CHARACTERIZATION OF THE INTERACTION BETWEEN PLASMINOGEN AND STAPHYLOKINASE

Binding parameters [association (k(a)) and dissociation (k(d)) rate constants, and affinity constants (K-a = k(a)/k(d))] for the interaction between recombinant staphylokinase (SakSTAR) and plasmin(ogen) were determined by real-time biospecific interaction analysis. The K-a value for binding of SakSTAR to native human Glu-plasminogen was 0.93X10(8)M(-1) as compared to 2.0X10(8)M(-1) and 1.6X10(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 K-a values of 4.0x10(8)M(-1) and 8.4X10(8)M(-1), respectively, whereas active-site blocked LMM-plasmin (a plasmin derivative lacking kringles 1-4) and the plasmin B-chain bound with K-a values of 1.0x10(8)M(-1) and 0.49X10(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 (K-a values of 1.2x10(5)M(-1) and 2.9x10(5)M(-1), respectively). The binding of these plasminogen derivatives to streptokinase occurred with similar relative K-a values. The K-a 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 K-a 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 Lysplasminogen 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.