Recombinant production and evaluation of a multifunctional haemostatic fusion protein
| dc.contributor.advisor | Pretorius, G. H. J. | |
| dc.contributor.advisor | Kotzé, H. F. | |
| dc.contributor.author | Van Zyl, Walda Brenda | |
| dc.date.accessioned | 2017-11-02T06:55:08Z | |
| dc.date.available | 2017-11-02T06:55:08Z | |
| dc.date.issued | 1999-10 | |
| dc.description.abstract | 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. | en_ZA |
| dc.description.sponsorship | Medical Research Council | en_ZA |
| dc.description.sponsorship | University of the Orange Free State, Central Research Fund | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/11660/7387 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | University of the Free State | en_ZA |
| dc.rights.holder | University of the Free State | en_ZA |
| dc.subject | Anticoagulants (Medicine) | en_ZA |
| dc.subject | Fibrinolytic agents | en_ZA |
| dc.subject | Hemostasis | en_ZA |
| dc.subject | Thesis (Ph.D. (Haematology and Cell Biology))--University of the Free State, 1999 | en_ZA |
| dc.title | Recombinant production and evaluation of a multifunctional haemostatic fusion protein | en_ZA |
| dc.type | Thesis | en_ZA |