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 (Kd) and the maximum number of binding sites (Bmax) were derived from linearized expression of the Langmuir isotherm equation. The Kd for the binding of Glu-plasminogen to intact fibrin was 0.99 +/- 0.17 microM and for degraded fibrin was 0.66 +/- 0.22 microM. The Kd for the binding of Lys-plasminogen to intact fibrin was 0.41 +/- 0.22 microM and for degraded fibrin was 0.51 +/- 0.12 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
Fleury
V
V
Institut National de la Santé et de la Recherche Médicale, Hôpital de Bicêtre, France.
Anglés-Cano
E
E
D000906:Antibodies; D001665:Binding Sites; D004586:Electrophoresis; D005337:Fibrin / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D010958:Plasminogen / Q000378:metabolism*; D011869:Radioligand Assay
D000906:Antibodies; D005971:Glutamates; D018698:Glutamic Acid; D005337:Fibrin; D010958:Plasminogen; D008239:Lysine