| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 84 === Streptokinase (SK) is a secretory single-peptide protein of
414 or 415 amino acid residues produced by various strains of ?-
hemolytic strptococcus. It is one of the plasminogen activators
that are clinically used as thrombolytic agents in the treatment
of myocardial infarction. Streptokinase is generally regarded as
a fibrin-nonspecific plasminogen activator, and it does not
distinguish the fibrin of a thrombus from the fibrin of a
hemostatic plug. Certain drawbacks such as systemic bleeding and
plasminemia have limited its overall usefulness. Our
investigations focus on designing lytic agents with greater
affinity for thrombus-bound plasminogen while reducing
activation of circulating zymogen. Since a thrombus contains
more thrombin than does a hemostatic plug, we choose thrombin as
a thrombus marker. Hirudin, an anticoagulant agent, is a
thrombin-specific inhibitor. Hirugen and hirulog are some
synthetic peptide fragments of hirudin, that have the same
properties as hirudin. To design a lytic agent with selectivity
for thrombi and inhibition of thrombin activity, we prepared SK
fusion proteins with hirugen or hirulog by gene cloning
techniques. Since arterial thrombi are very rich in activated
platelets, it might be useful to design a lytic agent with
specific affinity for the activated platelets. The ?-chain
peptide sequence (HHLGGAKQAGDV) of fibrinogen is responsible for
the binding to glycoprotein IIbIIIa on activated platelets. In
order to target the SK molecule to platelet-rich thrombus, ?-
chain peptide sequence was incorporated into the SK molecule.
The recombinant SK genes, hirugen-SK, hirulog-SK, and ?-chain-
SK, were prepared by PCR techniques using K58E-SK as a template.
The fusion proteins were expressed in pET expression system and
purified by anion exchang chromatography on DEAE-Sepharose CL-6B
or High Q support. The purified hirugen-SK and hirulog-SK
proteins could activate plasminogen more efficiently than native
SK, whereas ?-chain-SK had lower plasminogen activator activity.
Hirugen-SK and hirulog-SK, due to its hirugen or hirulog
residue, had affinity for thrombin. The hirugen-SK-plasminogen
or hirulog-SK-plasminogen complexes also had affinity for
thrombin as compared to native SK-plasminogen complex. Hirulog-
SK could inhibit the amidolytic activity of thrombin, and the
hirulog-SK-thrombin complex still could activate plasminogen.
Hirugen-SK and hirulog-SK were more effective in inducing
thrombin-rich clot lysis than native SK. ?-Chain-SK had high
affinity to bind platelet-rich plasma clot, and also induced
fibrinolysis of platelet-rich whole blood clot very efficiently.
In conclusion, SK can be efficiently targeted to the site of a
thrombus by conjugation to hirugen, hirulog, or ?-chain peptide
sequence, resulting in both more potent and more selective
thrombolysis.
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