Functional studies of streptokinase action on human plasminogen

Abstract

Fibrinolysis is performed by the serum protease plasmin (plm), which is normally present in the blood as a precursor, plasminogen (plg). The plg is activated to plm by the plg activators, such as tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). Streptokinase (SK), an extracellular protein produced by several strains of (-hemolytic streptococci, also activates plg to plm. SK is currently used as a therapeutic agent in treatment of the thromboembolic blockage, such as myocardial infarction. For the therapeutic use and the functional studies, a high-level expression plasmid for SK, pSK100, has been constructed. It contains a tac promoter, an ompA signal sequence, a SK structural gene (skc) and a rrnBT1T2 transcription terminator. E. coli JM109 carrying pSK100 produced about 5,000IU of SK per 1 ml of LB-ampicillin media. About 95% of the expressed SK were secreted into the periplasmic and extracellular fractions. The recombinant SK in high yield and purity may be a potential alternative source for the therapeutic agent. Because of its peculiar mechanism for plg activation, many researchers have long investigated SK. Unlike most of the plg activators, SK does not have proteolytic activity. It activates plg by the formation of an equimolar complex with plg. The SK-plg complex is an efficient proteolytic activator of plg. However, there are little reports on exact region of SK involved in activator complex formation with plg. In order to elucidate the interaction site or domain of SK with plg, C-terminal truncation analysis of SK was performed by exonuclease III method and PCR method. SK mutants, which lose 26, 33, 37, 40, 41, 46, 47, 50, 70, 97 or 124 amino acid residues from the C-terminus, were constructed. The truncated SKs were expressed in E.coli and purified. The 41 residue deletion (SKP373) from the C-terminus had not effect on the plg activation activity. However, the deletion of 46 amino acid residues (SKP368) resulted...