THE ACTIVATION-RESISTANT CONFORMATION OF RECOMBINANT HUMAN PLASMINOGEN IS STABILIZED BY BASIC RESIDUES IN THE AMINO-TERMINAL HINGE REGION

A. J. Horrevoets, A. E. Smilde, J. C. Fredenburgh, H. Pannekoek, M. E. Nesheim

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Fully activable recombinant human plasminogen (rPlg) was expressed in mammalian cells employing either recombinant vaccinia virus or stable lines coexpressing alpha(2)-plasmin inhibitor. A panel of eight variants of rPlg was constructed, in which progressively up to 6 basic amino acid residues in the hinge region of rPlg between the NH2-terminal acidic domain (''proactivation peptide'') and kringle 1 were substituted by neutral residues. Analysis of the cleavage rates of these variants by plasmin revealed that the peptide bond at Arg(68) is most susceptible, followed by Lys(62) and Lys(77). A variant with all 6 basic residues substituted was cleaved at Lys(20). Three of these variants, PlgB (R68A, R70A), PlgF (R68A, R70A, K77H, K78H), and PlgG (R61A, K62A, R68A, R70A, K77H, K78H), as well as rPlg, were analyzed in more detail. The conformation of these plasminogens was analzed by monitoring the change in intrinsic fluorescence upon binding of lysine analogs. This revealed that rPlg exhibits the native tight Glu(1)-plasminogen, conformation, whereas PlgB, PlgF, and Plg G display an open confirmation similar to Lys(78)-plasminogen, leading to an increased affinity for lysine analogs. This allowed a direct study of the impact of the activation-resistant conformation on the properties of Glu(1)-plasminogen. The open conformation of rPlg variants leads to an increased rate of activation by urokinase-type plasminogen activator and streptokinase and increased binding to a fibrin clot. Fibrin clot lysis mediated by tissue-type plasminogen activator was accelerated for the variants as a result of a lower K-m for tissue-type plasminogen activator-mediated plasminogen activation, resulting from the increased affinity of rPlg (variants) for intact fibrin. We conclude that the basic residues in the extremely plasmin susceptible hinge region of plasminogen are directly involved in maintaining the activation resistant Glu(1)-plasminogen conformation
Original languageEnglish
Pages (from-to)15770-15776
JournalJournal of biological chemistry
Volume270
Issue number26
DOIs
Publication statusPublished - 1995

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