Preparation of Recombinant Streptokinase Fusion Proteins with Specific Affinity to the Activated Platelets

• Main Authors: Shih, Yan-Ping, 石燕萍
• Other Authors: Shi, Guey-Yueh
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/16277200888270663595

碩士 === 國立成功大學 === 生物化學研究所 === 86 === Streptokinase (SK) is a secretory single-peptide protein of 414 or 415 amino acid residues produced by various strains of b-hemolytic streptococcus. It is one of the plasminogen activators that are clinicallly 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 a thrombus from 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 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 g-chain peptide sequence (HHLGGAKQAGDV) and RGD sequence of fibrinogen are resposible for the binding to glycoprotein IIbIIIa on activated platelets. In order to target the SK molecule to platelet-rich thrombus, g-chain and RGD peptide sequences were incorporated into the SK molecule. The recombinant SK genes, SK-K59E-g, SK-K59E-g-Glyn-RGD(n=4 or 7), SK-K59E-RGD, g-SK-K59E, and RGD-Gly7-g-SK-K59E, were prepared by PCR techniques using SK-K59E as a template. The fusion proteins were expressed in pET expression system and purified by anion exchange chromatography on High Q support. The purified SK-K59E, SK-K59E-g, SK-K59E-g-Glyn-RGD(n=4 or 7), and SK-K59E-RGD proteins could activate plasminogen as efficiently as native SK, whereas g-SK-K59E, and RGD-Gly7-g-SK-K59E had lower plasminogen activator activities. SK-K59E-g-Gly4-RGD, SK-K59E-g-Gly7-RGD, and RGD-Gly7-g-SK-K59E could specifically inhibit platelet aggregation. The clot lysis rates of the fusion proteins increased when the tests were performed with platelet-rich blood clots. In conclusion, the blood-clot specificity of SK as a thrombolytic agent in vitro might be improved by attachment of a platelet-specific ligand to SK molecule. Whether the fusion proteins have better clot specificity in vivo still has to be tested.