Abstract
Incubation of HTC rat hepatoma cells with the synthetic glucocorticoid dexamethasone rapidly inhibits plasminogen activator (PA) activity secondary to the induction of a specific acid-stable inhibitor of plasminogen activation (Cwikel, B. J., Barouski-Miller, P.A., Coleman, P.L., and Gelehrter, T.D. (1984) J. Biol. Chem. 259, 6847-6851). We have further characterized this inhibitor with respect to its interaction with both urokinase and tissue plasminogen activator, and its protease specificity. The HTC PA inhibitor rapidly inhibits urokinase and tissue plasminogen activator with an apparent second-order rate constant of 3-5 x 10(7) M-1 X s-1. The inhibitor forms stable covalent complexes with both urokinase and tissue plasminogen activator, with which plasmin, trypsin, and factor Xa apparently do not compete. Complex formation is saturable and requires the active site of the PA. The mass of the inhibitor-PA complex is 50,000 daltons greater than that of PA alone, consistent with an Mr for the PA inhibitor of 50,000 as demonstrated directly by reverse fibrin autography. The HTC PA inhibitor does not inhibit thrombin and differs in its kinetic and biochemical properties from protease nexin.
Highlights
From the Departments of Internal Medicine and Human Genetics, University of Michigan Medical School,Ann Arbor, Michigan 48109-0618
The HTCPA inhibitor (PA-I)is an acidtheticglucocorticoiddexamethasonerapidlyinhibits stable, 50,000-daltonprotein found in cytosol and inmedium plasminogen activator (PA) activity secondary to the conditioned by dexamethasone-treated cells [2,3,4,5]
HTC PA-I differs from protease nexin in its acid biochemical properties from proteasneexin
Summary
Time Course of Inhibition of PA-The time course of the inhibition of uPA and tPA by HTC PA-I was studied over a 10-fold range of inhibitor concentrations. The half-times of inhibition, determined graphically (Fig. l),were used to calculate an apparent second-order rate constant (Table I) of about 5 X lo M-'.s-' for uPA, and 3.7 k 0.3 X IO7M".s-'. Theseresults suggested that HTC PA-I forms an enzymatically inactive complex with uPA. The possibility that this represented acovalent complex. The concentration of PA-I indexamethasone conditioned medium (DexCM) was defined as described under "Experimental Procedures." The tIhof inhibition was determined graphically as shown in Fig. 1. k' 0.693/t~
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