Characterization of the Interaction Between Plasminogen and Staphylokinase

Abstract

Binding parameters [association (ka) and dissociation (kd) rate constants, and affinity constants (Ka = ka/kd)] for the interaction between recombinant staphylokinase (SakSTAR) and plasmin(ogen) were determined by real-time biospecific interaction analysis. The Ka value for binding of SakSTAR to native human Glu-plasminogen was 0.93 x 10(8) M-1 as compared to 2.0 x 10(8) M-1 and 1.6 x 10(8) M-1, respectively, for the binding to [S741A]recombinant plasminogen or Lys-[S741A]recombinant plasminogen (intact or proteolytically degraded plasminogen with the active site Ser741 replaced by alanine). Binding of SakSTAR to active plasmin or to active-site blocked plasmin occurred with Ka values of 4.0 x 10(8) M-1 and 8.4 x 10(8) M-1, respectively, whereas active-site blocked LMM-plasmin (a plasmin derivative lacking kringles 1-4) and the plasmin B-chain bound with Ka values of 1.0 x 10(8) M-1 and 0.49 x 10(8) M-1, respectively. Lysine-binding site I (a plasminogen derivative consisting of kringles 1-3) and lysine-binding site II (a plasminogen derivative consisting of kringle 4) bound with much lower affinity (Ka values of 1.2 x 10(5) M-1 and 2.9 x 10(5) M-1, respectively). The binding of these plasminogen derivatives to streptokinase occurred with similar relative Ka values. The Ka values for binding of the plasmin-SakSTAR complex to streptokinase and binding of the plasmin-streptokinase complex to SakSTAR, were, respectively, 44-fold and 30-fold lower than the values for free plasmin. The Ka for binding of plasminogen to the inactive mutants [M26R]Sak42D or [M26A]Sak42D (site-specific mutagenesis of Met26 to arginine or alanine) were 10-20-fold lower than that of native staphylokinase. These results indicate that: (a) the affinity of staphylokinase for Glu-plasminogen and Lys-plasminogen is comparable; (b) the active site in the plasmin molecule is not required for binding; (c) kringle structures 1-4 of plasminogen do not contribute significantly to plasminogen binding of staphylokinase; (d) Met26 in staphylokinase is important for its high-affinity binding to plasminogen; (e) the binding sites on plasmin for staphylokinase and streptokinase overlap at least partially.

Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

“…The first 8 amino acid residues of N-terminal sequence of the fragment P4 were V-V-G-G-C-V-A-H, which were identical with that of plasmin. With reference to the complete primary structure of human plasminogen [28], we deduce that the significant cleavage site was at R561-fl-V562, similar to t-PA [29] and staphylokinase [30], and that this cleavage site produced the catalytic domain (P4) of the activated plasmin. When human plasminogen was incubated with subtilisin Carlsberg, at least ten fragments from plasminogen were separated by SDS-PAGE (photographs not shown), but these fragments had no fibrinolytic activity in the fibrin plate.…”

Plasminogen/Plasmin Modulates Bone Metabolism by Regulating the Osteoblast and Osteoclast Function

“…Moreover, plasmin induced OPG expression in Plg Ϫ/Ϫ OBs (Fig. 3, I and J OBs lacking OPG expression was significantly higher than that co-cultured with Plg ϩ/ϩ OBs with or without IL-1␤ or PGE 2 . In addition, the number of TRAP-positive multinucleated cells co-cultured with Plg Ϫ/Ϫ OBs was decreased by plasmin (Fig.…”

“…PAI-1, the primary endogenous inhibitor of tPA or uPA, plays an important role in inhibiting arterial clot lysis (1). ␣2AP rapidly inactivates plasmin, resulting in the formation of a stable inactive complex, plasmin-␣2AP (2). Apart from the removal of fibrin, the fibrinolytic system also plays a pivotal role in such phenomena as embryogenesis, proliferation, migration, wound healing, fibrosis, and tumorigenesis (3)(4)(5)(6)(7)(8)(9).…”

Glycosylation of Asparagine-28 of Recombinant Staphylokinase with High-Mannose-type Oligosaccharides Results in a Protein with Highly Attenuated Plasminogen Activator Activity

“…The collected fractions from B were placed over a concanavalin A-Sepharose column. SakSTAR u was collected in the flow-through and wash in fractions [11][12][13][14][15][16]. After this, the column was washed with a solution of 10 mM sodium phosphate, 5 mM MgCl 2 , and 400 mM NaCl, pH 7.0.…”