The activation of human plasminogen (P) by two-chain tissue plasminogen activator (A) was studied in the presence of fibrin films (F) of increasing size and surface density. Initial rates of plasminogen activation (v) were determined as a function both of the plasminogen and fibrin concentration. The activation rate was strongly dependent on the presence of fibrin and plots of 1/v versus 1/ [p] or 1 /[F] yielded straight lines. The kinetic data were in agreement with the following reaction scheme.According to this model tissue plasminogen activator would bind to fibrin with a dissociation constant (KF of 0.2 µM and this complex fixes plasminogen with a Michaelis constant (Kp’) of 0.15 µM (Glu-plasminogen) or 0.02 µM (Lys-plasminogen) to form a ternary complex, converted to plasmin with a catalytic rate constant kcat = 0.05 s-1. This mechanism implies that both plasminogen and tissue plasminogen activator are concentrated on the fibrin surface through formation of a fibrin bridge. Activation of plasminogen in the absence of fibrin occurs with Km = 65 µM (Glu-plasminogen) or Km= 19 µM (Lys-plasminogen) and kcat = 0.05 s-1. Our data suggest that fibrin enhances the activation rate of plasminogen by tissue plasminogen activator by increasing the affinity of plasminogen for fibrin-bound tissue plasminogen activator and not by influencing the catalytic efficiency of the enzµMe. These data also support the hypothesis that fibrinolysis is both triggered by and directed towards fibrin.Generated plasmin was quantitated by measuring the rate of solubilization of 125I-labeled fibrin.

Tissue plasminogen activator from pig hearts may be isolated as a single-chain or as a two-chain molecule (Wallén et al., 1980). The present report deals with the two molecular forms of a plasminogen activator (tissue plasminogen activator-like) secreted by human melanoma cells in culture. A single-chain form was prepared by addition of aprotinin to the culture media and during the purification procedure while a two-chain form or a mixture of both was obtained in the absence of aprotinin.The fibrinolytic activities of the two forms were comparable on fibrin plates and in a clot lysis time system. Analysis of the molecular structure of 125I-labeled plasminogen activator by dodecyl-sulfate-gel electrophoresis, revealed that the single-chain form was converted into a two- chain form during the lysis of fibrin. The plasminogen activating properties of the two molecular forms were therefore measured in the presence of aprotinin, which prevents the conversion. Aprotinin (1,000 KIU/ml) was incorporated in a fibrin clot (1 mg/ml) containing plasminogen activator (20 ng/ml), 125I-labeled Glu-plasminogen and varying amounts of unlabeled Glu-plasminogen. The amount of plasmin formed after 30 min was quantitated by measuring the radioactivity migrating in the position of the plasmin B-chain on dodecyl-sulfate-gel electrophoresis under reducing conditions. The rate of plasmin formation obeyed Michaelis-Menten kinetics with Km = 2.5 μM and kcat = 0.6 s-1 for the single-chain form and Km = 1.1 μM and kcat = 0.3 s-1 for the two-chain form.Although the conversion of the single-chain tissue plasminogen activator into a two-chain form during fibrinolysis might have a regulatory function, these kinetic parameters of the plasminogen activation do not support this hypothesis.

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SummaryStaphylokinase (Sak), a 15.5-kDa bacterial protein, forms a complex with human plasmin, which in turn activates other plasminogen molecules to plasmin. Three recombinant DNA-based approaches, (i) site directed substitution with alanine, (ii) search for proximity relationships at the complex interface, and (iii) active-site accessibility to protease inhibitors have been used to deduce a coherent docking model of the crystal structure of Sak on the homology-based model of micro-plasmin (μPli), the serine protease domain of plasmin. Sak binding on μPli is primarily mediated by two surface-exposed loops, loops 174 and 215, at the rim of the active-site cleft, while the binding epitope of Sak on μPli involves several residues located in the flexible NH2-terminal arm and in the five-stranded mixed β-sheet. Several Sak residues located within the unique μ-helix and the β2 strand do not contribute to the binding epitope but are essential to induce plasminogen activating potential in the Sak:μPli complex. These residues form a topologically distinct activation epitope, which, upon binding of Sak to the catalytic domain of μPli, protrudes into a broad groove near the catalytic triad of μPli, thereby generating a competent binding pocket for micro-plasminogen (μPlg), which buries approximately 2500 Å of the Sak:μPli complex upon binding. This structural and functional model may serve as a template for the design of improved Sak-derived thrombolytic agents. Following the completion and presentation of the present study, the deduced Sak:μPli:μPlg complex was fully confirmed by X-ray crystallography, which further illustrates the power and potential of the present approach.

Plasminogen activators convert the proenzyme plasminogen to the active serine protease plasmin by hydrolysis of the Arg^560-Val^561 peptide bond. Physiological plasminogen activation is however regulated by several additional molecular interactions resulting in fibrin-specific clot lysis. Tissue-type plasminogen activator (t-PA) binds to fibrin and thereby acquires a high affinity for plasminogen, resulting in efficient plasmin generation at the fibrin surface. Single-chain urokinase-type plasminogen activator (scu-PA) activates plasminogen directly but with a catalytic efficiency which is about 20 times lower than that of urokinase. In plasma, however, it is inactive in the absence of fibrin. Chimeric plasminogen activators consisting of the NH(2)-terminal region of t-PA (containing the fibrin-binding domains) and the COOH-terminal region of scu-PA (containing the active site), combine the mechanisms of fibrin specificity of both plasminogen activators. Combination of t-PA and scu-PA infusion in animal models of thrombosis and in patients with coronary artery thrombosis results in a synergic effect on thrombolysis, allowing a reduction of the therapeutic dose and elimination of side effects on the hemostatic system.