Interaction of staphylokinase with different molecular forms of plasminogen

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Abstract
In order to obtain more information on the mechanism of plasminogen activation by staphylokinase (STA), we have studied the interaction between recombinant STA (STAR) and different molecular forms of human plasminogen, including Glu-plasminogen (native moiety), Lys-plasminogen (partially degraded moiety) and low-molecular-mass (LMM) plasminogen (moiety lacking kringles 1–4). Addition of 2 μM STAR to 0.4 μM Glu-plasminogen, Lys-plasminogen or LMM plasminogen resulted in the generation of proteolytic activity towards the chromogenic substrate D-Val-Leu-Lys-NH-PhNO2 (S-2251) corresponding to the exposure of 1 active center/plasminogen molecule. Complex formation was associated with conversion of the one-chain plasminogen moieties to two-chain plasmin, and with quantitative conversion of Glu-plasminogen to Lys-plasmin. The stoichiometry of the plasminogen-STAR complex, determined by binding of the complex to Lys-Sepharose and measurement of residual STAR, was found to be equimolar. The plasminogen-STAR complexes were inhibited by α2-antiplasmin with second-order rate constants of 2.4 ± 0.17 × 106 M−1 s−1 for Glu-plasminogen, 2.4 ± 0.21 × 106 M−1 s−1 for Lys-plasminogen and 9.4 ± 1.5 × 104 M−1 s−1 for LMM plasminogen. Glu-plasmin-STAR, Lys-plasmin-STAR and LMM plasmin-STAR had comparable catalytic efficiencies (kcat/Km) for the activation of Glu-plasminogen (0.24–0.29 μM−1 s−1), Lys-plasminogen (0.57–0.79 μM−1 s−1) or LMM plasminogen (0.11–0.16 μM−1 s−1). In a human plasma milieu in vitro STAR, Glu-plasmin-STAR, Lys-plasmin-STAR and LMM-plasmin-STAR were equally effective for the lysis of 125I-fibrin-labeled human plasma clots [50% clot lysis in 2 h (EC50) with 11–13 nM test compound] and equally fibrin-selective (residual fibrinogen levels of 72–84% after 2 h at EC50). Our results thus confirm that plasminogen and STAR form a 1:1 stoichiometric complex in which plasminogen is converted to plasmin and Glu-plasminogen to Lys-plasmin. The lysine-binding sites in kringles 1–4 of plasminogen are not required for the complex formation with STAR, nor for the enzyme activity of the complex with STAR in purified systems and in a human plasma milieu. The lysine-binding sites are, however, important for the rate of the inhibition of the complexes by α2-antiplasmin.