On The Fibrinolytic Properties Of Single-Chain And Two- Chain Human Tissue Plasminogen Activator

Tissue plasminogen activator from pig hearts may be isolated as a single-chain or as a two-chain molecule (Wallén et al., 1980). The present report deals with the two molecular forms of a plasminogen activator (tissue plasminogen activator-like) secreted by human melanoma cells in culture. A single-chain form was prepared by addition of aprotinin to the culture media and during the purification procedure while a two-chain form or a mixture of both was obtained in the absence of aprotinin.The fibrinolytic activities of the two forms were comparable on fibrin plates and in a clot lysis time system. Analysis of the molecular structure of 125I-labeled plasminogen activator by dodecyl-sulfate-gel electrophoresis, revealed that the single-chain form was converted into a two- chain form during the lysis of fibrin. The plasminogen activating properties of the two molecular forms were therefore measured in the presence of aprotinin, which prevents the conversion. Aprotinin (1,000 KIU/ml) was incorporated in a fibrin clot (1 mg/ml) containing plasminogen activator (20 ng/ml), 125I-labeled Glu-plasminogen and varying amounts of unlabeled Glu-plasminogen. The amount of plasmin formed after 30 min was quantitated by measuring the radioactivity migrating in the position of the plasmin B-chain on dodecyl-sulfate-gel electrophoresis under reducing conditions. The rate of plasmin formation obeyed Michaelis-Menten kinetics with Km = 2.5 μM and kcat = 0.6 s-1 for the single-chain form and Km = 1.1 μM and kcat = 0.3 s-1 for the two-chain form.Although the conversion of the single-chain tissue plasminogen activator into a two-chain form during fibrinolysis might have a regulatory function, these kinetic parameters of the plasminogen activation do not support this hypothesis.