MOLECULAR CONVERSIONS OF RECOMBINANT STAPHYLOKINASE DURING PLASMINOGEN ACTIVATION IN PURIFIED SYSTEMS AND IN HUMAN PLASMA

Recombinant staphylokinase (STAR) is produced as a 136 amino acid prot ein with NH2-terminal sequence Ser-Ser-Ser (mature STAR, HMW-STAR), wh ich may be converted to lower molecular weight forms (LMW-STAR) by rem oval of the first six residues (yielding STAR-DELTA6 with NH2-terminal Gly-Lys-Tyr-) or the first ten residues (yielding STAR-DELTA10 with N H2-terminal Lys-Gly-Asp-). In the present study the occurrence and eff ects of these conversions during plasminogen activation by HMW-STAR we re studied in purified systems and in human plasma. In stoichiometric mixtures of HMW-STAR and native human plasminogen (Glu-plasminogen), r apid and quantitative conversion of HMW-STAR to LMW-STAR occurred, con comitant with exposure of the active site in the plasmin-STAR complex. NH2-terminal amino acid sequence analysis revealed the sequence Lys-G ly-Asp- in addition to the known sequences of the Lys-plasmin chains, identifying STAR-DELTA10 as the derivative generated from HMW-STAR. In mixtures of catalytic amount of HMW-STAR and human plasminogen, plasm in generation occurred progressively, following an initial lag phase, during which HMW-STAR was converted to LMW-STAR. Plasmin-mediated conv ersion of HMW-STAR to LMW-STAR obeyed Michaelis-Menten kinetics with K (m) = 3.6 muM and k2 = 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 mul plasma clot submerged in 250 mul plasma, 80% clot lysis with in 1 h was obtained with 70 nM HMW-STAR. This was associated with fibr inogen depletion and conversion of 20% of the HMW-STAR to LMW-STAR. Ad dition of 100 nM HMW-STAR to human plasma in the absence of a clot did not induce significant fibrinogen breakdown (greater-than-or-equal-to 90% residual fibrinogen after 2 h), and was not associated with signi ficant coversion to LMW-STAR (less-than-or-equal-to 10% within 2 h). W ith 400 nM HMW-STAR, fibrinogen depletion in plasma occurred within 1 h, and 80% conversion to LMW-STAR was observed. Thus, at fibrinolytica lly 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.