Characterization of the Interaction Between Plasminogen and Staphylokinase

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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 0.93×108M‐1 as compared to 2.0×108M‐1 and 1.6×108M‐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 4.0×108M‐1 and 8.4×108M‐‐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 1.0×108M‐1 and 0.49×108M‐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 1.2×105M‐1 and 2.9×105M‐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 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.