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Activation of human plasminogen by staphylokinase. Direct evidence that
preformed plasmin is necessary for activation to occur
DK Grella and FJ Castellino
Department of Chemistry and Biochemistry, University of Notre Dame, IN
46556, USA.
To directly determine whether the mechanism of activation of human
plasminogen (HPg) by staphylokinase (Sak) required formation of an active
complex of Sak and HPg, recombinant (r) variants of HPg were examined that
allowed dissection of the steps involved in this activation. The rate of
activation of wild-type (wt) r-HPg by equimolar levels of Sak was enhanced
when small amounts of human plasmin (HPm) were included, suggesting that a
Sak-HPm complex was a more effective plasminogen activator than a putative
Sak-HPg complex. Incubation of equimolar Sak with a cleavage site resistant
mutant of HPg (r-[R581 A]HPg) did not result in generation of amidolytic
activity of the complex, in contrast to a similar experiment with
streptokinase (SK) in place of Sak, where substantial amidolytic activity
was generated. This result supplies evidence that an active complex of Sak
and HPg does not form, as is the case with SK. Another mutant, r-[D646
E]HPg, which, upon activation, would lead to a form of HPm defective in
enzymatic activity, is also not converted to its two-chain form by Sak, but
is converted to the inactive two-chain form by urokinase, a direct
plasminogen activator, and by equimolar complexes of SK or Sak with wtr-
HPm. This shows that the active site of HPm is the functional plasminogen
activator entity in the Sak-HPm complex. These results show that the
mechanism of activation of HPg by Sak proceeds in a distinctly different
manner than the similar activation by SK. Although SK does not require the
presence of HPm for this activation, a necessary condition for the
activation by Sak is formation of a small amount of HPm generated via
another activation pathway. These different mechanisms have significant
implications in production of the fibrinolytic state by these two indirect
bacterial plasminogen activators.
Volume 89,
Issue 5,
pp. 1585-1589,
03/01/1997
Copyright © 1997 by The American Society of Hematology
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