Histidine-rich glycoprotein binds fibrin(ogen) with high affinity and competes with thrombin for binding to the gamma'-chain

Abstract

Histidine-rich glycoprotein (HRG) is an abundant protein that binds fibrinogen and other plasma proteins in a Zn(2+)-dependent fashion but whose function is unclear. HRG has antimicrobial activity, and its incorporation into fibrin clots facilitates bacterial entrapment and killing and promotes inflammation. Although these findings suggest that HRG contributes to innate immunity and inflammation, little is known about the HRG-fibrin(ogen) interaction. By immunoassay, HRG-fibrinogen complexes were detected in Zn(2+)-supplemented human plasma, a finding consistent with a high affinity interaction. Surface plasmon resonance determinations support this concept and show that in the presence of Zn(2+), HRG binds the predominant γ(A)/γ(A)-fibrinogen and the γ-chain elongated isoform, γ(A)/γ'-fibrinogen, with K(d) values of 9 nm. Likewise, (125)I-labeled HRG binds γ(A)/γ(A)- or γ(A)/γ'-fibrin clots with similar K(d) values when Zn(2+) is present. There are multiple HRG binding sites on fibrin(ogen) because HRG binds immobilized fibrinogen fragment D or E and γ'-peptide, an analog of the COOH terminus of the γ'-chain that mediates the high affinity interaction of thrombin with γ(A)/γ'-fibrin. Thrombin competes with HRG for γ'-peptide binding and displaces (125)I-HRG from γ(A)/γ'-fibrin clots and vice versa. Taken together, these data suggest that (a) HRG circulates in complex with fibrinogen and that the complex persists upon fibrin formation, and (b) by competing with thrombin for γ(A)/γ'-fibrin binding, HRG may modulate coagulation. Therefore, the HRG-fibrin interaction may provide a novel link between coagulation, innate immunity, and inflammation.

FIGURE 1.

Effect of ZnCl₂ on the binding of HRG to γ_A/γ_A- or γ_A/γ′-fibrinogen. γ_A/γ_A-Fibrinogen (triangles) or γ_A/γ′-fibrinogen (circles) was immobilized to 6000–7000 RU on separate flow cells of a CM4 BIAcore chip. An unmodified flow cell served as control. HRG (0.2 μm) was injected into flow cells in the absence or presence of ZnCl₂ at the concentrations indicated. The RU at equilibrium (Req) was calculated and, after background correction, is plotted against the input ZnCl₂ concentrations. Data represent the mean ± S.D. of two experiments, and lines represent nonlinear regression analyses of the data.

FIGURE 2.

Determination of the affinity of HRG for γ_A/γ_A- or γ_A/γ′-fibrinogen in the presence of ZnCl₂. γ_A/γ_A-Fibrinogen (A) or γ_A/γ′-fibrinogen (B) was adsorbed to separate flow cells on a CM4 chip. HRG (0–700 nm) was injected into flow cells for 300 s in the presence of 20 μm ZnCl₂, and the cells were then washed with HBS buffer containing 2 mm CaCl₂ and 20 μm ZnCl₂ for 500 s to monitor dissociation. HRG concentrations in nm are indicated adjacent to each sensogram tracing. These are data from a single experiment, which was performed three times.

FIGURE 3.

Effect of the γ′-peptide-directed IgG on HRG binding to γ_A/γ_A- or γ_A/γ′-fibrinogen. Flow cells containing immobilized γ_A/γ_A-fibrinogen (triangles) or γ_A/γ′-fibrinogen (circles) were pretreated with (closed) or without (open) 0.5 μm γ′-peptide-directed IgG before injection of HRG (0–1.0 μm). Symbols represent the mean ± S.D. of two experiments, and lines represent nonlinear regression analyses of the data.

FIGURE 4.

Binding of HRG to γ′-peptide. A, the binding of 1.1 μm HRG to fluorescein-labeled γ′-peptide (50 nm) was monitored in the presence of ZnCl₂ (0–20 μm) at λ_ex = 492 nm and λ_em = 532 nm. Initial fluorescence was determined in the presence of HRG but in the absence of ZnCl₂ (I_o). Aliquots of ZnCl₂ were then added, and the fluorescence intensity (I) was measured after each addition. I/I_o values are plotted versus ZnCl₂ concentrations. B, binding of HRG to immobilized γ′-peptide was measured by SPR. Biotinylated γ′-peptide was adsorbed to a streptavidin-immobilized CM4 chip. Binding of HRG (0–700 nm) to γ′-peptide in the presence of 20 μm ZnCl₂ was then determined in the absence (open symbols) or presence (closed symbols) of the γ′-peptide-directed IgG. Corrected Req values are plotted against the input HRG concentrations. Symbols represent the mean ± S.D. of two experiments, and lines represent nonlinear regression analyses of the data.

FIGURE 5.

Binding of ¹²⁵I-HRG to γ_A/γ_A- or γ_A/γ′-fibrin clots. A, 0–1.25 μm γ_A/γ_A- (triangles) or γ_A/γ′-fibrinogen (circles) was added to microcentrifuge tubes, and the binding of ¹²⁵I-HRG (40 nm) to clots was assessed after thrombin addition. B, the binding of ¹²⁵I-HRG (20 nm) to 0.25 μm γ_A/γ_A- (triangles) or γ_A/γ′-fibrin clots (circles) was assessed in the presence of 0–4 μm γ′-peptide-directed Fab fragments (closed symbols) or a control sheep IgG (open symbols). Experiments were performed in TBS-Tween containing 20 μm ZnCl₂ and 2 mm CaCl₂ and clots were generated with 10 nm thrombin. After incubation at 23 °C for 45 min, fibrin clots were pelleted by centrifugation, and the amount of free ¹²⁵I-HRG in the supernatant was used to calculate the fraction bound. The percent of HRG bound to the clots is plotted versus fibrin or antibody concentrations. Symbols represent the mean ± S.D. of two experiments, each performed in duplicate, whereas the lines represent nonlinear regression analyses of the data.

FIGURE 6.

Dissociation of ¹²⁵I-HRG from preformed γ_A/γ_A- or γ_A/γ′-fibrin clots. Aliquots of 5 μm γ_A/γ_A- (A) or γ_A/γ′-fibrinogen (B) containing 2 mm CaCl₂, 20 μm ZnCl₂, 20 nm factor XIII, and 50 nm ¹²⁵I-HRG were clotted with 10 nm thrombin around plastic inoculation loops. After incubation at 23 °C for 45 min, clots were counted for radioactivity and incubated in tubes containing 5 ml of 2 m NaCl and 2 mm of EDTA (triangles), 2 mm CaCl₂ and 10 μm diethyldithiocarbamate trihydrate (squares), or 2 mm CaCl₂ and 20 μm ZnCl₂ (circles). At intervals, clots were removed, and residual radioactivity was used to determine the percent of ¹²⁵I-HRG that remained clot-associated. The symbols represent the mean ± S.D. of three experiments, whereas the lines represent nonlinear regression analyses of the data using two-component exponential decay model.

FIGURE 7.

Effect of FPRck-thrombin or prothrombin on the interaction of HRG with γ′-peptide. Biotinylated γ′-peptide was immobilized on a streptavidin-modified flow cell to 200 RU, and an unmodified flow cell served as a control. Arrows indicate injection of 1 μm HRG for 200 s followed by injections of FPRck-thrombin (panel A) or prothrombin (panel B) at the concentrations (μm) indicated. Injections were carried out in the presence of 20 μm Zn²⁺. These are data from a single experiment, which was performed three times.

FIGURE 8.

Effect of competitors on the binding of ¹²⁵I-HRG or ¹²⁵I-YPRck-thrombin to γ_A/γ_A- or γ_A/γ′-fibrin clots. A, ¹²⁵I-HRG (20 nm) was added to microcentrifuge tubes containing 2 μm γ_A/γ_A-fibrinogen (triangles) or γ_A/γ′-fibrinogen (circles) in the presence of FPRck-thrombin (0–10 μm). B, ¹²⁵I-YPRck-thrombin (20 nm) was added to microcentrifuge tubes containing 2 μm γ_A/γ_A-fibrinogen (triangles) or 0.25 μm γ_A/γ′-fibrinogen (circles) in the presence of HRG (0–2 μm). In both experiments 2 mm CaCl₂ plus 20 μm ZnCl₂ were present and clotting was initiated with 10 nm thrombin. After incubation at 23 °C for 45 min, fibrin was pelleted by centrifugation, and free ¹²⁵I-HRG or ¹²⁵I-YPRck-thrombin in the supernatant was used to calculate the bound fraction. The percent of fibrin-bound ¹²⁵I-HRG or ¹²⁵I-YPRck-thrombin is plotted versus the FPRck-thrombin or HRG concentration, respectively. The symbols represent the mean ± S.D. of two experiments, each performed in duplicate, whereas the lines represent nonlinear regression analyses of the data.