CHARACTERIZATION OF THE BINDING OF PLASMINOGEN TO FIBRIN SURFACES - THE ROLE OF CARBOXY-TERMINAL LYSINES

In the present study we have quantitatively characterized the interaction of purified human Glu- and Lys-plasminogen with intact and degraded fibrin by ligand-binding experiments using a radioisotopic dilution method and antibodies against human plasminogen. A fibrinogen monolayer was covalently linked to a solid support with polyglutaraldehyde and was treated with thrombin or with thrombin and then plasmin to respectively obtain intact and degraded fibrin surfaces. Under these conditions, a well-defined surface of fibrin is obtained (410 +/- 4 fmol/cm2) and, except for a 39-kDa fragment, most of the fibrin degradation products remain bound to the support. New binding sites for plasminogen were detected on the degraded surface of fibrin. These sites were identified as carboxy-terminal lysine residues both by inhibition of the binding by the lysine analogue 6-aminohexanoic acid and by carboxy-terminal end-group digestion with carboxypeptidase B. The binding curves exhibited a characteristic Langmuir adsorption isotherm saturation profile. The data were therefore analyzed accordingly, assuming a single-site binding model to simplify the analysis. Equilibrium dissociation constants (K(d) and the maximum number of binding sites (B(max)) were derived from linearized expression of the Langmuir isotherm equation. The K(d) for the binding of Glu-plasminogen to intact fibrin was 0.99 +/- 0.17-mu-M and for degraded fibrin was 0.66 +/- 0.22-mu-M. The K(d) for the binding of Lys-plasminogen to intact fibrin was 0.41 +/- 0.22-mu-M and for degraded fibrin was 0.51 +/- 0.12-mu-M. The K(i) for the inhibition of the binding by 6-aminohexanoic acid was 130 +/- 15-mu-M on intact fibrin and 109 +/- 18-mu-M on degraded fibrin. The total number of binding sites markedly increased upon degradation of the fibrin surface by plasmin: approximately 0.1 pmol of plasminogen was bound to intact fibrin while approximately 1 pmol was detected on the degraded surface. The plasminogen/fibrin ratio increased from 0.3 to 2.7 upon plasmin degradation. These data provide quantitative evidence of the amplification of fibrinolysis by carboxy-terminal lysine residues unveiled by plasmin on the surface of fibrin and support the concept of fibrinolysis as a surface-controlled process.