PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 INTERACTS EXCLUSIVELY WITH THE PROTEINASE DOMAIN OF TISSUE-PLASMINOGEN ACTIVATOR

Two different techniques have been used to study the complex formation of recombinant human plasminogen activator inhibitor type-1, PAI-1, with either recombinant human two-chain tissue plasminogen activator, tc tPA (EC 3.4.21.68), or the tPA deletion variants tc K2P, containing the kringle 2 domain and the proteinase domain, and P, containing only the proteinase domain. The same value for k(on), 2.10(7) M(-1)s(-1) for binding of PAI-1 was found for the three tPA forms by direct detection of the complex formation in real time by surface plasmon resonance, BIAcoreTM, or indirectly by monitoring the time course of the inhibition of tPA using the chromogenic substrate N-methylsulfonyl-D-Phe-Gly-Arg-4-pNA-acetate. Apparently, no conformational change is involved in the rate-limiting step, since the k(on) value was found to be independent of the temperature from 20 to 35 degrees C. By the BIAcoreTM technique, it was found that the complex between PAI-1 and tPA covalently coupled to the surface, was stable at 25 degrees C, since no dissociation was seen in buffer. However, extended treatment with 1 M NH4OH destroyed the complex with t(1/2) = 5 h. The same k(on) values and complex composition were found by measuring either the binding of tPA to PAI-1 captured on the monoclonal antibody MAI-11 or the binding of PAI-1 to tPA captured on the monoclonal antibody 2:2 B10. Quantification of the complex composition between PAI-1 captured on the monoclonal antibody MAI-11 with either tPA, K2P or P gave a one-to-one ratio with the fraction of active PAI-1, consistent with the results from SDS-PAGE and the specific activity of PAI-1. The complexes of the three tPA forms with PAI-1 captured on a large surface of MAI-11 dissociated more rapidly from MAI-11, with the same apparent k(off), k(dis), = 2.10(-3)s(-1), compared with 0.7.10(-3)s(-1) for the dissociation of PAI-1 alone. In consistence, the k(d), calculated from the direct determination of the k(on) and k(off) for the association of different forms of PAI-1 to a small surface of MAI-11, was found to be higher for PAI-1 in complex with tPA than for free active PAI-1. Apparently, upon complex formation, a change is induced in PAI-1 at the binding epitope for MAI-11. In conclusion, we have used two independent methods to study the interaction between tPA and PAI-1 and the data from both methods show that all relevant interaction sites in tPA are present in the P domain.