Fibrinolytic properties of one-chain and two-chain human extrinsic (tissue-type) plasminogen activator.

The fibrinolytic properties of two molecular forms of extrinsic (tissue-type) plasminogen activator, purified from human melanoma cells in culture, were compared. One form, obtained under protection of aprotinin, consisted of a single polypeptide chain with M, = 72,000 while the other form, obtained without aprotinin, consisted of two polypeptide chains with M, = 30,00040,000 each. The two forms had the same fibrinolytic activity (clot lysis time) in a purified system composed of fibrin and plasminogen, and both forms dissolved ‘261-fibrinogenlabeled plasma clots immersed in whole plasma at very similar rates. However, sodium dodecyl sulfate-gel electrophoresis revealed that ’“I-labeled one-chain plasminogen activator was converted into a two-chain form during the lysis of a purified fibrin clot. Therefore, the kinetic parameters of the activation of native (NH2 terminus Glu) plasminogen were determined in a system containing aprotinin (1,000 kallikrein inhibitor units/ml), which prevented the conversion of the onechain form into the two-chain form. The rate of plasmin formation was measured by using ‘2SI-labeled plasminogen and by quantitation of the plasmin B-chain by sodium dodecyl sulfate-gel electrophoresis. In the presence of fibrin (1 mg/ml) one-chain plasminogen activator had an apparent Michaelis constant of 2.42 PM and a catalytic rate constant of 0.22 s-’ corresponding to a second-order rate constant of 89 n”‘s-’. These kinetic parameters for the two-chain form were 1.07 PM, 0.10 s” and 94 11“‘s-l. In the absence of fibrin the apparent Michaelis constants were larger than 100 PM and the second-order rate constants 0.32 and 0.36