Summary
The relative contribution of plasmin, miniplasmin, PMN-elastase and cathepsin G to
the fibrin-gel dissolution is studied. The global kcat/KM ratios are determined as a measure of the fibrinolytic catalytic efficiency using
spectrophotometric kinetic analysis of the competition between fibrin and synthetic
peptide substrates for the proteases, turbi-dimetric assay for fibrin dissolution
and gel-filtration of the partially degraded fibrin. When the substrate is fibrin
polymerized in the presence of 3 mM Ca2+, the value of this ratio is 4.3 × 105 M-1·s-1 for plasmin, 1.9 × 105 M-1·s-1 for miniplasmin, 5.0 × 104 M-1·s-1 for PMN-elastase and 2.2 × 103 M-1·s-1 for cathepsin G. When fibrin is polymerized without addition of Ca2+, the kcat/KM values are increased by a factor of 2.3 for plasmin, 2.0 for miniplasmin and 1.6
for cathepsin G, whereas that of PMN-elastase is unchanged. Progressive crosslinking
of fibrin decreases the catalytic action of all studied proteases, but no change in
their relative contribution to fibrinolysis is observed. When plasmin inhibitor (at
physiological concentration) is also crosslinked to fibrin, the most efficient fibrinolytic
enzymes are miniplasmin and PMN-elastase. The effect of 6-aminohexanoate on the formation
of fibrin degradation products by plasmin and miniplasmin suggests that the high-affinity
lysine binding site in the N-terminal kringle domain of plasmin is involved in the
interactions with the native polymerized fibrin, whereas the fifth kringle found in
both enzymes participates in binding to newly exposed lysine residues. These results
provide a quantitative basis for the evaluation of fibrinolytic efficiency and support
the concept of synergistic fibrinolysis.
