Streptokinase contains two independent plasminogen-binding sites

Area of Science:

• Biochemistry
• Molecular Biology
• Thrombolysis

Background:

• Streptokinase (SK) is a thrombolytic agent that activates plasminogen (PG) indirectly.
• SK forms an equimolar complex with PG or plasmin (PN) to exert its effect.
• Understanding the PG/PN-binding sites in SK is crucial for its therapeutic application.

Purpose of the Study:

• To explore the location and nature of plasminogen (PG) and plasmin (PN) binding sites in Streptokinase (SK).
• To identify and characterize novel PG-binding regions within SK.
• To investigate the mechanism of PG binding by SK, independent of PG's Kringle domains.

Main Methods:

• Limited proteolysis of Streptokinase (SK) using immobilized trypsin.
• Western blotting with radiolabeled plasminogen (PG) after SDS-PAGE.
• High-performance liquid chromatography (HPLC) for fragment isolation.
• Enzyme-linked immunosorbent assay (ELISA) for binding property characterization.
• Synthetic peptide analysis to localize the N-terminal binding site.

Main Results:

• Three PG-binding fragments of MW 7 kD, 19 kD, and 31 kD were identified in tryptic digests of SK.
• A novel, high-affinity, independent PG-binding site was discovered in the N-terminal region (residues 1-59) of SK.
• This N-terminal binding site was further localized to residues 37-51 and does not involve PG's Kringle domains.
• The identified N-terminal sequence (LTSRPA) is also found in the PG-binding domain of human fibronectin.

Conclusions:

• Streptokinase (SK) possesses at least two distinct and independent plasminogen (PG) binding sites.
• The N-terminal region of SK (residues 37-51) contains a significant PG-binding site.
• This binding mechanism is independent of the Kringle domains of plasminogen (PG).