INTERACTION OF HISTIDINE PROLINE-RICH GLYCOPROTEIN WITH PLASMINOGEN - EFFECT OF LIGANDS, PH, IONIC-STRENGTH, AND CHEMICAL MODIFICATION

The association of plasma histidine-proline-rich glycoprotein (HPRG) with plasminogen (PLG) was examined using a sucrose density gradient assay in order to evaluate the effects of several relevant conditions on complex formation. Addition of PLG shifts the S-value of I-125-labeled HPRG from 4.8S to 6.8S, providing the first direct evidence that HPRG associates with the zymogen form of plasmin in solution. Complex formation is not sensitive to pH in the range of pH 6.5-8.5, but is abolished at high ionic strength (1 M NaCl). No species differences were found, as either rabbit or human HPRG bound readily to rabbit or human PLG. Of the Ligands of HPRG tested, mesoheme (20 mu M) but not heparin (M(r) 10 000, 10 mu M) inhibits the formation of the HPRG-PLG complex. Modification of lysine residues of HPRG or competitive binding by lysine and anti-fibrinolytic agents containing primary amino groups also inhibits association. Conversely, modification of arginine or histidine residues of HPRG has no effect on complex formation. These results indicate that HPRG has independent binding sites for heparin and PLG and confirm that one or more lysine residues of HPRG are involved in its recognition by PLG. The protein-protein interaction was also quantitatively characterized at thermodynamic equilibrium by analytical ultracentrifugation. The stoichiometry and dissociation constant (K-D) of the complex were determined from the equilibrium distribution of fluorescein-isothiocyanate-labeled PLC in the presence of HPRG. The experimental data were analyzed by nonlinear least-squares curve fitting and indicated that a heterodimer is formed. The binding is characterized by a K-D of 0.75 mu M, and heme (30 mu M) and epsilon-aminocaproic acid (5 mM) decrease the apparent affinity of the interaction by 12- and 4-fold, respectively. The temperature dependence of the association was also examined from 4 to 25 degrees C. A van't Hoff plot of the data was linear and yielded a small negative change in enthalpy (-7.6 kcal/mol) and a positive change in entropy (2.7 cal/mol . deg), suggesting that electrostatic forces play a prominent role in the interaction of HPRG with PLG.