Abstract
Histidine-proline-rich glycoprotein (HPRG), also known as histidine-rich glycoprotein, is a major plasminogen-binding protein. In this work we characterized extensively the circumstances under which HPRG accelerates plasminogen activation and the specificity of this effect. Soluble HPRG did not significantly influence plasminogen activation. In contrast, native HPRG bound to hydrazide or nickel chelate surfaces strongly stimulated the activation of plasminogen by tissue plasminogen activator, but not by urokinase or streptokinase. The efficiency of activation on surface-bound HPRG was increased for Glu-plasminogen (41-fold), Lys-plasminogen (17-fold), and cross-linked Glu-plasminogen (11-fold) but not for mini-plasminogen, and was mainly due to a decrease in the apparent Km. A reduced susceptibility to inhibition by chloride ions contributed to the higher activation rate of Glu-plasminogen on an HPRG surface. The immobilized N- and C-terminal domains, but not the histidine-proline-rich domain of HPRG, also bound plasminogen and stimulated its activation. HPRG-enhanced plasminogen activation was proportional to the quantity of HPRG immobilized and was abolished by anti-HPRG antiserum, by low concentrations of epsilon-aminocaproic acid, by methylation of lysine residues in HPRG, and by treatment of HPRG with carboxypeptidase B. Soluble HPRG and a plasminogen fragment, kringle 1-2-3, acted as competitive inhibitors by binding to plasminogen and immobilized HPRG, respectively. The interaction of the conserved C-terminal lysine of HPRG with the high affinity lysine binding site of plasminogen is necessary and sufficient to accelerate plasminogen activation. Unlike other stimulators of plasminogen activation, the effect of HPRG on fibrinolysis is modulated by factors that influence the equilibrium between solution and surface-bound HPRG.
Highlights
Plasmin, the primary enzyme of the fibrinolytic system, is formed from plasminogen by the action of plasminogen activators, such as tissue plasminogen activator (t-PA) or urokinase
Since the Histidine-proline-rich glycoprotein (HPRG)-plasminogen interaction does not appear to be species-specific, and since rabbit HPRG domains can be reproducibly prepared in large quantities by limited proteolysis with plasmin [14], we aimed to identify the conserved lysine residues of HPRG involved in the interaction with plasminogen
Since carboxypeptidase B (CPB) treatment abrogates plasminogen binding to the HPRG domains, the probable new plasminogen binding sites generated by plasmin digestion are Lys242 or Lys261 (Fig. 1), the putative C-terminal residue sites of the N-terminal domain [14]
Summary
The primary enzyme of the fibrinolytic system, is formed from plasminogen by the action of plasminogen activators, such as tissue plasminogen activator (t-PA) or urokinase. Proteins that interact with the LBS on plasminogen, most notably fibrin [4, 5] and proteins of the extracellular matrix [6] or cell surface [7], greatly stimulate plasminogen activation, and this effect is competitively inhibited by ⑀-aminocaproic acid (⑀-ACA) or other lysine analogues. Many of these effectors bind the plasminogen activator, thereby acting as a template that brings the enzyme and its substrate in close proximity. We investigated how immobilized HPRG accelerates plasminogen activation by comparing the activation rates of different plasminogen forms under various experimental conditions
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