The effects of fibrinogen and its cleavage products on the kinetics of plasminogen activation by urokinase and subsequent plasmin activity.

Abstract

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)