Summary
Recombinant staphylokinase (STAR) is produced as a 136 amino acid protein with NH2-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-Δ6 with NH2-terminal Gly-Lys-Tyr-) or the first ten residues (yielding STAR-Δ10 with NH2-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. NH2-terminal amino acid sequence analysis revealed the sequence Lys-Gly-Asp- in addition
to the known sequences of the Lys-plasmin chains, identifying STAR-Δ10 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 μM 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 μl plasma clot submerged in 250 μl 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 (≥ 90% residual fibrinogen after 2 h), and was not associated
with significant coversion to LMW-STAR (≤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.
