Influence of Glutathione and its Derivatives on Fibrin Polymerization

Jun 21, 2008 - Carri B. Geer, Nathan A. Stasko, Ioana A. Rus, Susan T. Lord and Mark H. Schoenfisch*. Department of Chemistry and Department of ...
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Influence of Glutathione and its Derivatives on Fibrin Polymerization Carri B. Geer, Nathan A. Stasko, Ioana A. Rus, Susan T. Lord, and Mark H. Schoenfisch* Department of Chemistry and Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Received February 11, 2008; Revised Manuscript Received April 25, 2008

A complex relationship exists between reduced, oxidized, and nitrosated glutathione (GSH, GSSG, and GSNO, respectively). Although previous studies have demonstrated S-nitrosoglutathione (GSNO) has potent antiplatelet efficacy, little work has examined the role of GSNO and related species on subsequent aspects of coagulation (e.g., fibrin polymerization). Herein, the effects of GSH, GSSG, and GSNO on the entire process of fibrin polymerization are described. Relative to normal fibrinogen, the addition of GSH, GSSG, or GSNO leads to prolonged lag times, slower rates of protofibril lateral aggregation and the formation of clots with lower final turbidities. Dose-dependent studies indicate the influence of GSH on fibrin formation is a function of both GSH and fibrinogen concentration. Studies with AR251 recombinant fibrinogen (lacking RC regions) showed GSH had no influence on its polymerization, suggesting the glutathione species interact within the RC region of fibrinogen.

Introduction The complex relationship between glutathione (GSH) and its derivatives (GSX) found in the blood has been used to diagnose and monitor a number of disease states.1–4 For example, increased glutathione (GSH) found in the lungs has been associated with cystic fibrosis, chronic obstructive airway disease, and asthma.5 Conversely, decreased glutathione and elevated levels of oxidized glutathione (GSSG) are a reliable biomarker of oxidative stress.3,4 The nitrosated analogue, S-nitrosoglutathione (GSNO), is a key transporter of nitric oxide (NO) and has been explored as a treatment for cardiovascular disorders due to its antiplatelet6–11 and vasodilatory effects.12–15 The physiological interconversion between oxidized, reduced, and nitrosated glutathione is shown in Figure 1. Glutathione is converted to GSNO via nitrosative agents (e.g., N2O3), through redox mechanisms involving transition metal centers, or via direct transnitrosation.16 Nitrosothiols are subject to a number of decomposition pathways, one of which involves the direct decomposition via free GSH to form disulfides (e.g., GSSG) and multiple NOx species.17,18 Oxidized glutathione generated from glutathione peroxidase in response to oxidative stress is reduced via glutathione reductase to continuously replenish the antioxidant supply of free GSH. S-Nitrosothiol signal transduction is central to this cycle. The direct transfer of NO from one thiol to another, or transnitrosation, is responsible for much of the NO transport in vivo and establishes a dynamic relationship between the high serum concentrations of GSH (500 µM) and the transient RSNO species in blood (2 µM).19 The transfer of NO from low molecular weight thiols to protein bound thiols (e.g., GSNO to a free cysteine residue of serum albumin) results in stable protein-SNO complexes that serve as the ultimate sink for circulating NO.19,20 In a number of these reactions, transnitrosation involving GSNO has been shown to result in S-thiolation, forming mixed disulfides between proteins and glutathione.21 Recently, a number of examples have demonstrated that * To whom correspondence should be addressed. Tel.: (919) 843-8714. Fax: (919) 962-2388. E-mail: [email protected].

Figure 1. Interconversion between GSH, GSSG, and GSNO species.

nitrosothiols can modulate protein structure and function through S-thiolation.22–25 One of the most significant examples is the glutathionylation of protein disulfide isomerase (PDI) following exposure to GSNO.26–28 Glutathionylation of PDI on the platelet surface releases NO that diffuses across the plasma membrane and inhibits platelet activation via a guanylyl cyclase-dependent mechanism.6 The mixed disulfide formed also plays a role in inhibiting platelet aggregation by interfering with the enzymatic function of PDI.26–28 Due to GSNO’s ability to mitigate platelet adhesion and aggregation, a number of animal and clinical studies have been conducted using GSNO and other nitrosothiols as novel antithrombotic agents.8–11 Because activated platelets initiate the coagulation cascade, platelets have been an excellent target for RSNO intervention. However, little emphasis has been placed on the effects of nitrosothiols or their decomposition products on subsequent aspects of coagulation, specifically fibrin formation and fibrinogen. In vivo, fibrin serves as the structural scaffolding of a blood clot by stabilizing the initial platelet plug and localizing

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Lag time, Vmax, and final optical densities (OD) for the polymerization of fibrin were examined as a function of treatment with each glutathione species. Studies were also conducted to examine the rates of fibrinopeptide cleavage and the polymerization of an engineered recombinant fibrinogen (AR251) to elucidate the mechanism of the glutathione species’ influence on fibrin formation.

Experimental Methods

Figure 2. Illustration of the multistep process of fibrin formation.

important wound healing and inflammation factors to the site of injury. Fibrinogen, the precursor to fibrin, is a 340 kDa plasma protein present in blood at concentrations ranging from 2-4 mg/mL (6-12 µM). Fibrinogen is composed of two copies each of three polypeptide chains, AR, Bβ, and γ (Figure 2).29 Fibrin polymerization is initiated by the enzyme thrombin, which cleaves fibrinopeptide A (FpA) from the N-terminus of fibrinogen’s AR chains, exposing a new amino acid sequence (the “A” knob) and generating fibrin monomer29 (Figure 2). The “A” knobs interact with holes “a” in other fibrin molecules to form double-stranded, half-staggered protofibrils. Thrombin also cleaves fibrinopeptide B (FpB) from the N-termini of the Bβ chains, exposing the “B” knobs that interact with holes “b” to form “B-b” interactions. Upon removal of FpB, protofibrils lengthen, branch, and laterally aggregate to form thicker fibers. Protofibril lateral aggregation is thought to be mediated predominantly by “B-b” interactions and intermolecular RC-RC interactions between the C-termini of the AR chains, although the exact mechanism remains controversial.30,31 Clot structure is highly sensitive to the kinetics of clot formation. Alterations to the multistep process of fibrin formation may lead to clots with different structural and functional properties. Exploring the mechanism by which physiologically relevant thiols (e.g., GSH, GSSG) or novel nitrosothiol-based therapeutics (e.g., GSNO) affect the rate of fibrin formation is critical to understanding their role in hemostasis. Previously, Mutus and co-workers examined the effects of GSNO on the initial rate of fibrin formation and found 4.0 ( 1.0 µM GSNO was sufficient to inhibit the initial rate of polymerization by 50%.32 GSNO-dependent structural changes in fibrinogen were also observed using circular dichroism and tryptophan fluorescence spectroscopy. The authors concluded that the observed inhibition was due to conformational changes in the C-terminal region of fibrinogen’s R-chain induced by GSNO binding.32 Herein, we investigated the effects of the reduced, oxidized and nitrosated derivatives of glutathione on the entire process of fibrin formation with the goal of expanding the previous work beyond the exploration of the initial rate of fibrin formation.

Materials. Reagents were of analytical grade and purchased from Sigma (St. Louis, MO) unless noted otherwise. Human plasma fibrinogen (FIB 1) and R-thrombin (HT 2970PA) were purchased from Enzyme Research Laboratories (Southbend, IN). Fibrinogen was stored at -80 °C, thawed at 37 °C for 10 min, and maintained at ambient temperature. The buffer for all experiments was 20 mM HEPES, 150 mM NaCl, 1 mM CaCl2, pH 7.4 (HBSC). Protein concentration was determined using an extinction coefficient of 1.51 or 1.60 at 280 nm for a 1 mg/mL solution of normal fibrinogen and AR251 fibrinogen, respectively.33 Recombinant normal and AR251 fibrinogen were prepared as described previously.33,34 Thrombin aliquots were freshly thawed, diluted to 10 U/mL and stored on ice. Immediately prior to experiments, thrombin was diluted to 0.1 U/mL. Reduced and oxidized glutathione solutions, GSH and GSSG, respectively, were dissolved in HBSC and stored on ice. Synthesis of S-Nitrosoglutathione (GSNO). GSNO was synthesized according to a previously reported procedure.35 Briefly, GSH (100 mg) was dissolved in 10 mL of 2 M HCl at 0 °C and treated with a 2:1 molar excess of NaNO2. After stirring for 30 min in the dark, acetone (25 mL) was added to the red solution and stirred for an additional 10 min on ice. The pink GSNO precipitate was recovered via filtration, washed with cold water (2 × 10 mL) and cold ether (2 × 10 mL), dried under vacuum, and stored at -20 °C in the dark. To minimize thermal decomposition, GSNO was kept on ice in the dark and diluted immediately prior to use for each experiment. The percent conversion of GSH to GSNO was calculated from the total nitric oxide released (µmol NO/mg) upon reacting GSNO with copper (Cu2+), a nitrosothiol decomposition trigger.16,17 Briefly, solid GSNO (1-3 mg) was added to a reaction flask containing 10 mM phosphate buffered saline (PBS, pH ) 7.4) and 200 µM CuBr2. The ensuing NO was measured using a Sievers 280i Chemiluminescence Nitric Oxide Analyzer (Boulder, CO).36,37 Based on the NO measured (2.92 µmol NO/mg), a percent conversion of 98.3% was calculated (theoretical amount was 2.97 µmol NO/mg). Fibrin Polymerization Assays. Experiments were performed in triplicate using medium-binding 96-well microtiter plates. Fibrinogen (100 µL) and either GSH, GSNO, or GSSG (100 µL) were added to each well and incubated at 37 °C for 10 min. Next, thrombin (100 µL) was added simultaneously to each well to initiate fibrin polymerization. Changes in optical density (OD) were monitored at 405 nm using a SpectraMax plate reader. Final concentrations varied from 0.07-2 mg/ mL (0.2-6 µM) fibrinogen and 0.03-500 µM GSH, GSSG, or GSNO. A final thrombin concentration of 0.03 U/mL (0.3 nM) was used for all polymerization experiments. Analysis of Polymerization Results. The maximum velocity of polymerization (Vmax) was determined as the steepest part of the polymerization curve by finding the maximum value of the first derivative. Lag time was measured as the time of initial increase in optical density (OD) and the final OD was the turbidity value at the plateau in the polymerization curve. A p value of 0.05 was considered significant according to unpaired t tests. Fibrinopeptide Release. To initiate fibrinopeptide release, thrombin (10 µL of 1 U/mL) was added to fibrinogen (2 mL of 0.7 mg/mL), mixed by inversion, and pipetted into 240 µL aliquots. All protein manipulations were completed within 1 min after thrombin addition. The polymerization reactions were quenched by placing each tube in boiling water for 15 min at 2, 5, 10, 20, 40, 80, or 120 min after thrombin addition. The boiled samples were stored on ice for the

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Figure 3. Turbidity curves monitored spectrophotometrically at 405 nm, comparing the polymerization of 0.07 mg/mL fibrinogen and 0.03 U/mL thrombin with and without 500 µM GSH. The three phases of the polymerization curves are as follows: (A) Lag time is the time at which an increase in optical density was observed; (B) Maximum rate of change in optical density; (C) Final optical density of the turbidity curves.

duration of the experiment. Fibrinogen (240 µL) not treated with thrombin and fibrinogen (240 µL) treated for 60 min with an excess of thrombin (1 µL of 2360 U/mL) served as controls for no fibrinopeptide release and complete fibrinopeptide release, respectively. Samples were spun for 10 min, and the supernatant containing fibrinopeptides was stored at -80 °C before analysis via reverse-phase HPLC monitored at 210 nm, as previously described.38 The percent of FpA and FpB released was calculated relative to the amount of FpA or FpB detected after excess thrombin treatment. The rates of FpA and FpB release were determined using kinetic equations described previously.39

Results and Discussion Due to the abundance of GSH in plasma (∼500 µM), the therapeutic utility of GSNO and the role of GSSG as a predicative marker for oxidative stress, the mechanistic effects of these species on fibrin formation were explored. Representative polymerization curves for normal, untreated fibrinogen and fibrinogen treated with GSX species are shown in Figure 3. Based on methods described previously, three parameters relating to critical aspects of fibrin formation were obtained from the polymerization curves.40 The lag time, representative of the rate of protofibril formation (Figure 3A), was measured as the time when an increase in turbidity was first detected. Factors that influence the lag time include the rate of FpA release and the ability of the activated fibrin monomers to interact with one another.40 In the next phase of polymerization, the protofibrils laterally aggregate to form fibers, leading to an increase in optical density that reflects the rate of protofibril assembly into fibers (Figure 3B). Both FpB release (leading to “B-b” interactions) and RC-RC intermolecular interactions have been shown to influence the lateral aggregation of protofibrils.30,41 The maximum rate of lateral aggregation or Vmax is the steepest part of the polymerization curve. The final optical density (Figure 3C) is related to the number and size of fibers, and may provide insight into the structure of the resulting clot.40 Dose-Dependent Effects of GSH on Fibrin Formation. The concentration of GSH in blood has been shown to fluctuate in response to numerous disease states.4,42–45 For example, GSH levels increase in response to the oxidative stress induced by cigarette smoking.43,44 Consequently, investigation of the concentration dependence of GSH’s influence on fibrin poly-

Figure 4. Vmax, lag time, and final OD calculated relative to normal (no glutathione) fibrin polymerization of 0.07 mg/mL fibrinogen with 0.03 U/mL thrombin as a function of glutathione concentration. The values obtained for normal were defined as 100% and are denoted by the dashed lines.

merization may prove useful in understanding the broader impact of the up- or down-regulation of this important antioxidant. The polymerization of 0.07 mg/mL fibrinogen was examined over a range of GSH concentrations (32 nM to 500 µM). As shown in Figure 3, the addition of GSH (500 µM) interfered with the entire process of fibrin formation as evidenced by the delay in clotting, reduced rate of turbidity increase and lower final optical density relative to normal. The dose-dependent effects of GSH on each aspect of polymerization obtained from the complete set of fibrin polymerization curves are shown in Figure 4. As expected, the greatest inhibition was observed at the largest GSH

Glutathione Species and Fibrin Formation

concentration (500 µM) where the lag time was prolonged by ∼20% and the Vmax slowed by 30%, indications that GSH impaired protofibril formation and lateral aggregation, respectively. Of note, 100 and 500 µM GSH had the same effect on fibrin formation, suggesting maximal inhibition was achieved at 100 µM GSH. The three lowest concentrations of GSH (32, 160, and 800 nM) did not significantly affect fibrin formation. The dose-dependent influence of GSH on fibrin polymerization suggests changes in glutathione levels in response to oxidative stress or disease could also affect fibrin clot formation. GSX Influence on Polymerization. S-Nitrosoglutathione and other nitrosothiols have been previously reported to inhibit the initial rates of fibrin polymerization at low micromolar concentrations, although the exact mechanism remains unclear.32 Herein, the dose-dependent effects of GSNO were evaluated for the entire process of fibrin polymerization to gain a deeper understanding of which phases of fibrin formation are affected by nitrosothiol species. Similar to results described above for GSH, inhibition of fibrin formation was observed in the presence of GSNO with a maximum inhibition occurring at 500 µM (Figure 5). In contrast to the 50% inhibition of the rate polymerization reported by Akhter et al.,32 no inhibition was observed at 4 µM GSNO. Our experiments are distinct from those of Akhter et al. in two regards. First, the polymerization assays that we describe examine the entire polymerization process instead of only the first 100 s. As well, the concentrations of fibrinogen and thrombin are less. The lower concentration of thrombin was carefully selected to allow observation of the early phases of fibrin formation that are difficult to observe at higher thrombin concentrations. Oxidized glutathione disulfide (GSSG) is a major product following thermal decomposition of GSNO at 37 °C. To explore the possibility that this nitrosothiol byproduct or common in vivo oxidative marker may influence fibrin formation, the concentration dependent effects of GSSG on polymerization were also tested. The three parameters of fibrin polymerization in the presence of 500 µM GSH, GSSG, and GSNO relative to normal conditions with no glutathione species added are shown in Figure 5. For clarity the results are reported as percentages of normal polymerization without any glutathione species. The addition of GSH, GSSG, and GSNO resulted in a ∼20% increase in lag time compared to normal. The rate of protofibril aggregation and Vmax were also reduced. Compared to GSNO, the inhibition of Vmax was greater for GSH and GSSG, but this difference was not significant in an unpaired t test (p ) 0.11 and p ) 0.16, respectively). The addition of GSH or GSSG led to a ∼30% reduction in turbidity, in contrast to only ∼20% for GSNO. The small difference in GSNO’s influence on final OD was significant compared to GSH (p ) 0.05) but not significant compared to GSSG (p ) 0.12). The final optical densities and Vmax values of all the clots formed in the presence of glutathione and its derivatives were reduced compared to controls (i.e., normal), suggesting a thiol-dependent mechanism. Based on the results obtained from the polymerization curves, GSH, GSNO, and GSSG each influence fibrin formation to a similar extent, contradicting the work Akhter et al. who reported only GSNO and not GSSG influenced the initial rates of fibrin formation.32 The influence of S-nitrosoglutathione on fibrinogen either proceeds via S-thiolation following a transnitrosation reaction or the GSNO species acts as a reducing agent on a solvent accessible fibrinogen disulfide. An additional nitrosothiol/thiol pair was tested to confirm the thiol-dependent mechanism. Similar behavior was observed for the exogenous RSNO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) and the free thiol

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Figure 5. Maximum rate of lateral aggregation (Vmax), clotting onset time (lag time), and final clot turbidity (OD) for 0.07 mg/mL fibrinogen, 0.03 U/mL thrombin, and 500 µM GSH, GSNO, or GSSG relative to fibrin polymerization with no added GSH, GSNO, or GSSG. The values obtained for normal were defined as 100% and are denoted by the dashed lines.

N-acetyl-DL-penicillamine (NAP; Table 1). The structurally related N-acetyl-DL-cysteine (NAC) also inhibited fibrin formation at concentrations up to 600 µM suggesting a relationship between the presence of reactive thiols and the inhibition of fibrin formation (Table 1). N-acetyl-DL-serine (NAS), an alcohol instead of a thiol, with the same N- and C-terminus as NAC showed no effect on fibrin formation. Akhter et al. reported a similar phenomenon (no inhibition in fibrin polymerization) in the presence of a carboxymethylated glutathione derivative containing a protected, unreactive thiol.32 Because the concentration of GSH in blood is greater than GSSG and GSNO, the remainder of our studies focused on fibrin formation in the presence of GSH. These studies expand the significance of GSH influence on fibrin formation and suggest glutathione in blood may serve a regulatory role with respect to fibrin formation.

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Table 1. Comparison of the Effects of 670 µM S-Nitrosothiol and Thiol Species on Final Optical Density and Vmax Valuesa final

normal SNAP NAP NAC NAS a

OD

Vmax

raw (O.D.)

ratio to normal

0.065 ( 0.007 0.026 ( 0.012 0.030 ( 0.008 0.032 ( 0.004 0.064 ( 0.004

1 0.50 ( 0.18 0.53 ( 0.15 0.48 ( 0.08 0.99 ( 0.11

-1

raw (O.D. s

)

1.1 × 10-4 ( 1.9 × 10-5 5.4 × 10-5 ( 4.6 × 10-6 5.0 × 10-5 ( 2.0 × 10-6 5.0 × 10-5 ( 1.8 × 10-6 1.1 × 10-4 ( 4.5 × 10-6

ratio to normal 1 0.62 ( 0.08 0.54 ( 0.10 0.44 ( 0.06 0.96 ( 0.12

During the polymerization of 0.07 mg/mL fibrinogen with 0.03 U/mL thrombin.

Table 2. Rate of Thrombin-Catalyzed FpA and FpB Releasea rate of FpA release (10-2 M-1 s-1)

rate of FpB release (10-2 M-1 s-1)

expt

-GSH

+GSH

-GSH

+GSH

1 2 3 avg

2.10 ( 0.16 1.66 ( 0.08 1.68 ( 0.06 1.81 ( 0.25

1.56 ( 0.12 1.51 ( 0.05 2.10 ( 0.10 1.72 ( 0.33

1.57 ( 0.79 1.23 ( 0.06 1.42 ( 0.05 1.41 ( 0.17

1.35 ( 0.07 0.96 ( 0.04 1.55 ( 0.08 1.29 ( 0.30

a With and without 500 µM GSH, as detected by HPLC (0.07 mg/mL fibrinogen, 0.005 U/mL thrombin).

Effect of GSH on Thrombin Activity. Previous studies have demonstrated that lag time is affected by the rate of FpA release (e.g., thrombin’s activity) and fibrin monomer assembly into protofibrils. Similarly, the rate of lateral aggregation is influenced by FpB release and/or the assembly of protofibrils into branched, thicker fibers. To determine if GSH impaired thrombin’s ability to activate fibrinogen, thrombin activity was assayed directly via the detection of FpA and FpB using reverse-phase HPLC. Table 2 summarizes the kinetic constants for fibrinopeptide release with and without 500 µM GSH. No significant difference in the rate of FpA or FpB release from fibrinogen was observed in the presence of GSH compared to controls (i.e., its absence), indicating GSH’s influence on thrombin activity is minimal. These data agree with previous studies that examined thrombin activity in the presence of GSNO using a colorimetric assay.32 Because fibrinopeptide release was normal under these conditions, the activation of fibrinogen to fibrin monomer and the release of FpB from protofibrils are assumed to be unaffected. As such, the activation of fibrinogen is not influenced by GSH. More likely, the effects of GSH on fibrin polymerization are the result of changes in the fibrin molecule that preclude its interaction with other fibrin molecules during fibrin formation. Influence of Fibrinogen Concentration. The normal rates of fibrinopeptide cleavage observed in the presence of GSH suggest that structural changes in fibrinogen are responsible for the inhibited rates of protofibril formation/aggregation. To determine the influence of fibrinogen concentration on polymerization, the concentration of fibrinogen was varied from 0.07 to 2 mg/mL in the presence of 500 µM GSH. The results of the polymerization curves and parameters of polymerization relative to the normal polymerization of fibrinogen at each concentration are shown in Figure 6. The effect of GSH on fibrin polymerization was minimal at 0.25 mg/mL fibrinogen. At fibrinogen concentrations 0.25 mg/mL, the effect of GSH was the opposite of that observed at lower GSH concentrations: slightly greater Vmax, shorter lag times, and greater final ODs compared to normal clots at each concentra-

Figure 6. Vmax, lag time, and final OD values calculated relative to normal (no glutathione) fibrinogen polymerization for each fibrinogen concentration with 0.03 U/mL thrombin and 500 µM glutathione. The values obtained for normal were defined as 100% and are denoted by the dashed lines.

tion. Based on these results, GSH’s influence on fibrin formation is also dependent on fibrinogen concentration. Furthermore, the inhibition appears to be minimized at elevated levels of fibrinogen.

Glutathione Species and Fibrin Formation

Mechanism of GSH Effect on Fibrin Formation. Mutus and co-workers proposed several regions rich in aromatic residues within the C-terminus of fibrinogen’s R-chain (e.g., RC regions) as potential sites of interaction with GSNO.32 Because no substantive differences were observed between GSNO, GSH, and GSSG in our study, the impairment of fibrin formation likely does not result from specific interactions between GSNO and aromatic residues within fibrinogen. Rather, GSX species act on fibrinogen in a thiol-specific manner potentially reducing disulfide bonds that lead to structural and functional alterations. Indeed, fibrinogen contains 29 disulfide bonds, two of which are located in the highly solvent-accessible RC regions of the molecule (Figure 2). Due to their flexible structure and location (on the exterior of fibrinogen), the C-termini of the R-chains are readily susceptible to reduction and degradation. Furthermore, two RC regions exist on the symmetric fibrinogen structure (Figure 2), correlating with the 2:1 stoichiometry for the interaction of GSNO with fibrinogen observed by Akhter et al. in tryptophan fluorescence quenching and isothermal titration calorimetry experiments.32 The role of RC regions in lateral aggregation is well documented,30,33,46,47 but the exact mechanism remains controversial. It has been hypothesized that the two RC regions interact intramolecularly prior to the removal of fibrinopeptides A and B and switch to intermolecular interactions during fibrin formation.30,47 Weisel and co-workers measured the force of the interaction between two RC regions.48 As well, Collet et al. reported direct evidence that the presence of the RC regions accelerates fibrin polymerization and makes the ultimate clot structure more stable and resistant to fibrinolysis.49 Further evidence for the intermolecular interaction between RC regions was provided by Medved and co-workers who established the NMR solution structure of bovine RC fragments and observed their concentration-dependent, thermodynamically driven selfoligomerization.50 These results led to the hypothesis that AR423Cys-AR453Cys interactions play a critical role in fibrin polymerization. Disruption to the analogous disulfide in human fibrinogen via GSX species may decrease the affinity of the two independent RC regions, slowing the rate protofibril lateral aggregation. To determine if GSH alters fibrin formation by influencing the function of the RC regions, native fibrinogen was replaced with AR251 recombinant fibrinogen for the fibrin polymerization experiments. AR251 fibrinogen consists of normal Bβ and γ chains, but only the first 251 residues of the AR chain.33 Thus, AR251 lacks the RC regions necessary for assisting in protofibril lateral aggregation. AR251 polymerization in the presence and absence of 500 µM GSH after exposure to thrombin, as studied via turbidity measurements, is shown in Figure 7. As evidenced by the identical turbidity curves, GSH had no measurable effect on the polymerization of AR251. It is thus reasonable to conclude that reductions in the disulfide bridge in the RC regions induced by GSH, GSNO, or GSSG may lead to changes in fibrin formation similar to those observed when functional RC regions are not present.

Conclusions The studies reported herein represent the first examination of the influence of reduced, oxidized and nitrosated glutathione on the entire process of fibrin formation. Based on turbidity measurements, each of the glutathione derivatives inhibited fibrin formation to nearly the same degree. While it is possible that GSH, GSSG, and GSNO inhibit polymerization by different

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Figure 7. Turbidity curves monitored spectrophotometrically at 405 nm comparing the polymerization of 0.05 mg/mL AR251 recombinant fibrinogen and 0.03 U/mL thrombin without glutathione (black trace) and 500 µM glutathione (gray trace).

mechanisms, a more likely explanation for the reduced polymerization is that the glutathione species undergo thiolation reactions with fibrinogen to alter the protein structure that subsequently leads to changes in fibrin formation. Studies using AR251 recombinant fibrinogen indicated that the exposed, solvent-accessible disulfides between AR442Cys-AR472Cys are critical to normal fibrin polymerization. Understanding the interactions of glutathione and its derivatives with fibrinogen and their role in fibrin formation may provide greater insight to the physiological roles of nitrosothiols and related decomposition products. Additionally, these findings suggest glutathione or other physiological thiols have an added role in hemostasis. A recent report by Weisel and co-workers illustrates the effects of homocysteine on clot formation, further implicating a regulatory role of physiological thiols in vascular biology.51 Studies on the exact location of S-thiolation and subsequent effects on protein structure represent important future studies. Acknowledgment. This work was supported by the National Institutes of Health (NIH EB000708 and HL31048). Special thanks to Oleg Gorkun for valuable discussions.

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