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

Abstract

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, = 30,000-40,000 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 plasminogen activating properties of both molecular forms of plasminogen activator are similar.The two molecular forms adsorbed to a similar extent to purified fibrin clots: 50% binding occurred at a concentration of about 0.14 mg fibrin/ml.Plasma clots immersed in mixtures of whole human plasma and '261-labeled one-chain plasminogen activator (150 ng/ml) dissolved slowly (approximately 50% lysis in 5 h).Sodium dodecyl sulfate-gel electrophoresis revealed that during the fibrinolytic process the plasminogen activator bound to the fibrin clot consisted almost exclusively of a two-chain form, in contrast to the plasminogen activator in the surrounding plasma.This suggested that one-chain plasminogen activator is quickly converted to a two-chain form on the fibrin surface and therefore that physiological fibrinolysis induced by native one-chain plasminogen activator nevertheless occurs mainly via a two-chain derivative.However, at present this conversion does not seem to play a role in the regulation of fibrinolysis. *-ls-', respectively.These findings suggest that the Ondenoeksacties (project 80-85/3).The costs of