PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 INTERACTS EXCLUSIVELY WITH THEPROTEINASE 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, w ith either recombinant human two-chain tissue plasminogen activator, t c 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) f or binding of PAI-1 was found for the three tPA forms by direct detect ion of the complex formation in real time by surface plasmon resonance , BIAcoreTM, or indirectly by monitoring the time course of the inhibi tion of tPA using the chromogenic substrate N-methylsulfonyl-D-Phe-Gly -Arg-4-pNA-acetate. Apparently, no conformational change is involved i n the rate-limiting step, since the k(on) value was found to be indepe ndent of the temperature from 20 to 35 degrees C. By the BIAcoreTM tec hnique, it was found that the complex between PAI-1 and tPA covalently coupled to the surface, was stable at 25 degrees C, since no dissocia tion was seen in buffer. However, extended treatment with 1 M NH4OH de stroyed the complex with t(1/2) = 5 h. The same k(on) values and compl ex composition were found by measuring either the binding of tPA to PA I-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 anti body 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 a nd the specific activity of PAI-1. The complexes of the three tPA form s with PAI-1 captured on a large surface of MAI-11 dissociated more ra pidly 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 o f 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 com plex with tPA than for free active PAI-1. Apparently, upon complex for mation, 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 int eraction between tPA and PAI-1 and the data from both methods show tha t all relevant interaction sites in tPA are present in the P domain.