The effects of intact recombinant streptokinase (rSK) and of a 17-kDa fragment, consisting of residues Val143 through Lys293 (17-kDa rSK), on the interaction of recombinant staphylokinase (SakSTAR) with plasminogen was evaluated. Determination of affinity constants (K-A) by biospecific interaction analysis revealed that a complex between active site blocked plasmin (VFK-plasmin) and 17-kDa rSK bound to SakSTAR with an affinity which was only 3-fold lower than that of VFK-plasmin (K-A of 1.5 X 10(8) M(-1) and 4.4 x 10(8) M(-1), respectively). Similarly, the VFK-plasmin-SakSTAR complex bound to 17-kDa rSK with a comparable affinity as VFK-plasmin (K-A of 0.92 X 10(8) M(-1) and 2.0 x 10(8) M(-1), respectively). In contrast, the VFK-plasmin-rSK complex virtually did not bind to 17-kDa rSK or to SakSTAR (K-A < 0.001 x 10(8) M(-1)). Binding of biotinylated SakSTAR to VFK-plasmin coated on microtiter plates, was not displaced by addition of up to 20-fold molar excess of 17-kDa rSK, Gel filtration suggested the formation of a ternary complex in equimolar mixtures of VFK-plasmin, 17-kDa rSK and SakSTAR. Binding of 17-kDa rSK to plasminogen resulted in enhancement of the rate of active site exposure upon addition of equimolar amounts of SakSTAR and also enhanced the activation rate by catalytic amounts of SakSTAR. These data indicate that, whereas the binding sites for rSK and SakSTAR on plasmin(ogen) overlap at least partially, 17-kDa rSK and SakSTAR interact with independent binding sites, Binding of 17-kDa rSK to plasminogen apparently induces a conformational change which facilitates subsequent complex formation with SakSTAR and active site exposure.