Quercetin-3-rutinoside Inhibits Protein Disulfide Isomerase by Binding to Its b'x Domain

Abstract

Quercetin-3-rutinoside inhibits thrombus formation in a mouse model by inhibiting extracellular protein disulfide isomerase (PDI), an enzyme required for platelet thrombus formation and fibrin generation. Prior studies have identified PDI as a potential target for novel antithrombotic agents. Using a fluorescence enhancement-based assay and isothermal calorimetry, we show that quercetin-3-rutinoside directly binds to the b' domain of PDI with a 1:1 stoichiometry. The binding of quercetin-3-rutinoside to PDI induces a more compact conformation and restricts the conformational flexibility of PDI, as revealed by small angle x-ray scattering. The binding sites of quercetin-3-rutinoside to PDI were determined by studying its interaction with isolated fragments of PDI. Quercetin-3-rutinoside binds to the b'x domain of PDI. The infusion of the b'x fragment of PDI rescued thrombus formation that was inhibited by quercetin-3-rutinoside in a mouse thrombosis model. This b'x fragment does not possess reductase activity and, in the absence of quercetin-3-rutinoside, does not affect thrombus formation in vivo. The isolated b' domain of PDI has potential as an antidote to reverse the antithrombotic effect of quercetin-3-rutinoside by binding and neutralizing quercetin-3-rutinoside.

Keywords: PDI; coagulation factor; fibrin; isoquercetin; mouse; platelet; protein-disulfide isomerase; quercetin-3-rutinoside; thrombosis; thrombus formation.

FIGURE 1.

Interaction of quercetin-3-rutinoside and isoquercetin with thiol isomerases. A and C, direct molecular interaction of PDI with quercetin-3-rutinoside (A) and isoquercetin (C) monitored by the fluorescence emission spectra of the complex. In the presence of PDI (18 μm), quercetin-3-rutinoside (55 μm) and isoquercetin (55 μm) emission maxima are enhanced and blue-shifted from 550 to 530 nm when excited at 430 nm. ····, quercetin-3-rutinoside or isoquercetin alone; – – –, PDI alone; ——, complex. B, the binding affinity, IC₅₀, of PDI and quercetin-3-rutinoside (Q-3-R), measured by monitoring the perturbation of the emission fluorescence, was 12.3 μm. D, the binding affinity, IC₅₀, of PDI and isoquercetin, measured by monitoring the perturbation of the emission fluorescence, was 14 μm. E, quercetin-3-rutinoside binds directly to PDI (●), but not to ERp5 (■), ERp57 (▴), or ERp72 (▾), as detected by monitoring the emission fluorescence of quercetin-3-rutinoside. TI, thiol isomerase. F, binding isotherm of quercetin-3-rutinoside to PDI-AGHA, an enzymatically inactive mutant. The IC₅₀ was 15.5 μm. G, analysis of a mixture of PDI and quercetin-3-rutinoside at a concentration of 100 μm at a 1:1 molar ratio by gel filtration on Superdex 200. Black, PDI and quercetin-3-rutinoside; gray, PDI alone; light gray, quercetin-3-rutinoside alone. H, isothermal calorimetry measurement of the binding between quercetin-3-rutinoside and PDI. A dissociation constant of 18.3 μm, ΔH = −9.6 kcal/mol, and ΔS = −10.7 cal/mol/K were determined. Error bars indicate ± S.E.

FIGURE 2.

Recombinant PDI and PDI fragments: purification and characterization. A, PDI contains four domains (a, b, b′, a′), with a linker region (x) between b′ and a′ domains, and an extension c right after the a′ domain. Residues 1–117 (a domain), 118–218 (b domain), 219–331 (b′ domain), 332–351 (x′ linker), 342–462 (a′ domain), and 463–491 (c extension) comprise the mature PDI protein. B, His-tagged full-length human PDI (abb′xa′c) and each of its fragments (a, b, b′x, a′, ab, bb′, b′xa′, abb′, abb′x, bb′xa′, and abb′xa′) were cloned into a pET-15b vector at the restriction site of NedI and BamHI and transformed into E. coli Origami B (DE3) cells. The recombinant proteins were isolated by affinity chromatography with the cOmplete His-Tag purification resin (Roche Applied Science) and further purified by gel filtration chromatography on a Superdex 200. C, the purified proteins appeared as single bands on reduced SDS-PAGE. D, thiol reductase activity was measured in each of these expressed proteins using the insulin reductase assay. a, ▾; b, □; b′x, ; a′, ▵; ab, ♦; bb′, ■; b′xa′, *; abb′, ▴; abb′x, +; bb′xa′, ♢; abb′xa′, ○; abb′xa′c, ●; buffer control, −. Error bars indicate ± S.E. A650, optical density at 650 nm.

FIGURE 3.

Direct binding measurements of quercetin-3-rutinoside with different recombinant PDI fragments by fluorescence assay. The fragments a, a′, b, and ab do not bind to quercetin-3-rutinoside (Q-3-R). All fragments that contain the b′ domain have a similar binding affinity for quercetin-3-rutinoside. A–L: IC₅₀ values for PDI or fragments of PDI binding to quercetin-3-rutinoside were: abb′xa′c, 12.3 μm (A); abb′xa′, 10.1 μm (B); abb′, 20.8 μm (C); abb′x, 10.2 μm (D); bb′xa′, 8.6 μm (E); b′xa′, 11.6 μm (F); bb′, 10.4 μm (G); ab, N. M. (H); a, N. M. (I); b, N. M. (J); b′x, 11.5 μm (K); and a′, N. M. (L). N. M. = not measurable. Error bars indicate ± S.E.

FIGURE 4.

Reductase activity of PDI fragments in the presence and absence of quercetin-3-rutinoside. A–I, reduction of insulin by PDI fragments in the absence (gray) and presence (black) of quercetin-3-rutinoside (100 μm): a′ (A); a (B); ab (C); b′xa′ (D); abb′ (E); abb′x (F); bb′xa′ (G); abb′xa′ (H); and abb′xa′c (I). Error bars indicate ± S.E.

FIGURE 5.

PDI·quercetin-3-rutinoside structure. A, comparison of the SAXS molecular envelopes of PDI (magenta) and PDI·quercetin-3-rutinoside (PDI·Q-3-R) complex (yellow), overlapped with the crystal structure of PDI (Protein Data Bank code 4KEZ), in two orthogonal orientations (top view and side view). B, SAXS profiles of PDI (yellow) and the PDI·quercetin-3-rutinoside complex (blue) show that the scattering profile of PDI·quercetin-3-rutinoside complex is shifted upward (blue), from free PDI (taupe). The observed profiles were compared with those calculated from the restored molecular shapes (red). C, the P(r) plot showing that the size of PDI indicates contraction of PDI (taupe) when complexed with quercetin-3-rutinoside (blue).

FIGURE 6.

Rescue of thrombus formation following quercetin-3-rutinoside-mediated PDI inhibition using the b′x fragment of PDI. A–C, top panels: representative images as a function of time of platelet thrombus formation and fibrin generation as monitored by intravital microscopy over 120 s using platelet-specific anti-CD42b antibody conjugated to DyLight 649 and a mouse monoclonal anti-fibrin-specific antibody conjugated to Alexa Fluor 488. A, rescue with b′x fragment of PDI. Lane 1, thrombus formation following vessel wall injury. Lane 2, after infusion of quercetin-3-rutinoside to a final plasma concentration of ∼10 μm (0.5 μg/g of body weight), vessel wall injury does not lead to thrombus formation. Lane 3, if PDI fragment b′x is infused to an estimated final plasma concentration of 40 μm, there is full recovery of thrombus formation following vessel wall injury. RFU, relative fluorescence units. B, infusion of b fragment of PDI has no effect on thrombus formation. Lane 1, thrombus formation following vessel wall injury. Lane 2, after infusion of quercetin-3-rutinoside to a final plasma concentration of ∼10 μm (0.5 μg/g of body weight), vessel wall injury does not lead to thrombus formation. Lane 3, if fragment b of PDI is infused to an estimated final plasma concentration of 40 μm, there is no recovery of thrombus formation following vessel wall injury. C, infusion of b′x fragment of PDI, in the absence of quercetin-3-rutinoside, has no effect on thrombus formation. Lane 1, thrombus formation after infusion of PDI fragment b′x to an estimated plasma concentration of 40 μm. A–C, bottom panels: kinetics of thrombus formation for the median relative fluorescence units of ∼30 thrombi. Red, platelets,; green, fibrin. The results of aggregate thrombi generation experiments from three or four mice for each set of experiments are depicted.

FIGURE 7.

Statistical analysis of the rescue of thrombus inhibition induced by quercetin-3-rutinoside using the b′x fragment of PDI. A, thrombus formation was fully inhibited following quercetin-3-rutinoside (Q-3-R) infusion when compared with untreated control mice. Platelets, p < 0.0001; fibrin, p < 0.0001. Gray, control (no quercetin-3-rutinoside or PDI fragment); black, quercetin-3-rutinoside plus no PDI fragment, b′x fragment of PDI or b fragment of PDI. RFU, relative fluorescence units. B, infusion of the b′x fragment of PDI rescued thrombus formation in mice treated with quercetin-3-rutinoside. Platelets, p = 0.77; fibrin, p = 0.93. C, infusion of the b domain of PDI did not rescue thrombus formation in mice treated with quercetin-3-rutinoside. Platelets, p < 0.0001; fibrin, p < 0.0001. In contrast to the representative images of a single experiment shown in Fig. 6, these analyses are based on the results from 30–34 vascular injuries.