Inactivation of single-chain urokinase (pro-urokinase) by thrombin and
thrombin-like enzymes: relevance of the findings to the interpretation of
fibrin-binding experiments
V Gurewich and R Pannell
Whereas crude bovine thrombin activated single-chain urokinase-type
plasminogen activator (scu-PA), otherwise called pro-urokinase (pro- UK),
purified human thrombin converted pro-UK (scu-PA) to a two-chain form that
had no amidolytic activity. The two chains (Mr approximately 33,000 and
22,000) were disulfide linked and resistant to subsequent activation by
plasmin. By contrast, thrombin did not inactivate tissue plasminogen
activator or two-chain urokinase. The enzyme from snake venom Agkistrodon
contortrix, relatively specific for fibrinopeptide B, had an effect similar
to thrombin, whereas the enzyme from Agkistrodon rhodostoma (ancrod),
specific for fibrinopeptide A, did not. When pro- UK (scu-PA) was present
during thrombin clotting of fibrinogen, degradation of 125I-pro-UK (scu-PA)
in the clot supernatant was seen, whereas virtually full recovery (95%) of
radioactivity was found. A loss of latent amidolytic activity in the clot
supernatant was also found, the extent of which could be correlated with
the degree of degradation of the radiolabeled probe. It was concluded that
thrombin inactivation of pro-UK (scu-PA) accounts for the loss of
amidolytic activity in the clot supernatant, which has been attributed to
fibrin binding. Further confirmation was obtained from experiments in which
ancrod was used as the clotting agent. Full recovery of both radioactivity
and latent amidolytic activity of pro-UK (scu-PA) in the supernatant was
obtained under these conditions. These findings indicate that thrombin may
introduce an artifact in the results of certain experiments designed to
study the fibrin affinity or fibrinolytic effect of pro-UK (scu-PA).
Volume 69,
Issue 3,
pp. 769-772,
03/01/1987
Copyright © 1987 by The American Society of Hematology
