Epitopes on plasminogen activator inhibitor type-1 important for binding to tissue plasminogen activator.
The molecular details of the rapid complex formation between tissue plasminogen activator (tPA, E.C. 3.4.21.68) and plasminogen activator inhibitor type-1 (PAI-1) are still not fully elucidated. We have used surface plasmon resonance (SPR), the BIAcore, to characterize the binding of a large panel of monoclonal antibodies to four forms of recombinant human PAI-1, including active and latent PAI-1 as well as the complex between PAI-1 and recombinant human tc tPA or the protease part of tPA, the B-chain. Antibodies that discriminate between these different forms of PAI-1 have been identified, which is reflected by differences in k(a), k(d) as well as in Kd. In addition, in a chromogenic assay with PAI-1 and tPA we determined the IC50-values for these antibodies, i.e., studied their ability to inhibit the decrease in tPA-activity caused by PAI-1. In a competition assay using SPR, we have also been able to study whether concurrent binding of these antibodies to PAI-1 was possible. We could thereby assign the antibodies to five groups according to their binding areas. Furthermore, by using this technique, we have for the first time been able to identify three distinct epitopes on PAI-1, which are all of importance for the interaction and complex-formation with tPA. Since the antibodies that bind to one of these areas all have very poor affinity for the complex between PAI-1 and tPA, we suggest that this not previously described epitope must be located near the final binding site for tPA in this complex. Altogether, this also supports the theory of a multistep reaction between PAI-1 and tPA, in which tPA interacts with different parts of the PAI-1-molecule.
Björquist
P
P
Biochemistry, Preclinical R and D, Astra Hässle AB, Mölndal, Sweden.
Ehnebom
J
J
Inghardt
T
T
Deinum
J
J
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D001667:Binding, Competitive; D000939:Epitopes / Q000032:analysis* / Q000276:immunology; D007700:Kinetics; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000276:immunology*; D010959:Tissue Plasminogen Activator / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D017395:Plasminogen Activator Inhibitor 1; D010959:Tissue Plasminogen Activator