Doctoral Degrees (Haematology and Cell Biology)

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Browsing Doctoral Degrees (Haematology and Cell Biology) by Author "Meiring, S. M."

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    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.
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    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.