ROLES OF INDIVIDUAL KRINGLE DOMAINS IN THE FUNCTIONING OF POSITIVE AND NEGATIVE EFFECTORS OF HUMAN PLASMINOGEN ACTIVATION

In order to identify the indivudal contributions of the kringle (K) domains of human plasminogen (Pg) to the epsilon-aminocaproic acid (EACA) induced stimulation of Pg activation by low-molecular-weight urokinase-type plasminogen activator (LMW-uPA) and inhibition of this same activation by Cl-, we constructed the most conservative recombinant- (r-) Pg mutants possible that would greatly reduce the strength of the EACA binding site in the omega-amino acid binding kringles, [K1(Pg)]([D-139-->N]r-Pg), [K4(Pg)] ([D-413-->N]r-Pg), and [K5(Pg)]([D-518-->N]r-Pg). In each case, this involved mutation of a critical Asp (to Asn) within these three kringle domains in intact Pg. The three r-mutants were expressed in r-baculovirus-infected lepidopteran insect (Trichoplusia ni) cells. In the presence of Cl-, the positive activation effector, EACA, first stimulated and then inhibited the LMW-uPA-catalyzed initial activation of wild-type (wt) r-[Glu(1)]Pg and, to a lesser extent, the [K5(Pg)] mutant, [D-518-->N/Glu(1)]r-Pg. The concentration of EACA that produced 50% stimulation of activation (C-50) occurred at 3.3 mM for wtr-[Glu(1)]Pg and at 0.7 mM for [D-518-N/Glu(1)]r-Pg. Subsequent inhibition by EACA occurred with a C-50 of approximately 15 mM and is likely due to inhibition of the amidolytic activity of plasmin generated during,o the activation. Similar initial activation rates of both [D-139-->N]r-Pg and [(DN)-N-413]r-Pg did not display this initial EACA-mediated stimulatory phase but did undergo ultimate inhibition with a C-50 for this process that was similar to wtr-[Glu(1)]Pg and [D-518-->N/Glu(1)]r-Pg. The initial activation rates of both wtr-[Glu(1)]Pg and, to a lesser degree, [D-518-->N/Glu(1)]r-Pg were inhibited by Cl-, but those same rates of [D-139-->N]r-Pg and [D-413-->N]r-Pg were not inhibited by this anion. These results demonstrate that both K1 and K4 contain functionally relevant binding sites for the activation effecters, EACA and Cl-, respectively, and that the integrity of both of these Ligand binding sites is required to produce the EACA-induced stimulation and Cl--mediated inhibition of [Glu(1)]Pg activation. On the other hand, the binding site of omega-amino acids on K5 is not of similar importance, since its elimination only partially affects the influence of these effecters on Pg activation.