The effects of
fibrinogen and its plasmic cleavage fragments on the activation of Glu-, Lys-, and Val442-
plasminogen by
urokinase were investigated. A possible explanation for the large variations in the published steady state parameters for
Glu-plasminogen activation is the undetected formation of
Lys-plasminogen and its subsequent more rapid activation to
plasmin. When
Lys-plasminogen formation was avoided, the Km for
Glu-plasminogen activation by
urokinase was 2.5 microM with or without
lysine present and the catalytic rate constant (kcat) was 3.4 min-1 in the absence of
lysine, but increased to 49.0 min-1 in its presence. For
Lys-plasminogen activation, both the Km of 2.7 microM and the kcat of 57.8 min-1 were only slightly increased by
lysine. With Val442-plasminogen, the absence of the first 4 kringle structures of
Lys-plasminogen resulted in a 6-fold higher Km and a 3-fold higher kcat, both of which were relatively unchanged by
lysine. The specificity of
urokinase for Val442-plasminogen, as measured by the quotient kcat/Km was thus half that for
Lys-plasminogen.
Fibrinogen, Fragment D, and Fragment E enhanced the rate of activation of
Glu-plasminogen to
Glu-plasmin as measured by the irreversible binding of
plasmin to fluorescently labeled
bovine pancreatic trypsin inhibitor. Both
fibrinogen and Fragment D increased the value of kcat/Km about 4-fold whereas Fragment E caused a 2-fold enhancement. In contrast to
Glu-plasminogen activation, the
urokinase activation of
Lys-plasminogen was not affected by
fibrinogen or its fragments, yet a marked inhibition of
Lys-plasmin autolysis occurred in their presence, with the half-life of
plasmin being increased 13-fold by
fibrinogen, 5-fold by Fragment D, and 3-fold by Fragment E. The K4 kringle region may be particularly involved in the
plasmin-
plasmin interaction that results in
autolysis, since it significantly reduced degradation when incubated with
Lys-plasmin. Val442-plasmin displayed essentially no
autolysis, which further implicates the first 4 kringles in the autolytic reactions. In addition to these effects, the rate of
Glu-plasminogen conversion to
Lys-plasminogen by
plasmin was increased 4-fold by
fibrinogen or Fragment E, but only 2-fold by Fragment D. This augmentation was not merely due to inhibition of
Lys-plasmin autolysis since Fragment D has a greater effect in that regard. The sum of these interactions indicates that
Glu-plasminogen binds to the Fragment D region of
fibrinogen/
fibrin through its low affinity binding site(s) and, as when
lysine binds at these sites, the activation to
Glu-plasmin is then accelerated.(ABSTRACT TRUNCATED AT 400 WORDS)