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 I-125-t-pA has a dissociation constant (K-d) of 0.36 +/- 0.08 mu M 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 K-d (1.1 +/- 0.2 mu M) 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 K-d (3.2 +/- 0.6 mu M). 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 (K-m) of 37 +/- 2 nM. Deletion of either the K2 or F domain results in an increase of the K-m 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. ''K-m-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