Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin.
The kinetics of the activation of Glu-plasminogen and Lys-plasminogen (P) by a two-chain form of human tissue plasminogen activator (A) were studied in purified systems, and in the presence of fibrinogen (f) and of fibrin films (F) of increasing size and surface density. The activation in the purified systems followed Michaelis-Menten kinetics with a Michaelis constant of 65 microM and a catalytic rate constant of 0.06 s-1 for Glu-plasminogen as compared to 19 microM 0.2 s-1 for Lys-plasminogen. In the presence of fibrinogen plots of 1/v versus 1/[P] or 1/v versus 1/[f] yielded straight lines with an apparent Michaelis constant at infinite [f] of 28 microM and a catalytic rate constant of 0.3 s-1 for Glu-plasminogen as compared to 1.8 microM and 0.3 s-1 for Lys-plasminogen. In the systems with fibrin, plasmin was estimated from the rate of release of 125I from 125I-labeled fibrin films. The initial rate of activation (v) was calculated and Lineweaver-Burk plots of 1/v versus 1/[P] or 1/v versus 1/[F] yielded straight lines. Activation occurred with an intrinsic Michaelis constant of 0.16 microM and a catalytic rate constant of 0.1 s-1 for Glu-plasminogen as compared to 0.02 microM and 0.2 s-1 for Lys-plasminogen. The kinetic analysis suggested that the activation in the presence of fibrin occurs through binding of an activator molecule to the clot surface and subsequent addition of plasminogen (sequential ordered mechanism) to form a cyclic ternary complex. The Low Michaelis constant in the presence of fibrin allows efficient plasminogen activation on a fibrin clot, while its high value in the absence of fibrin prevents efficient activation in plasma.
D004789:Enzyme Activation; D005337:Fibrin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D010958:Plasminogen / Q000378:metabolism*; D010960:Plasminogen Activators / Q000302:isolation & purification / Q000502:physiology*
D005337:Fibrin; D010958:Plasminogen; D010960:Plasminogen Activators