Recombinant production and evaluation of a multifunctional haemostatic fusion protein
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Date
1999-10
Authors
Van Zyl, Walda Brenda
Journal Title
Journal ISSN
Volume Title
Publisher
University of the Free State
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.
Description
Keywords
Anticoagulants (Medicine), Fibrinolytic agents, Hemostasis, Thesis (Ph.D. (Haematology and Cell Biology))--University of the Free State, 1999