SELECTIVE SCREENING OF A LARGE PHAGE DISPLAY LIBRARY OF PLASMINOGEN-ACTIVATOR INHIBITOR-1 MUTANTS TO LOCALIZE INTERACTION SITES WITH EITHERTHROMBIN OR THE VARIABLE REGION-1 OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR

Phage display technology has been exploited to study in detail the int eraction between plasminogen activator inhibitor 1 (PAI-1) and either thrombin or an essential positively charged ''loop'' of tissue-type pl asminogen activator (t-PA), denoted variable region 1 (VR1). For this purpose, a PAI-1 mutant phage library was used that served as a reserv oir of PAI-1 proteins potentially deficient in the interaction with ei ther VR1 or thrombin. A stringent two-step selection procedure was dev eloped. (i) A negative selection was performed by incubating the pComb 3/PAI-1 mutant library with an excess of a thrombin mutant with its VR 1 domain substituted with that of t-PA (thrombin-VR1). (ii) The remain ing phages were complexed with t-PA (positive selection) and selected by panning with an immobilized anti-t-PA monoclonal antibody. Four con secutive panning rounds yielded an enrichment of pComb3/PAI-1 mutant p hages of similar to 50-fold. Sequence analysis of 16 different cDNAs, encoding PAI-1 mutants that are hampered in the binding to thrombin-VR 1, revealed the following mutations. Four independent variants share a mutation of the P4' residue (Glu(350) --> Lys). Nine independent PAI- 1 variants share a substitution of P1' (Met(347) --> Lys), whereas thr ee others share a P2 substitution (Ala(345) --> Asp). Kinetic analysis of representative PAI-1 mutants provides evidence that the P4' residu e is essential for the interaction with the VR1 domain, consistent wit h the data of Madison et al. (Madison, E. L., Goldsmith, E. J., Gethin g, M. J., Sambrook, J. F., and Gerard, R. D. (1990) J. Biol. Chem. 265 , 21423-21426), whereas the P1' and P2 residues confer thrombin specif icity. Concordant with the design of the selection procedure, mutants were obtained that inhibit thrombin-VR1 at least 100-fold slower than wild-type PAI-1, identifying residues that are central to the interact ion with either thrombin or VR1. This study demonstrates that phage te chnology can be used to analyze large numbers of mutants defective in their interaction with other (domains of) proteins, provided an adequa te selection scheme is devised.