The specific roles of finger and kringle 2 domains of tissue-type plasminogen activator during in vitro fibrinolysis.
The specific roles of the finger (F) and kringle 2 (K2) domains of tissue-type plasminogen activator (t-PA) were quantified with regard to fibrin binding and kinetic parameters for plasminogen activation by employing domain-deletion variants. On an intact fibrin clot, active site-blocked 125I-t-PA has a dissociation constant (Kd) of 0.36 +/- 0.08 microM and a single binding site per fibrin monomer (n = 1.1 +/- 0.1). Deletion of the K2 domain results in a 3-fold increase of the Kd (1.1 +/- 0.2 microM) and a 2-fold increase of the binding sites per fibrin monomer (n = 2.0 +/- 0.3). Deletion of the F domain results in nonsaturable binding with high Kd (3.2 +/- 0.6 microM). These results indicate that the high affinity binding of t-PA to intact fibrin is the resultant of the cooperation of two low affinity binding sites assembled on intact t-PA. Furthermore, fibrin clot lysis experiments were performed, using polymerized fibrin and plasminogen. Enzymatic activity of t-PA (variants) was assessed by following the decrease in turbidity of the polymerized fibrin. Intact recombinant t-PA exhibited a Michaelis constant for plasminogen activation (Km) of 37 +/- 2 nM. Deletion of either the K2 or F domain results in an increase of the Km for plasminogen of 4- and 16-fold, respectively. We interpret these kinetic parameters in terms of the ternary complex model: binding of t-PA to fibrin, mediated simultaneously by both the F and K2 domain, is essential for a correct orientation of the enzyme on the fibrin polymer to yield the optimal "Km-driven" stimulation of fibrin on the activation of plasminogen. The different potencies of various deletion mutants in a plasma clot are explained by decreased affinity of the enzymes both for fibrin and for the substrate plasminogen.
Horrevoets
A J
AJ
Department of Biochemistry, University of Amsterdam, Academic Medical Center, The Netherlands.
Smilde
A
A
de Vries
C
C
Pannekoek
H
H
D001665:Binding Sites; D002460:Cell Line; D005337:Fibrin / Q000378:metabolism; D005342:Fibrinolysis / Q000502:physiology*; D005353:Fibronectins / Q000502:physiology*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D018082:Kringles / Q000502:physiology*; D010959:Tissue Plasminogen Activator / Q000737:chemistry*
D005353:Fibronectins; D005337:Fibrin; D010959:Tissue Plasminogen Activator