Inactivation of single-chain urokinase (pro-urokinase) by thrombin and thrombin-like enzymes: relevance of the findings to the interpretation of fibrin-binding experiments.
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).
D000721:Ancrod / Q000494:pharmacology; D001777:Blood Coagulation; D003435:Crotalid Venoms / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005189:False Positive Reactions; D005337:Fibrin / Q000378:metabolism*; D005340:Fibrinogen / Q000494:pharmacology; D005341:Fibrinolysin / Q000494:pharmacology; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015849:Plasminogen Inactivators; D011489:Protein Denaturation; D013917:Thrombin / Q000494:pharmacology*; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000378:metabolism
C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D015849:Plasminogen Inactivators; D005337:Fibrin; D005340:Fibrinogen; D000721:Ancrod; D010960:Plasminogen Activators; D013917:Thrombin; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator