Oxidative inactivation of plasmin and other serine proteases by copper and
ascorbate
SE Lind, JR McDonagh and CJ Smith
Experimental Medicine Division, Brigham and Women's Hospital, Boston, MA
02115.
Fibrin thrombi form at sites of injury, where leukocytes release a variety
of oxidants. To determine whether oxidants might affect proteins of the
fibrinolytic system, we examined the effects of various oxidants on
plasmin. Plasmin was not inhibited by micromolar concentrations of
hypochlorous acid, chloramine T, or H2O2. Neither Fe nor Cu affected
plasmin alone or in the presence of H2O2. However, incubation of plasmin
with 5 mumol/L Cu(I or II) in the presence of the reducing agent ascorbic
acid resulted in a loss of its hydrolytic activity towards proteins as well
as towards small synthetic substrates. The addition of EDTA, but not
mannitol, prevented its inactivation. Inactivation was prevented by the
addition of catalase and accelerated by hydrogen peroxide. Preincubation of
plasmin with the competitive inhibitor alpha-N-acetyl-L-lysine methyl ester
prevented inactivation by Cu(II) and ascorbate. These results together
suggest site-specific oxidation of plasmin's active site. Treatment of the
plasminogen activators tissue plasminogen activator and two-chain
urokinase-type plasminogen activator, as well as trypsin, neutrophil
elastase, and thrombin with Cu(II) and ascorbate resulted in a loss of
their amidolytic and proteolytic activity, indicating the general
susceptibility of serine proteases to this type of oxidation. Oxidation of
the zymogens Glu-plasminogen and single-chain urokinase-type plasminogen
activator by Cu(II) and ascorbate resulted in the failure of these
molecules to generate active enzymes when treated with plasminogen
activators or plasmin, respectively. The active site His residue may be the
target of oxidative inactivation, as evidenced by the partial protection
afforded plasmin by the addition of Zn(II), histidine, or the platinum
derivative, platinum(II) (2,2':6',2"- terpyridine) chloride. Because
platelets contain micromolar concentrations of Cu and leukocytes are rich
in ascorbate, Cu-dependent site-specific oxidation might play a role in
modulating proteolytic events and the life span of thrombi formed at sites
of tissue injury.
Volume 82,
Issue 5,
pp. 1522-1531,
09/01/1993
Copyright © 1993 by The American Society of Hematology
