Development of an Arterial Thrombosis Model and Study on the Antigenicity of Streptokinase Mutants

• Main Authors: Jo-chi wang, 王若琦
• Other Authors: H.L.Wu
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/80812931668406348064

碩士 === 國立成功大學 === 生物化學研究所 === 89 === Abstract Streptokinase(SK), a single-chain polypeptide of 414 amino acids, is used as a clinical thrombolytic agent. The stoichiometric complex of SK and human plasminogen(HPlg) is considered to be an activator for converting HPlg to human plasmin(HPlm), which is capable of dissolving thromboembolic clots in blood vessels. SK itself does not have proteolytic activity. SK forms a 1:1 molar complex with HPlg, altering its conformation to expose, without protelytic cleavage, the active site in the HPlg moiety, named “virgin enzyme”. The SK-HPlg complex is then converted to SK-HPlm complex, both of which can act as an effective HPlg activator to catalyze the hydrolysis of the activating peptide bond of substrate HPlg. Several recombinant SK fusion proteins have been constructed in our lab. According to the previous studies in vitro, these proteins could effectively induce clot-lysis on platelet-rich thrombus. In order to examine the thrombolytic potency of the SK proteins in vivo, a platelet-rich arterial thrombus model was set up and was tested to monitor the thrombolysis process. We have developed a novel double-opposing inverted— sutures model to crease a platelet-rich thrombus in the femoral artery of rabbits. The blood flow was measured with a real-time ultrasonic flow meter. The results demonstrated that tissue-type plasminogen activator can cause more rapid dissolution than native SK. This in vivo method is not only simple but also reproducible and non intrusive. The development of this procedure will provide a good testing model for analysis of newly developed drugs. Another goal of the study is to determine the epitope of the antigenicity of SK. Because SK is a foreign protein, the injection of this drug might induce allergic reaction in the patient who have been exposed to Streptococcus infection. This immunologic reaction might reduce the potency of SK and induce allergic reaction, so that it becomes a major concern in the administration of SK in patients. We have constructed several SK mutants with point mutations in different locations on the protein. The SK proteins with mutations to alanine in regions A, C and D near SK N-terminal were expressed in E.coli system in soluble forms and were purified by precipitating with ammonium sulfate and High-Q chromatography. However, SK proteins with mutation to alanine in regions B, A+B and C+D near SK N-terminal were insoluble. These insoluble proteins were dissolved and denatured with 6M urea and renatured slowly in to a soluble form. From these procedures, mutant SK proteins were prepared with high purity as assayed by SDS-PAGE. All these mutant SK proteins could effectively activate HPlg with 60﹪to 80﹪activity of that of SK(16-378). In the direct binding assay of SK, A+B and C+D had slightly less antigenicity as compared with SK(16-378). In the neutralizing activity titers of SK, anti-wild-type SK(16-378) polyclonal antibody had a 30% neutralizing effect on C+D as compared with wild-type-SK(16-378). In conclusion, these residues in the N-terminal region of SK are associated with immune response. It is possible to design a SK with alteration in this region that has less immunogenicity. This study could provide to develope an alternative treatment choice for patients suffering from myocardial infarction.