EPITOPES ON PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 IMPORTANT FOR BINDING TO TISSUE-PLASMINOGEN ACTIVATOR

The molecular details of the rapid complex formation between tissue pl asminogen activator (tPA, E.C, 3.4.21.68) and plasminogen activator in hibitor type-1 (PAL-1) are still not fully elucidated. We have used su rface plasmon resonance (SPR), the BIAcore(TM), to characterize the bi nding of a large panel of monoclonal antibodies to four forms of recom binant human PAI-1, including active and latent PAL-1 as well as the c omplex between PAI-1 and recombinant human tc tPA or the protease part of tPA, the B-chain. Antibodies that discriminate between these diffe rent forms of PAI-1 have been identified, which is reflected by differ ences in k(a), k(d) as well as in K-d. In addition, in a chromogenic a ssay with PAL-1 and tPA we determined the IC50-values for these antibo dies, i.e., studied their ability to inhibit the decrease in tPA-activ ity caused by PAL-1. In a competition assay using SPR, we, have also b een able to study whether concurrent binding of these antibodies to PA L-1 was possible. We could thereby assign the antibodies to five group s according to their binding areas. Furthermore, by using this techniq ue, we have for the first time been able to identify three distinct ep itopes on PAI-1, which are all of importance for the interaction and c omplex-formation with tPA. Since the antibodies that bind to one of th ese areas all have very poor affinity for the complex between PAI-1 an d tPA, we suggest that this nor previously described epitope must be l ocated near the final binding site for tPA in this complex. Altogether , this also supports the theory of a multistep reaction between PAL-1 and tPA, in which tPA interacts with different parts of the PAI-l-mole cule. (C) 1997 Elsevier Science B.V.