The mechanism of plasminogen activation by streptokinase

• Main Authors: Young Kung-Chia, 楊孔嘉
• Other Authors: Wu Hua-Lin
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/19736392908023263805

博士 === 國立成功大學 === 基礎醫學研究所 === 86 === Streptokinase (SK), a single-peptide secretory protein of 414 amino acid residues, is produced from various strains of b-hemolytic Streptococci in Lancefield group A, C and G. SK can activate human plasminogen (HPlg) to human plasmin (HPlm) potently, therefore, it is used as a thrombolytic agent to dissolve thromboembolic clots in blood vessels, such as acute myocardial infarction. SK itself does not have proteolytic activity. The activation of HPlg by SK-type activator may be via a unique mechanism involving the modulation of HPlg molecule. The role of SK moiety in HPlg activation has been proposed to have two components: (a) SK may open the active center on one HPlg, thereby inducing it to serve as an activator HPlg; (b) SK may modify the geometry near the Arg560-Val561 bond of the substrate HPlg for an efficient cleavage. In this study, the goals aimed to clarify more precisely the mechaism of HPlg activation by SK, in searching of the functions of various domains of SK and the interaction modes between SK and HPlg. A series of recombinant truncated SK peptides with deletion of NH2 and/or COOH-terminal amino acid residues were prepared by gene expression and protein purification from Escherichia coli system. In assaying of the enzymatic activities, SK(16-414) and SK(16-378) were as acitve as native SK in HPlg activation. SK(60-414), with the modification by SK(1-59), could also serve as a HPlg activator. SK(91-414), SK(127-414) and SK(158-414), at catalytic concentrations, all failed to activate HPlg. However, these truncated SK peptides retained the ability to expose amidolytically "virgin enzyme"-active center in a 1:1 stoichiometric complex with HPlg. SK(16-316) could neither transform HPlg to "virgin enzyme", nor catalyze the HPlg activation, but the HPlg moiety was slowly converted to HPlm in equimolar HPlg-SK(16-316) complex. Although SK(16-251) and SK(220-414) alone didn''t have any enzymatic activity, combination of SK(16-251) and SK(220-414) allowed the two peptides in complement with each other to form an efficient HPlg activator. In addition, SK(16-251) dose-dependently enhanced the activation of HPlg by SK(16-414), while the HPlg activation by SK(16-414) was inhibited by SK(220-414). Based on the concentration-dependent binding assay and competitive binding assay, three interaction modes between SK and HPlg domains were demonstrated as follows: SK(220-414) and catalytic domain of HPlg, SK(16-251) and HPlg kringle domain as well as SK(220-414) and HPlg kringle domain. Furthermore, SK(16-251) and SK(220-414) could compete for binding on the same or nearby locations on HPlg kringle domain. In the sandwich-binding assay, a HPlg-specific ternary complex in form of HPlg-SK-HPlg was detected. Formation of the ternary complex may represent an enzyme-substrate intermediate for HPlg activation. In conclusion, one SK can interact with two HPlg molecules simultaneously. The C-terminal domain of SK is responsible for the formation of enzymatic center in complexing with HPlg, whereas the NH2-terminal domain of SK acts as a substrate modulator. As bridged with SK, the HPlg that binds to the C-terminal domains of SK functions as an enzyme to efficiently catalyze the conversion of substrate HPlg that binds to the NH2-terminal domain of SK to human plasmin.