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Thromb Haemost 1993; 70(03): 495-499
DOI: 10.1055/s-0038-1649612
Original Article
Fibrinolysis
Schattauer GmbH Stuttgart

Molecular Conversions of Recombinant Staphylokinase During Plasminogen Activation in Purified Systems and in Human Plasma

S Ueshima
The Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
,
K Silence
The Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
,
D Collen
The Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
,
H R Lijnen
The Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
› Author Affiliations
Further Information

Publication History

Received 29 December 1992

Accepted after revision 05 April 1993

Publication Date:
05 July 2018 (online)

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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.

 

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