Haematology and Cell Biology

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Browsing Haematology and Cell Biology by Subject "Anticoagulants (Medicine)"

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    ItemOpen 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.
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    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.
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    ItemOpen 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.