Biochemical properties of natural and recombinant staphylokinase

Abstract

The biochemical properties of natural staphylokinase (STAN), purified from the culture broth of a selected Staphylococcus aureus strain, were compared with those of different molecular species of recombinant staphylokinase (STAR), produced by expression of a genomic DNA fragment of the Staphylococcus aureus strain in E. coli. STAR was obtained from the supernatant (STAR-S) or periplasmic fraction (STAR-P) of the transformed E. coli either as a low molecular weight form lacking the 10 NHZ-terminal amino acids (STA-Δ10) (peak I following chromatography of STAR-S or STAR-P on CM-Sephadex, STA-CM-I) or as a mixture of mature STA (STA-M) and a proteolytic derivative lacking the 6 NH 2-terminal amino acids (STA-Δ6) (peak II following chromatography of STAR-S or STAR-P on CM-Sephadex, STA-CM-II), or it was obtained from the cytosol fraction (STAR-C) as STA-Δ10. STAN, STA-CM-I, STA-CM-II and STAR-C were found to be similar with respect to following properties: rate and extent of complex formation with plasminogen, plasminogen activating potential in the absence or the presence of a fibrin-like stimulator and inhibition rate of the plasminogen-STA complex by a2-antiplasmin, and fibrinolytic and fibrinogenolytic potential in a human plasma milieu in vitro. In a human plasma milieu in vitro, STAN had a somewhat lower fibrinolytic activity versus platelet-rich plasma ((PRP); 4 × 10 5 platelets/μl) clots than versus platelet-poor plasma ((PPP); <5 × 10 3 platelets/μl) clots, with equi-effective concentrations (causing 50% clot lysis in 2h; C 50) of 0.37±0.02 μg/ml or 0.58±0.07 μg/ml (mean±SEM; n=6), respectively (p=0.02). In contrast, no significant lysis of PRP clots was obtained using up to 20 μg/ml of streptokinase, whereas its C 50 for PPP clots was 2.1±0.13 μg/ml. Clot lysis was inhibited by platelets in a dose-dependent way: 50% inhibition occurred with 1.4×10 5/μl platelets for streptokinase, and with 13 × 10 5/μI platelets for STAN. When clot retraction was inhibited with a synthetic platelet GPIIb/IIIa receptor antagonist, the thrombolytic potency of STAN towards a platelet-rich clot was unaffected, whereas that of streptokinase was increased to a level similar to that observed with PPP clots. Mechanically compressed human plasma clots, submerged in a human plasma milieu in vitro, were equally sensitive to lysis with STAN (C 50 of 0.34±0.03 μg/ml) as PPP clots, but were very resistant to lysis with streptokinase (<10% lysis with 20 μg/ml). Thus, STAN, STA-CM-I, STA-CM-II and STAR-C are functionally indistinguishable, and in a human plasma milieu STA is relatively more potent towards retracted or compressed plasma clots than streptokinase.