore FVlla is a suitable candidate for the development of novel antithrombotics. We selected inhibitors to FVlla using the technique of phage display. A repeated selection of phages from a cyclic heptapeptide and a linear 12-mere phage display library resulted in the enrichment of phages that bind to human FVlla. We selected twelve colonies (6 from each library) that showed the strongest binding to FVlla. The colonies from the cyclic 7-mere library showed a higher affinity binding for FVlla than the colonies from the linear 12- mere library. TF also prevents the binding of one of the cyclic colonies to FVlla. This colony as well as one colony from the linear library showed lengthening of the prothrombin time (PT) as well as the thrombin time (TT) in a dose-dependent manner. A cyclic heptapeptide was synthesised with the corresponding sequence as the sequence displayed on the cyclic 7-mere colony. The peptide showed lengthening of the PT and TT in a dose-dependent manner with a more pronounced effect on the PT than the TI. We also studied the effect of this peptide on platelet adhesion on human vascular endothelial cell matrix, collagen and TF under both venous and arterial shear stresses. The peptide inhibits platelet adhesion to HMEC-1 under both shear stresses. The effect on arterial shear is however more pronounced. It does not inhibit platelet adhesion to collagen, but has a dose-dependent inhibitory effect on platelet adhesion TF at arterial shear. Kinetic analysis of the peptide showed that this peptide is a competitive inhibitor of FVlla by altering the Km-values but not the Vmax-values. The Lineweaver-Burk plot also indicates a competitive inhibition, because the slope of the graphs increased with increasing inhibitor concentrations. The Ki-value was determined at 0.1232 mM. In summary, this peptide inhibits thrombus formation by preventing FVlla from binding to TF and therefore preventing the activation of FX by the FVlla/TF complex. This study suggests that inhibitors to FVlla provide a novel therapeutic approach to prevent thrombosis.Item Open Access Comparison of platelet receptors P2Y12, GPIIB/IIIA, GPVI, and GPIBα between the Cape chacma baboon and the human(University of the Free State, 2015) Janse van Rensburg, Walter James; Badenhorst, Philip N.; De Kock, AndréEnglish: Background: Acute coronary syndrome is globally a major cause of morbidity and mortality. Treatment and prevention involve the use of an anti-platelet agent. The current available agents have either side-effects or are relatively ineffective. Therefore, there exists a need to develop safer and more effective agents. Platelet receptors are a target for anti-platelet agents and new generation agents function on a molecular level. The Cape chacma baboon (Papio ursinus) has been a popular model for the pre-clinical evaluation of anti-platelet agents. However, limited molecular data are available for these animals, restricting its translational value. The aim of this study was to characterize four common platelet receptors in the Cape chacma baboon and compare the results to human data. Methods: The platelet receptors P2Y12, glycoprotein (GP) VI, GPIIb/IIIa and GPIbα were selected for this study. Light transmission platelet aggregometry was performed to assess baboon platelet function; receptor number quantification was performed by flow cytometry; and Sanger sequencing was done on genomic baboon DNA. All results were compared to normal human data. Results: Baboon ADP-induced platelet aggregation results were significantly different from normal human results, even at ADP levels four times (40 μM) the highest human concentration of 10 μM. Baboon collagen-induced aggregation remained significantly different at twice (8 μg/ml) the highest human concentration of 4 μg/ml. However, the differences in collagen-induced aggregation results were not clinically relevant from the human results, because all except one result (at 8 μg/ml) fell within the normal human reference range. At double the highest human concentration for ristocetin (2.5 mg/ml) baboon platelets gave statistically similar results. At double the highest human concentration (1 mg/ml) arachidonic acid results remained significantly different between baboons and human. Baboon quantification results showed a 37% increase in GPIIb, 27% increase in GPIIIa and 25.5% increase in GPIbα. GPVI quantification failed due to non-reactive monoclonal antibodies. P2Y12 quantification was not possible, as no commercial monoclonal antibodies exist for it. The P2Y12 protein sequence was 98.8% similar. It differed by only four amino acids, none of which have been described as functionally essential. The GPVI protein sequence showed 95% similarity. It included a 14 amino acid difference and a three amino acid deletion. One change was at a region where an amino acid change has been implicated in reduced collagen-induced platelet aggregation in humans. Two differences were directly adjacent to a collagen-binding amino acid. The deletion was within the signalling region of GPVI. Exon 28 of GPIIb could not be sequenced. The GPIIb protein sequence for exon 1-27 was 98.2% similar and for exons 29-30 there was 98.3% similarity. There was an 18 amino acid difference. One amino acid change was in the ligand-binding region. The GPIIIa protein sequence was 99.6% similar, with three amino acid changes. One change was in the ligand-binding region. 54 amino acid changes were found in GPIbα. The protein sequences of the signal peptide, VWF-binding-, PEST / macroglycoprotein-, transmembrane- and cytoplasmic domains showed 93.8%, 89.4%, 57.9%, 90.5% and 95.0% similarity, respectively. 246 bases of GPIbα failed to sequence. Discussion and Conclusion: Sequentially and functionally baboon P2Y12, GPIIb/IIIa and GPIbα is comparable to humans. The higher agonist-levels needed for baboon platelet aggregation may be attributed to the increase in surface receptor numbers. However, receptor-number, optimal agonist concentrations and potentially inhibiting amino acid changes should be noted for future studies. Non-reactive antibodies and changes in critical amino acids caused the baboon GPVI to be not comparable to humans. The Cape chacma baboon (Papio ursinus) is therefore, deemed a suitable animal model for the evaluation of human-targeted anti-platelet agents directed against the receptors P2Y12, GPIIb/IIIa and GPIbα, but not for the evaluation of human-targeted anti-GPVI agents.Item Open Access Monitoring of genetically modified food products in South Africa(University of the Free State, 2010-12) Marx, Gertruida M.; Viljoen, C. D.English: Globally, South Africa is the eighth largest producer of GM crops and also imports GM food. In addition to the promise of increased agricultural production, the introduction of GM crops is also having an impact on society in terms of consumer acceptance and trade. As a result, most countries manage GMOs in terms of development, use and application as well as require mandatory GM labelling for consumer preference. With an increase in GM developments, monitoring the food chain in terms of GM labelling and unapproved GM events will continue to pose a regulatory challenge. The aims of this thesis were the following: 1. To determine the uptake of GM food into the food chain; 2. To study the application of voluntary GM labelling; 3. To investigate the impact of mandatory GM labelling; and 4. To establish a monitoring system to detect illegal GMOs in South Africa. Until 2005 it was assumed that there were only low levels of GM crop in the food chain, based on production volumes. However, results from this thesis have shown that 76% of food products tested positive for the presence of GM in 2005. There was also no consideration of mandatory GM labelling as it was thought that voluntary GM labelling was successfully being applied in South Africa. Despite this, 31% of products labelled to indicate an absence of GM, such as “GMO free”, “non-GM” and “organic”, contained genetic modification above 1%, and 20% of these contained more than 5% genetic modification. These results demonstrated the extent of GM in the food chain in South Africa and highlighted the fact that voluntary GM labelling does not protect consumers against misleading claims. In 2008, the Consumer Protection Act mandated the labelling of GM in food products and ingredients. However, there was a lot of uncertainty as to how this would impact the food industry. The subsequent research on the impact of mandatory GM labelling in South Africa determined that 67% of maize and 54% of soybean products will have to be labelled for GM content. In addition to this, GM was also detected in 50% of products labelled to indicate an absence of GM. Furthermore, results indicated that the use of either a 1% or 5% threshold does not make a considerable difference in terms of the number of products implicated. The use of the term “may contain genetic modification” as suggested by draft regulations to the Consumer Protection Act may provide a cost effective manner in which GM labelling can be applied in a developing country similar to South Africa, as it would reduce costs in terms of GM detection. The draft regulations for the Consumer Protection Act also make provision to indicate the absence of GM below a threshold that does not included terminology such as ”GMO free” or “non-GM”. Furthermore, the draft regulations do not require third party verification and compliance will mainly be self-regulating. The implication of this is that consumers or consumer groups will become responsible for policing the application of GM labelling in South Africa. Finally, this thesis presents a GM monitoring scheme for unapproved GMOs, that have not been proven safe for human health and/or the environment. The scheme has the advantage of being cost effective and can be applied to the regulatory situation in any country, taking approved GM events into consideration. The scheme was applied to off-the-shelf food products in South Africa to determine the presence of illegal GMOs. Even though no unapproved GM events were detected, a potential illegal import of GM soybean event A2704-12 was found. It was also found that an approved GM soybean event was comingled with rice and wheat products, although not indicated in the ingredients. The research emanating from this thesis has contributed to inform discussions that have resulted in the inclusion of mandatory GM labelling in the Consumer Protection Act 68 of 2008. It is hoped that the research on the application of mandatory GM labelling and the monitoring for unapproved GM events in the food chain will have a similar impact on the regulatory system in South Africa.