Long range communication between exosites 1 and 2 modulates thrombin function

Abstract

Although exosites 1 and 2 regulate thrombin activity by binding substrates and cofactors and by allosterically modulating the active site, it is unclear whether there is direct allosteric linkage between the two exosites. To begin to address this, we first titrated a thrombin variant fluorescently labeled at exosite 1 with exosite 2 ligands, HD22 (a DNA aptamer), gamma'-peptide (an analog of the COOH terminus of the gamma'-chain of fibrinogen) or heparin. Concentration-dependent and saturable changes in fluorescence were elicited, supporting inter-exosite linkage. To explore the functional consequences of this phenomenon, we evaluated the capacity of exosite 2 ligands to inhibit thrombin binding to gamma(A)/gamma(A)-fibrin, an interaction mediated solely by exosite 1. When gamma(A)/gamma(A)-fibrinogen was clotted with thrombin in the presence of HD22, gamma'-peptide, or prothrombin fragment 2 there was a dose-dependent and saturable decrease in thrombin binding to the resultant fibrin clots. Furthermore, HD22 reduced the affinity of thrombin for gamma(A)/gamma(A)-fibrin 6-fold and accelerated the dissociation of thrombin from preformed gamma(A)/gamma(A)-fibrin clots. Similar responses were obtained when surface plasmon resonance was used to monitor the interaction of thrombin with gamma(A)/gamma(A)-fibrinogen or fibrin. There is bidirectional communication between the exosites, because exosite 1 ligands, HD1 (a DNA aptamer) or hirudin-(54-65) (an analog of the COOH terminus of hirudin), inhibited the exosite 2-mediated interaction of thrombin with immobilized gamma'-peptide. These findings provide evidence for long range allosteric linkage between exosites 1 and 2 on thrombin, revealing further complexity to the mechanisms of thrombin regulation.

FIGURE 1.

Binding of exosite-directed ligands to fluorescently labeled Quick I-thrombin. After determining the initial fluorescence intensity (I_o) of 50 nm 5-IAF-Quick I-thrombin (λ_ex 492, λ_em 532 nm), fluorescence (I) was monitored as the sample was titrated with aliquots of either 200 μm γ′-peptide (▴), 66.7 μm heparin (♦), or 1 mm HD22 (inset). Values of I/I_o were then calculated and plotted as a function of the titrant concentration, and the K_d values were determined by nonlinear regression analysis (lines). Data represent the means ± S.E. of three experiments.

FIGURE 2.

Effect of exosite-directed ligands on thrombin clotting times. γ_A/γ_A-Fibrinogen (2 μm) containing 2 mm CaCl₂ was clotted with 2 nm α-thrombin in the absence or presence of exosite 2-directed ligands, HD22 (●), γ'-peptide (▴), or F2 (■) (A), or exosite 1-directed ligands, HD1 (○) or hirudin-(54–65) (▵), exosite 1-directed ligands (B). Absorbance was determined spectrophotometrically at 405 nm, and the time to reach half-maximal turbidity (clot time) was normalized with respect to that measured in the absence of ligands. Symbols represent the means ± S.E. of three experiments, each done in duplicate, while the lines represent non-linear regression analysis of the data.

FIGURE 3.

The effect of exosite 2-directed ligands on ¹²⁵I-YPR-α-thrombin binding to γ_A/γ_A-fibrin clots. 20 nm ¹²⁵I-YPR-α-thrombin and 5 nm α-thrombin were added to microcentrifuge tubes containing 2 μm γ_A/γ_A-fibrinogen, 2 mm CaCl₂, and HD22 (●), γ′-peptide (▴), or F2 (■) at the indicated concentrations. After incubation for 45 min, clots were pelleted by centrifugation, and free ¹²⁵I-YPR-α-thrombin in the supernatant was used to calculate the bound fraction. Data are plotted as the relative amount of thrombin bound to the clot versus the concentration of exosite-directed ligand and represent the means ± S.E. of three experiments, each done in duplicate, while the lines represent non-linear regression analysis of the data. HD22 was the most potent of the three ligands, having a K_iobs of 673 ± 83 nm. γ′-Peptide and F2 are less potent than HD22, and reduce thrombin binding with K_iobs values of 15.6 ± 3.8 and 18.5 ± 3.3 μm, respectively.

FIGURE 4.

Effect of HD22 on the affinity of thrombin for γ_A/γ_A-fibrin clots. 20 nm ¹²⁵I-YPR-α-thrombin and 5 nm α-thrombin were added to a series of microcentrifuge tubes containing 0–12.5 μm γ_A/γ_A-fibrinogen and 2 mm CaCl₂ in the absence (♦) or presence of 20 μm HD22 (●). After incubation for 45 min, fibrin was pelleted by centrifugation, and free ¹²⁵I-YPR-α-thrombin in the supernatant was used to calculate the bound fraction. Data are plotted as the relative amount of thrombin bound to the fibrin clot versus the γ_A/γ_A-fibrin concentration and represent the means ± S.E. of three experiments, each done in duplicate, while the lines represent non-linear regression analysis of the data. The affinity of ¹²⁵I-YPR-α-thrombin for γ_A/γ_A-fibrin is 6-fold lower in the presence of HD22 than it is in its absence (K_d values of 12.6 ± 1.4 and 2.1 ± 0.7 μm, respectively).

FIGURE 5.

SPR analysis of the effect of exosite 2-directed ligands on the binding of thrombin to immobilized γ_A/γ_A-fibrinogen or fibrin. A, γ_A/γ_A-fibrinogen was immobilized on a CM-5 flow cell to ∼10000 RU. To control for nonspecific binding of thrombin, ovalbumin was immobilized in a separate flow cell. Increasing concentrations of HD22 (●), γ′-peptide (▴), or F2 (■), exosite 2-directed ligands, were injected at flow rates of 20 μl/min in the presence of 2 μm FPR-α-thrombin. Binding of FPR-α-thrombin was determined by the maximum RU values, and data were plotted as the relative amount of thrombin bound versus the concentration of exosite-directed ligand. Symbols represent the means ± S.E. of three experiments, while the lines represent non-linear regression analysis of the data. HD22, γ′-peptide, and F2 disrupt FPR-α-thrombin binding to fibrinogen with K_iobs values of 3.9 ± 1.8, 7.1 ± 1.1, and 19.7 ± 5.2 μm, respectively. B, after γ_A/γ_A-fibrinogen was converted to fibrin by treatment with thrombin, 2 μm FPR-α-thrombin was injected in the absence or presence of exosite 2-directed ligands. HD22, γ′-peptide, and F2 disrupt the binding of FPR-α-thrombin to immobilized γ_A/γ_A-fibrin with K_iobs values of 2.5 ± 0.2, 10.1 ± 0.2, and 11.7 ± 3.8 μm, respectively.

FIGURE 6.

Dissociation of ¹²⁵I-YPR-α-thrombin from preformed γ_A/γ_A-fibrin clots in the absence or presence of exosite-directed ligands. γ_A/γ_A-Fibrinogen (9 μm) containing 2 mm CaCl₂ was clotted around plastic inoculation loops by addition of 100 nm α-thrombin in the presence of 17 nm ¹²⁵I-YPR-α-thrombin and 15 nm factor XIII. After incubation for 45 min, clots were counted for radioactivity and placed in 50-ml conical tubes containing either 5 ml of HBS (♦), 2 m NaCl (▴), 5 μm HD1 (○), or 20 μm HD22 (●). At intervals, the clots were removed and counted for residual radioactivity to determine the amount of ¹²⁵I-YPR-α-thrombin that remained bound. Symbols represent the means ± S.E. of three experiments, while the lines represent non-linear regression analysis of the data by two-component exponential decay.

FIGURE 7.

SPR analysis of the effect of exosite ligands on thrombin binding to immobilized γ′-peptide. Biotinylated γ′-peptide was adsorbed to the flow cell of a streptavidin-CM5 chip to ∼700 RU. To control for nonspecific binding, a flow cell containing only streptavidin was used. A, 1 μm FPR-α-thrombin was injected in the presence of increasing concentrations of HD1 (○), HD22 (●), or hirudin-(54–65) (▵) at a rate of 20 μl/min, and maximal RU values determined at each concentration of exosite ligand were corrected by subtracting the background RU from the control flow cell. Symbols represent the means ± S.E. of three experiments, while the lines represent non-linear regression analysis of the data. HD22, HD1, and hirudin-(54–65) inhibit FPR-α-thrombin binding with K_iobs values of 2.6 ± 0.4, 0.7 ± 0.1, and 1.3 ± 0.3 μm, respectively. B, the experiment was repeated using 2 μm γ-thrombin in place of FPR-α-thrombin. Hirudin-(54–65) has no effect on γ-thrombin binding, whereas the K_iobs values for HD1 and HD22 are 20.8 ± 2.1 and 3.2 ± 0.6 μm, respectively.