Zinc as a Cofactor for Heparin Neutralization by Histidine-rich Glycoprotein

Abstract

We have studied the ability of histidine-rich glycoprotein (HRG) to neutralize the anticoagulant activity of heparin in plasma and in a purified component clotting assay. Addition of HRG to plasma or to the purified component assay did not neutralize the anticoagulant activity of heparin unless micromolar concentrations of zinc were present. Higher zinc concentrations were required for citrated than for heparinized plasmas due to competition of citrate with HRG for zinc binding. Zinc concentrations as low as 1.25 M revealed HRG to be a powerful competitor of antithrombin for heparin in the purified component assays. HRG binding of heparin also was shown by affinity chromatography of HRG from immobilized heparin in the presence and absence of zinc. In the absence of zinc, HRG was eluted by 0.1 M NaCl, but, in the presence of zinc, elution of HRG required 1.0 M NaCl. Investigation of other divalent cations (copper and magnesium) indicated that augmentation of heparin binding by HRG in the presence of antithrombin was restricted to zinc. The HRG⅐Zn complex effectively competes with antithrombin for heparin, which restricts the availability of heparin to bind antithrombin and allows thrombin-mediated fibrinogenesis to proceed unimpeded. This could be initiated by zinc released from activated platelets.

Activated Platelets Provide a Functional Microenvironment for the Antiangiogenic Fragment of Histidine-Rich Glycoprotein

“…Proteolytic fragments of discrete mass are consistently found in purified fractions of HRG from human plasma (23). We have previously shown that one of these fragments, a 30-kDa peptide derived from the histidine-and proline-rich (His/Pro-rich) domain of HRG, mediates the antiangiogenic effect of HRG and that this peptide must be released from the full-length protein to exert its effect as an inhibitor of angiogenesis (18).…”

Characterization of Plasmin-mediated Activation of Plasma Procarboxypeptidase B

“…Batroxobin, goat anti-human Plg IgG, and mouse anti-␣ 2 -antiplasmin (␣ 2 AP) monoclonal IgG were from American Diagnostica, Inc. (Greenwich, CT). Human histidine-rich glycoprotein was purified from fresh citrated plasma as described elsewhere (9). Potato carboxypeptidase inhibitor (PCI), UH from porcine intestinal mucosa (grade II; 167 USP unit/mg), low molecular weight heparin (M r 3000), heparan sulfate (i.e.…”

The Anti-angiogenic His/Pro-rich Fragment of Histidine-rich Glycoprotein Binds to Endothelial Cell Heparan Sulfate in a Zn2+-dependent Manner

“…HRGP binds to heparin in a Zn 2ϩ -dependent manner (7,35). To investigate the contribution of the His/Pro-rich region to this interaction, we determined the heparin-binding properties of full-length HRGP and a truncated form corresponding to the N-terminal cystatin domains (HRGP1-240), i.e.…”

“…2ϩ is a known ligand for HRGP, and its importance for the binding of HRGP to heparin (7,35) H]heparin oligosaccharides were tested for their ability to interact with HRGP335. The peptide was incubated with equimolar amounts of 3 H-labeled oligosaccharides, and those bound to the peptide were trapped along with the peptide on a nitrocellulose filter (Fig.…”