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

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 varian ts of rPlg was constructed, in which progressively up to 6 basic amino acid residues in the hinge region of rPlg between the NH2-terminal ac idic 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 suscepti ble, followed by Lys(62) and Lys(77). A variant with all 6 basic resid ues substituted was cleaved at Lys(20). Three of these variants, PlgB (R68A, R70A), PlgF (R68A, R70A, K77H, K78H), and PlgG (R61A, K62A, R68 A, R70A, K77H, K78H), as well as rPlg, were analyzed in more detail. T he conformation of these plasminogens was analzed by monitoring the ch ange in intrinsic fluorescence upon binding of lysine analogs. This re vealed that rPlg exhibits the native tight Glu(1)-plasminogen, conform ation, whereas PlgB, PlgF, and Plg G display an open confirmation simi lar to Lys(78)-plasminogen, leading to an increased affinity for lysin e analogs. This allowed a direct study of the impact of the activation -resistant conformation on the properties of Glu(1)-plasminogen. The o pen conformation of rPlg variants leads to an increased rate of activa tion by urokinase-type plasminogen activator and streptokinase and inc reased binding to a fibrin clot. Fibrin clot lysis mediated by tissue- type plasminogen activator was accelerated for the variants as a resul t of a lower K-m for tissue-type plasminogen activator-mediated plasmi nogen activation, resulting from the increased affinity of rPlg (varia nts) for intact fibrin. We conclude that the basic residues in the ext remely plasmin susceptible hinge region of plasminogen are directly in volved in maintaining the activation resistant Glu(1)-plasminogen conf ormation.