No 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.

Streptokinase reacts very rapidly with human plasmin (rate constant 5.4 × 107 M−1 s−1) forming a 1:1 stoichiometric complex which has a dissociation constant of 5 × 10−11 M. This plasmin‐streptokinase complex is 105 times less reactive towards α2‐antiplasmin than plasmin, the inhibition rate constant being 1.4 × 102 M−1 s−1. The loss of reactivity of the streptokinase‐plasmin complex towards α2‐antiplasmin is independent of the lysine binding sites in plasmin since low‐Mr plasmin, which lacks these sites, and plasmin in which the sites have been blocked by 6‐amino‐hexanoic acid, are both equally unreactive towards α2‐antiplasmin on reaction with streptokinase. The plasmin‐streptokinase complex binds to Sepharose‐lysine and Sepharose‐fibrin monomer in the same fashion as free plasmin, showing that the lysine binding sites are fully exposed in the complex. Bovine plasmin is rapidly inhibited by human α2‐antiplasmin (k1= 1.6 × 106 M−1 s−1) and similarly loses reactivity towards the inhibitor on complex formation with streptokinase (50% binding at 0.4 μM streptokinase).