Molecular conversions of recombinant staphylokinase during plasminogen activation in purified systems and in human plasma
Ueshima, S.; Silence, K.; Collen, D.; Lijnen, H.R.
Thrombosis and Haemostasis 70(3): 495-499
1993
ISSN/ISBN: 0340-6245 PMID: 8259556 Document Number: 406174
Recombinant staphylokinase (STAR) is produced as a 136 amino acid protein with NH,-terminal sequence Ser-Ser-Ser (mature STAR, HMW-STAR), which may be converted to lower molecular weight forms (LMW-STAR) by removal of the first six residues (yielding STAR-DELTA-6 with NH-2-terminal Gly-Lys-Tyr-) or the first ten residues (yielding STAR-DELTA-10 with NH-2-terminal Lys-Gly-Asp-). In the present study the occurrence and effects of these conversions during plasminogen activation by HMW-STAR were studied in purified systems and in human plasma. In stoichiometric mixtures of HMW-STAR and native human plasminogen (Glu-plasminogen), rapid and quantitative conversion of HMW-STAR to LMW-STAR occurred, concomitant with exposure of the active site in the plasmin-STAR complex. NH-2-terminal amino acid sequence analysis revealed the sequence Lys-Gly-Asp- in addition to the known sequences of the Lys-plasmin chains, identifying STAR-DELTA-10 as the derivative generated from HMW-STAR. In mixtures of catalytic amount of HMW-STAR and human plasminogen, plasmin generation occurred progressively, following an initial lag phase, during which HMW-STAR was converted to LMW-STAR. Plasmin-mediated conversion of HMW-STAR to LMW-STAR obeyed Michaelis-Menten kinetics with K-m = 3.6 mu-M and k-2 = 0.38 s-1. The specific clot lysis activities of HMW-STAR (122,000 +- 8,000 units/mg) and LMW-STAR (129,000 +- 8,000 units/mg) were indistinguishable. In an in vitro system consisting of a 60 mu-l plasma clot submersed in 250 mu-l plasma, 80% clot lysis within 1 h was obtained with 70 nM HMW-STAR. This was associated with fibrinogen depletion and conversion of 20% of the HMW-STAR to LMW-STAR. Addition of 100 nM HMW-STAR to human plasma in the absence of a clot did not induce significant fibrinogen breakdown ( gtoreq 90% residual fibrinogen after 2 h), and was not associated with significant coversion to LMW-STAR ( ltoreq 10% within 2 h). With 400 nM HMW-STAR, fibrinogen depletion in plasma occurred within 1 h, and 80% conversion to LMW-STAR was observed. Thus, at fibrinolytically active concentrations which do not cause fibrinogen breakdown, no significant conversion of HMW-STAR to LMW-STAR occurs in human plasma in the absence of a clot. These findings indicate that the conversion of HMW-STAR to LMW-STAR may occur in association with clot lysis, but is not required to induce it.