We provide evidence that NAC is an effective and safe alternative to currently available antithrombotic agents to restore vessel patency after arterial occlusion.

We demonstrated that intravenous NAC administration promotes lysis of arterial thrombi that are resistant to conventional approaches such as recombinant tissue-type plasminogen activator, direct thrombin inhibitors, and antiplatelet treatments. Through in vitro and in vivo experiments, we provide evidence that the molecular target underlying the thrombolytic effects of NAC is principally the VWF that cross-link platelets in arterial thrombi. Coadministration of NAC and a nonpeptidic GpIIb/IIIa inhibitor further improved its thrombolytic efficacy, essentially by accelerating thrombus dissolution and preventing rethrombosis. Thus, in a new large-vessel thromboembolic stroke model in mice, this cotreatment significantly improved ischemic lesion size and neurological outcome. It is important to note that NAC did not worsen hemorrhagic stroke outcome, suggesting that it exerts thrombolytic effects without significantly impairing normal hemostasis.

Experimental models of thrombotic stroke induced by either intra-arterial thrombin injection or ferric chloride application followed by measurement of cerebral blood flow using a combination of laser Doppler flowmetry and MRI were performed to uncover the effects of NAC on arterial thrombi. To investigate the effect of NAC on larger vessels, we also performed ferric chloride–induced carotid artery thrombosis. In vitro experiments were performed to study the molecular bases of NAC thrombolytic effect, including platelet aggregometry, platelet-rich thrombi lysis assays, thromboelastography (ROTEM), and high-shear VWF string formation using microfluidic devices. We also investigated the putative prohemorrhagic effect of NAC in a mouse model of intracranial hemorrhage induced by in situ collagenase type VII injection.

Platelet cross-linking during arterial thrombosis involves von Willebrand Factor (VWF) multimers. Therefore, proteolysis of VWF appears promising to disaggregate platelet-rich thrombi and restore vessel patency in acute thrombotic disorders such as ischemic stroke, acute coronary syndrome, or acute limb ischemia. N -Acetylcysteine (NAC, a clinically approved mucolytic drug) can reduce intrachain disulfide bonds in large polymeric proteins. In the present study, we postulated that NAC might cleave the VWF multimers inside occlusive thrombi, thereby leading to their dissolution and arterial recanalization.

Introduction

Editorial, see p 661

Clinical Perspective What Is New? This study shows that N -acetylcysteine has thrombolytic effects in the arterial circulation by reducing the size of von Willebrand Factor multimers, a key constituent of platelet-rich thrombus.

N -Acetylcysteine exerts its thrombolytic effect essentially by reducing the disulfide bridges in large von Willebrand Factor multimers, leading to their fragmentation and subsequent platelet disaggregation.

In 3 different experimental models of acute ischemic stroke, intravenous injection of N -acetylcysteine induces arterial recanalization and improves ischemic lesion size and neurological deficits.

Coadministration of N-acetylcysteine and an antagonist of platelet GpIIb/IIIa further increases its thrombolytic efficiency by preventing and possibly reversing platelet reaggregation. What Are the Clinical Implications? N -Acetylcysteine may constitute an alternative to currently available thrombolytic agents (such as tissue-type plasminogen activator and its derivatives), with potential applications in ischemic stroke, acute myocardial infarction, and acute limb ischemia.

The large availability, low cost, and apparent safety of N -acetylcysteine may improve the access to thrombolytic therapy in low-income countries with possible dramatic improvement in the management of acute thrombotic disorders at a global level.

The mechanistic interpretation of the effects of high-dose N-acetylcysteine administration in clinical trials deserves to be reevaluated in light of its thrombolytic properties.

In acute thrombotic disorders, prompt reestablishment of vessel patency can alleviate tissue injuries triggered by the thrombus-induced ischemia. Current pharmacological strategy to promote reperfusion relies on the use of tissue-type plasminogen activator (tPA) or its derivatives to activate the fibrinolytic cascade.1 However, despite unquestionable clinical utility, tPA remains mainly active on fibrin and does not significantly affect the other components of the thrombus. As a result, in stroke for instance, the rate of early arterial recanalization after tPA administration is low (≈30%),2 especially when occlusive thrombi are platelet rich (≈6%).3 Moreover, tPA promotes life-threatening brain hemorrhages, thereby impairing the benefit/risk ratio of thrombolysis. There is therefore a clinical need for thrombolytic agents that can disaggregate arterial thrombi without significantly increasing bleeding.

Arterial thrombi often originate from atherosclerotic lesions, where they constitute under high shear rates. It is interesting to note that we recently demonstrated that high shear arterial thrombosis involves mainly von Willebrand Factor (VWF)–dependent platelet cross-linking.4–6 Thus, proteolysis of VWF could be an efficient strategy to disaggregate arterial and platelet-rich thrombi.7,8N-Acetylcysteine (NAC) is a widely used mucolytic drug with a simple molecular structure containing a free thiol group. Thanks to this free thiol group, NAC can reduce the disulfide bonds inside multimeric proteins, such as VWF.9

In the present study, we hypothesized that NAC may reduce the size of VWF multimers that cross-link platelets inside arterial thrombi. Thus, systemically administered NAC may promote thrombus dissolution and arterial recanalization, especially in case of platelet-rich thrombi. If right, the use of NAC will provide a new, effective, and cheap thrombolytic treatment.

Methods

An extended methods section is available in the online-only Data Supplement.

Animals

Experiments were performed on male Swiss mice (35±2 g; CURB, Caen, France), VWF-deficient mice10 (VWF–/–) and C57BL/6 wild-type mice (15–18 weeks old). All experiments were performed in accordance with the French (Decree 87/848) and the European Communities Council (2010/63/EU) guidelines and were approved by institutional review board (French ministry of Research). All the experiments were validated by the local ethical committee of Normandy (CENOMEXA) registered under the reference CENOMEXA-0113-03 and received the agreement number N/03-01-13/03/01 to 18. Anesthesia was induced by using 5% isoflurane (Aerrane, Baxter) and maintained by using 2% isoflurane in a mixture of O 2 /N 2 O (30%/70%).

In Situ Middle Cerebral Artery Occlusion

Three methods were used to induce occlusion of the middle cerebral artery (MCA). In the first method, a piece of Whatman filter paper strip soaked in freshly prepared FeCl 3 (20%, the lowest concentration to achieve occlusive and stable cerebral blood flow [CBF] reduction under 20% of baseline; Sigma-Aldrich) was placed on the intact dura mater over the endothelium of the MCA for 4 minutes.11 CBF in the MCA territory was determined by laser Doppler flowmetry (Oxford Optronix) recording. Occlusion time was defined as the time between FeCl 3 application and CBF diminution <20% of baseline. In the second approach, the coagulation cascade was triggered by injection of thrombin, as previously described.12 In this model, 1 µL of murine α-thrombin (1.0 IU) was injected in the MCA by using a glass micropipette. The last method was direct electrocoagulation of the MCA.13

Large-Vessel Thromboembolic Stroke Model

Mice were placed on the back, and a midline incision was performed in the neck. The right common carotid artery was isolated. A piece of Whatman filter paper strip soaked in freshly prepared FeCl 3 (20%) was placed on the artery for 5 minutes and then removed to allow the formation of a thrombus. Thereafter, ischemic stroke was induced by mechanically promoting embolization of FeCl 3 -triggered thrombi to the internal carotid artery (as evidenced by the recoloration of the previously thrombosed common carotid artery). Then anesthesia was stopped after analgesia was delivered (0.1 mg/kg of subcutaneously injected buprenorphine). Neurological assessment was performed at 1 hour and 24 hours blinded to the experimental data. Only mice presenting a neuroscore >1 were randomly assigned. This model obtained specific institutional review board approval by the local ethic committee CENOMEXA under the title “Modèle murin d’infarctus cérébral thrombo-embolique à point de départ carotidien.”

Intracranial Hemorrhage Model

A unilateral striatal injection of collagenase type VII14 (0.1 U in 0.5 µL of saline) was performed after placing Swiss male mice in a stereotaxic frame (coordinates: 0.5 mm anterior, 2.0 mm lateral, –3 mm ventral to the bregma). Solutions were injected by the use of a glass micropipette to minimize hemorrhage-unrelated tissular damage. Neurological evaluation was performed blind to the experimental groups using the neuroscore scale, as previously described.14

Treatments

For NAC injection, mice received 400 mg/kg of NAC in phosphate-buffered saline. Administration of NAC was performed as a slow intravenous bolus (60 seconds) through a tail vein catheter. For fibrinolysis, animals under isoflurane anesthesia (this anesthetic regimen does not influence ischemic lesion size15), were injected intravenously (tail vein, 200 µL, 10% bolus, 90% infusion over 40 minutes) with tPA (Actilyse, 10–30 mg/kg), either early (20 minutes) or late (4 hours) after thrombus formation. Intra-arterial administration of tPA was performed according to the same protocol as for intravenous injection but after placement of a catheter in the right common carotid artery. For the blockade of platelet GpIIb/IIIa receptors, mice were injected intravenously as a bolus with GR144053 trihydrochloride (GR, 10 mg/kg; Tocris), a nonpeptidic GpIIb/IIIa inhibitor. Aspirin (100 mg/kg, Sigma-Aldrich) was dissolved in dimethyl sulfoxide and phosphate-buffered saline (10%/90%) and injected intravenously as a slow bolus (60 seconds). To achieve anticoagulation, argatroban (a direct thrombin inhibitor, 10 mg/kg, Sigma-Aldrich) or unfractionated heparin (200 U/kg) were injected intravenously in bolus.

Statistical Analyses

Results are expressed as mean±SD. When comparing multiple groups, statistical analyses were performed using the Kruskal-Wallis test followed by the Mann-Whitney test and the P values were adjusted for multiple testing. When comparing only 2 groups, the Mann-Whitney test was used. Differences were considered statistically significant if probability value P<0.05.

Results

Ferric Chloride-Induced Thrombi Are Platelet Rich and tPA Resistant

We first aimed to develop an ischemic stroke model in mice involving platelet-rich thrombi. To this aim, we selected 2 experimental models to induce in situ occlusive thrombus formation in the middle cerebral artery (MCA): direct intra-arterial injection of recombinant thrombin, as described by Orset et al,12 and topical application of FeCl 3 , as described by Karatas et al.11 We first characterized these 2 models in terms of thrombus composition by immunohistofluorescence. Brain samples were collected 20 minutes after thrombus formation and immunohistological analyses of the arterial thrombi were performed. Our results indicate that FeCl 3 induced the formation of merely pure platelet thrombi, whereas thrombin induced the formation of mixed thrombi, containing significant amounts of both platelets and fibrin (Figure 1A).

Figure 1. Thrombi induced by FeCl 3 in the middle cerebral artery (MCA) are platelet rich and resistant to tPA-mediated thrombolysis.A, Left, Representative immunohistological images of thrombi in the MCA 20 minutes after thrombin- or FeCl 3 -induced MCA thrombosis. Right, Quantitative analysis of fibrinogen-fibrin (Fg-Fn) and platelets contents of thrombi 20 minutes after thrombosis (n=3 per group). Thrombin induced fibrin-rich thrombi, whereas FeCl 3 induced platelet-rich thrombi. B, Representative images of thionin-stained brain sections, 24 hours post-MCA occlusion (MCAo) induced by either thrombin or FeCl 3 (dotted lines represent the ischemic lesions). Subsequently, 20 minutes (early) or 4 hours (late) after MCAo, mice received an intravenous infusion of tPA (10 mg/kg). C, Mean lesion size 24 hours after MCAo in thrombin- and FeCl 3 -induced stroke models, with or without tPA administration (n=10 per group). Although, in the thrombin model, early and late tPA were beneficial and deleterious, respectively (because of the deleterious effect of late tPA-mediated reperfusion), tPA had no significant effect in the FeCl 3 model. D, Mean angiographic score (see Methods) of longitudinally studied mice after thrombin- or FeCl 3 -induced MCAo (n=5 per group) showing spontaneous and tPA-induced recanalization in the thrombin model. No recanalization occurred in the FeCl 3 model, with or without tPA treatment. E, Representative MRI ΔR2* maps of mice immediately (20 minutes) or 24 hours after MCAo in thrombin- and FeCl 3 -induced stroke models. Black arrows indicate areas of perfusion defect, confirming the lack of reperfusion in the FeCl 3 model. F, Quantitative assessment of perfusion index (see Methods) in the 2 models (n=4–6 per group). *P<0.05. DAPI indicates 4′,6-diamidino-2-phenylindole; ns, nonsignificant; and tPA, tissue-type plasminogen activator.

Because clinical evidence suggests that tPA-mediated thrombolysis is less efficient in platelet-rich thrombi, we investigated whether the sensitivity to intravenous tPA differs in these 2 models. In line with clinical studies, early intravenous administration of tPA (10 mg/kg, 20 minutes after occlusive thrombus formation) diminished the lesion size by 26.2% in the thrombin model, whereas it failed to influence the lesion size in the FeCl 3 model (Figure 1B and 1C). The lack of efficacy of tPA in the FeCl 3 model was also observed when started 4 hours (late) after thrombus formation (Figure 1B and 1C).We consistently demonstrated that tPA promoted recanalization and subsequent reperfusion in the thrombin model, as assessed at different time points (20 minutes and 4 hours) by magnetic resonance angiography and perfusion-weighted imaging, respectively, whereas it failed to do so in the FeCl 3 model (Figure 1D through 1F).

Because human tPA has a lower efficacy in mouse than in human plasma, we also investigated whether higher doses of tPA (up to 30 mg/kg) or local delivery (intra-arterial administration) improved vessel patency and stroke outcome in the FeCl 3 model. As shown in Figure IA through IC in the online-only Data Supplement, neither classical nor higher doses of intravenously administered tPA induced arterial recanalization in the FeCl 3 model during the 40-minute monitoring period. In line with this resistance to tPA, there were no significant differences in ischemic lesion sizes between control (saline-treated) and tPA-treated mice as assessed by MRI 24 hours after stroke onset (Figure ID and IE in the online-only Data Supplement). Similarly, intra-arterial administration of tPA (10 mg/kg) also failed to induce reperfusion and improve stroke outcome in this model (Figure II in the online-only Data Supplement).

Altogether, these results demonstrated that FeCl 3 -induced thrombi in the mouse MCA are platelet rich and resistant to classical thrombolytic treatment.

NAC Induces Arterial Recanalization After Acute Thrombosis

We thereafter investigated whether NAC could disaggregate FeCl 3 -induced platelet-rich thrombi. To this aim, we injected 400 mg/kg NAC (we chose this dose because it is the lowest dose that has been shown to reduce VWF multimers in vivo in mice9) 20 minutes after occlusive thrombus formation (Figure 2A). NAC administration led to a rapid and significant reperfusion reaching up to 53% of the baseline CBF, as measured by laser Doppler flowmetry (Figure 2B and 2C). This reperfusion was transient and followed by rapid rethrombosis and subsequent cyclic flow variations. Thus, NAC as a monotherapy induces transient arterial recanalization in the FeCl 3 model.

Figure 2. NAC restores vessel patency after occlusive thrombosis and improves thrombotic stroke outcome.A, Schematic representation of the experiments performed in B and C. B, Representative Doppler flowmetry after FeCl 3 injury on the middle cerebral artery (monitoring during 1 hour) of saline- and NAC (400 mg/kg)-treated animals. The arrow indicates time to saline or NAC intravenous injection. C, Mean value of cerebral blood flow in the last 10 minutes of monitoring. D, Schematic representation of the experiments performed in E, F, and G. E, Graphs: Mean lesion size in saline- and NAC-treated animals (400 mg/kg, 20 minutes after arterial occlusion). Twenty-four hours after middle cerebral artery occlusion in thrombin (E)-, FeCl 3 (D)-, and electrocoagulation (permanent) (E)-induced stroke models as assessed by T2-weighted imaging. Top, Representative T2-weighted images of saline- and NAC-treated animals. * means significant versus saline. CBF indicates cerebral blood flow; GR, GR144053 trihydrochloride; NAC, N-acetylcysteine; and tPA, tissue-type plasminogen activator.

Then, we investigated whether other clinically available antithrombotic agents influence arterial patency in the FeCl 3 model (Figure 2A). As shown on Figure 2C, neither tPA (10 mg/kg), unfractioned heparin (200 UI/kg), aspirin (100 mg/kg), nor GR-144053 (a fast-acting reversible GpIIb/IIIa inhibitor, analog to tirofiban, 10 mg/kg) influenced the CBF in this model. These data suggest that the thrombolytic effects of NAC are independent of plasmin generation, anticoagulation, or platelet activation inhibition.

To further assess the potential of NAC as an acute ischemic stroke treatment, we injected NAC 20 minutes after ischemic onset in 3 different stroke models (Figure 2D): electrocoagulation (permanent ischemia), in situ thrombin injection (mixed thrombi with significant amount of VWF, Figure III in the online-only Data Supplement), or topical FeCl 3 application (platelet-rich thrombi). NAC significantly reduced ischemic lesion sizes in both thrombin-induced (–39%, P<0.05, Figure 2E) and FeCl 3 -induced (–57%, P<0.05, Figure 2F) stroke models as assessed by MRI 24 hours after stroke. In contrast, NAC failed to reduce the ischemic lesion size in the electrocoagulation model (–10%, P=0.57, Figure 2G), suggesting that its brain-protective effects in our experimental conditions are related to arterial recanalization rather than direct neuroprotection. These results demonstrate that NAC administration is beneficial in acute ischemic stroke because of its thrombolytic properties.

Overall, these results demonstrate that NAC acts as a thrombolytic in the presence of platelet-rich (FeCl 3 model) and mixed thrombi (thrombin model) and improves ischemic stroke outcome when injected as a monotherapy.

NAC Prevents the Formation of Large VWF Fibers, Reduces the Size of VWF Multimers, and Displays Direct Thrombolytic Effects on VWF- and Platelet-Rich Thrombi

To get further insights into the mechanism of action of NAC underlying its ability to induce arterial recanalization, we performed additional in vitro and in vivo experiments aiming at studying NAC-mediated VWF cleavage and the impact of NAC on coagulation and tPA-mediated fibrinolysis. As shown on Figure IVA in the online-only Data Supplement, concentrations of NAC as low as 5 mmol/L were able to induce VWF multimers cleavage in vitro, with a stronger effect at 10 and 20 mmol/L, confirming previous studies.9 In another experiment, we investigated the effect of an intravenous injection of NAC (400 mg/kg) on circulation VWF multimers in mice. It is interesting to note that 1 hour after NAC administration, we observed a reduced concentration of circulating high-molecular-weight multimers of VWF in mice (Figure IVB in the online-only Data Supplement). It is noteworthy that NAC at concentrations ≤20 mmol/L had no effect on the apparent size after electrophoretic migration of fibronectin and fibrinogen, 2 other proteins involved in platelet aggregation (Figure V in the online-only Data Supplement).

Then, we performed a microfluidic assay allowing us to observe the formation of VWF fibers under high shear rates.16,17 When NAC was added to citrated platelet-free plasma and perfused over collagen at 30 000 s–1 for 5 minutes, fewer and smaller VWF fibers formed on the collagen than in the saline-treated control (Figure VIA in the online-only Data Supplement). When examining the distribution of fiber sizes, the addition of NAC resulted in a downward shift in the size distribution of VWF fibers (Figure VIB and VIC in the online-only Data Supplement). It was also observed that, in the presence of NAC, the total length of all fibers formed was less than half that of the control. These results confirm that NAC can alter the structure of VWF and suggests that NAC could inhibit platelet adhesion on injured vessels by preventing VWF fiber formation on collagen.

Even if NAC alters the structure of VWF, whether this is sufficient to induce disaggregation of platelet thrombi remains unproven. To test this hypothesis, we performed additional experiments in vitro, investigating the effect of NAC on in vitro–formed platelet aggregates. VWF-dependent platelet agglutination was induced by using ristocetin in an aggregometer (Figure 3A). Once the platelet agglutinates were stably formed, we added different concentrations of NAC (0, 2.5, 5, 7.5, or 10 mmol/L) and observed whether this induced platelet disagglutination. As shown on Figure 3B and 3C, NAC at 10 mmol/L induced complete disagglutination of the platelet thrombi, with a partial but significant effect starting at the 5 mmol/L dose. We completed this experiment by performing microscopic analyses of the resulting platelet thrombi at the end of the monitoring period in the 0 mmol/L (saline), 5 mmol/L, and 10 mmol/L samples. As shown on Figure 3D and 3E, we confirm that NAC has a thrombolytic effect on these VWF-rich platelet thrombi. At the highest tested dose (10 mmol/L), virtually no platelet agglutinate was visible anymore.

Figure 3. NAC has a direct thrombolytic effect on platelet thrombi after ristocetin-induced agglutination.A, Schematic representation of the platelet agglutination experiments. Once platelet agglutinates were stably formed, either saline or NAC (2.5, 5, 7.5, or 10 mmol/L) was added to the solution to observe platelet disagglutination. The small increase in platelet agglutination at the time of treatment addition (purple dotted line) is related to the small dilution of the samples attributable to the 30-µL volume increase. B, Representative agglutination and disagglutination curves of lyophilized platelets in the presence of ristocetin before and after either saline or NAC treatment. C, Corresponding quantification (n=3–5/group) at the end of the monitoring period (450 s). D, Schematic representation (Left) and 3 representative bright-field microscopic images (Right) of the resulting platelet agglutinates after either saline or NAC treatment. White line=20 µm. E, Corresponding quantification (n=4/group). The size of resting lyophilized platelets and fully agglutinated platelets are shown in blue and orange, respectively, for comparison purposes. * means significant versus saline. NAC indicates N-acetylcysteine; and VWF, von Willebrand Factor.

It is important to note that further experiments performed using platelets from platelet-rich plasma after ADP or ristocetin-induced platelet aggregation also support a direct effect of NAC on the stability of VWF-rich platelet aggregates (Figure VII in the online-only Data Supplement). We first performed platelet aggregation experiments using ADP as a platelet agonist. ADP induces platelet activation and therefore triggers the formation of GpIIb/IIIa-fibrinogen and, to a lesser extent, GpIIb/IIIa-VWF–dependent platelet aggregates. Accordingly, NAC treatment (0, 5, or 10 mmol/L) induced partial disaggregation of the platelet aggregates (up to 43% using 10 mmol/L NAC in comparison with control saline-treated platelets). Second, we performed platelet aggregation experiments using ristocetin as a platelet agonist (Figure VIID through VIIF in the online-only Data Supplement). Ristocetin induces changes in conformation of VWF, leading to GpIbα-VWF interactions followed by platelet activation and subsequent formation of GpIIb/IIIa-fibrinogen and, to a lesser extent, GpIIb/IIIa-VWF–dependent platelet aggregates. As expected, and in line with the results obtained using ADP, NAC treatment (0, 5, or 10 mmol/L) induced partial disaggregation of the platelet aggregates after ristocetin treatment (up to 27% using 10 mmol/L NAC in comparison with control saline-treated platelets). In both ADP-induced and ristocetin-induced platelet aggregation experiments, we hypothesized that the resistant platelet aggregates were cross-linked by GpIIb/IIIa-fibrinogen interactions. To test this hypothesis, we induced platelet aggregation using ristocetin in the presence of a GpIIb/IIIa antagonist (GR, 50 µg/mL). In these conditions, platelet cross-linking is only dependent on GpIbα-VWF interactions (Figure VIIG through VIII in the online-only Data Supplement). Accordingly, NAC treatment (0, 5, or 10 mmol/L) induced almost complete disaggregation of the platelet aggregates (up to 85% using 10 mmol/L NAC in comparison with control saline-treated platelets).

Thereafter, we looked for potential effects of NAC on coagulation and fibrinolysis. As shown in Figure VIII in the online-only Data Supplement, NAC affected coagulation testing and tPA-mediated fibrinolysis in plasma clot-lysis assays18 only at the highest studied concentration (20 mmol/L), without significant effects at lower concentrations. Similar results were obtained using thromboelastography (ROTEM, Figure IX in the online-only Data Supplement). In line with these results, NAC failed to induce cleavage of plasminogen and tPA (Figure X in the online-only Data Supplement). It is interesting to note that according to pharmacokinetic studies, the plasmatic concentration of NAC after a bolus of 400 mg/kg is expected to reach a peak of 9.1 mmol/L and, thus, is able to induce VWF cleavage and VWF-rich thrombi disaggregation without significantly affecting coagulation or endogenous fibrinolysis.

NAC Impacts Thrombus Stability but Cannot Prevent Occlusive Thrombus Formation

Then we investigated whether NAC could prevent occlusive thrombus formation when injected before application of FeCl 3 (Figure XIA in the online-only Data Supplement). This hypothesis was supported by our in vitro studies showing that NAC prevents the formation of large VWF fibers and also by additional in vivo experiments revealing that VWF knockout mice (VWF KO19) were protected from thrombosis in this model (Figure XIB in the online-only Data Supplement). To this aim, we injected either NAC or saline 10 minutes before topical FeCl 3 application on the MCA (Figure XIA in the online-only Data Supplement). As shown on Figure XIC through XIF in the online-only Data Supplement, saline-pretreated mice presented stable thrombi with a resulting CBF never exceeding 20% of its baseline value during the monitoring period. NAC pretreatment failed to prevent occlusive thrombus formation because all mice presented a sudden drop in CBF with visual evidence of complete thrombosis (Figure XID in the online-only Data Supplement). However, the resulting thrombi were unstable, leading to spontaneous recanalization and cyclic flow variations in most NAC-treated mice. These findings demonstrate that, when injected before topical FeCl 3 application, NAC impacts thrombus stability but cannot prevent complete thrombosis.

Synergistic Effect of NAC and GpIIb/IIIa Inhibitors on Thrombolytic Efficiency

As demonstrated earlier, NAC treatment induces arterial recanalization but cannot prevent rethrombosis. Thus, we hypothesized that coinjection of NAC with an antithrombotic agent would prevent rethrombosis after NAC-induced recanalization and, in consequence, allow a faster and more complete reperfusion. To test this hypothesis, we injected either NAC, a GpIIb/IIIa inhibitor (GR144053), or a combination of the 2 drugs following FeCl 3 -induced thrombosis of the MCA (Figure 4A). In line with the previous results, administration of NAC alone induced reperfusion followed by cyclic flow reductions resulting in significantly reduced ischemic lesion sizes, as assessed by MRI at 24 hours (Figure 4B through 4E). As previously demonstrated,4 when injected alone, GpIIb/IIIa inhibitors failed to promote reperfusion and improve stroke outcome in this model. It is remarkable that coadministration of NAC with GpIIb/IIIa inhibitors induced a rapid and almost complete recanalization of the MCA. Consequently, ischemic lesion sizes were reduced by a mean of 73% in NAC+Anti-GpIIb/IIIa–treated mice in comparison with saline-treated mice. Thus, coadministration of NAC and a GpIIb/IIIa inhibitor presents a synergistic effect on thrombolytic efficiency and subsequent improvement of stroke outcome.

Figure 4. Adjunctive treatment with GpIIb/IIIa inhibitors further improves N-acetylcysteine–induced reperfusion.A, Schematic representation of the performed experiments. B, Representative Doppler flowmetry after FeCl 3 injury on the middle cerebral artery (monitoring during 30 minutes) of saline-, N-acetylcysteine (400 mg/kg)–, or N-acetylcysteine +Anti-GpIIb/IIIa (400 mg/kg, 10 mg/kg)–treated mice. The arrow indicates time-to-treatment injection. C, Mean value of cerebral blood flow in the last 5 minutes of monitoring (n=8 per group). D, Representative T2-weighted images 24 hours after middle cerebral artery occlusion. E, Quantification of the lesion size (n=8 per group). * means significant versus saline or otherwise indicated. CBF indicates cerebral blood flow; GR, GR144053 trihydrochloride; and NAC, N-acetylcysteine.

NAC Restores Vessel Patency After Large Artery Thrombosis

To determine whether our findings would be valid for larger vessels, we performed a model of common carotid artery thrombosis induced by the topical application of 10% FeCl 3 (Figure 5A). When injected before thrombus formation, NAC-pretreated mice showed delayed time to first occlusion (614 s versus 491 s) and higher residual CBF (45.6% versus 18.0%) than saline-pretreated animals (both P<0.05, Figure 5B through 5D). Ten minutes after FeCl 3 application, intravenous injection of 400 mg/kg NAC induced significant reperfusion of the common carotid artery as assessed by Doppler laser flowmetry (45.8% versus 21.3% of the baseline CBF, in comparison with saline injection, P<0.05; Figure 5E and 5F). Last, we injected NAC 20 minutes after FeCl 3 application, when the thrombus was stabilized. As shown on Figure 5G and 5H, NAC slightly enhanced the blood flow, but rethrombosis frequently occurred, so that the mean blood flow at the end of the measurement was not significantly improved in comparison with control mice (Figure 5G and 5H). It is interesting to note that coadministration of NAC and a GpIIb/IIIa inhibitor led to a persistent recanalization with significantly improved blood flow, further supporting the synergistic effect of NAC and GpIIb/IIIa inhibitors to restore arterial patency.

Figure 5. NAC restores vessel patency after common carotid artery thrombosis.A, Schematic representation of the performed experiments. B, Representative Doppler flowmetry after FeCl 3 -induced injury on the common carotid artery (monitoring during 1 hour). Mice received either NAC (400 mg/kg) or an equivalent volume of saline 10 minutes after arterial injury, when the artery was already occluded. C, Corresponding time to first occlusion. D, Mean blood flow in the last 10 minutes of monitoring (n=5 per group). E, Representative Doppler flowmetry after FeCl 3 -induced injury on the common carotid artery (monitoring during 1 hour). Mice received either NAC (400 mg/kg) or an equivalent volume of saline 10 minutes before arterial injury. The time to first occlusion was measured (drop of the blood flow to <30% of baseline) and the monitoring lasted 1 hour after FeCl 3 treatment. F, Mean blood flow in the last 10 minutes of monitoring (n=5 per group). G, Representative Doppler flowmetry after FeCl 3 -induced injury on the common carotid artery (monitoring during 1 hour). Mice received either NAC (400 mg/kg) or an equivalent volume of saline 20 minutes after arterial injury, when the artery was stably occluded. H, Corresponding mean blood flow at the end of the monitoring period. * means significant versus saline. CBF indicates cerebral blood flow; and NAC, N-acetylcysteine.

NAC Improves Ischemic Lesion Size and Neurological Outcome in a New Large-Vessel Thromboembolic Stroke Model in Mice

To investigate whether the reduction in ischemic lesion size translates into an improved neurological outcome after stroke, we developed an original model of large-vessel thromboembolic stroke. Indeed, we failed to measure consistent neurological deficits in the thrombin and FeCl 3 models, which involve distal occlusion of the MCA and induce only small cortical strokes. To overcome this limitation, we developed and characterized an original thromboembolic stroke model induced by intracranial embolization of a thrombus formed at the surface of the common carotid artery (Figure 6A through 6D). Therefore, it mimics part of the pathophysiology of human large-artery atherosclerotic stroke: ie, carotid atherosclerotic plaque rupture followed by thrombus embolization and subsequent intracranial arterial occlusion.

Figure 6. Thrombolytic treatment using NAC and an anti-GpIIb/IIIa improves neurological outcome in a mode of large-vessel thromboembolic stroke.A, Schematic representation of the experiments in mice. Only mice presenting a neuroscore >1 were randomly assigned. The other mice were excluded. B, Pictures from the operator view (Upper) and schematic representation (Lower) of the different steps of the thromboembolic model. First, the right common carotid artery (CCA) is isolated. Then, FeCl 3 (20%, mass/volume) is applied on the CCA for 3 minutes, leading to an endothelial lesion. Thereafter, clot formation on the endothelial lesion induces CCA occlusion. Last, mechanical pressure is applied on the occluded CCA to detach the thrombus, thereby inducing its migration toward the intracranial circulation. C, Representative immunohistological images of the right CCA 5 minutes after thrombus formation revealing a large occluding platelet-rich thrombus. D, Left, Representative magnetic resonance angiogram showing a patent left middle cerebral artery (green), but the absence of flow in the right middle cerebral artery (red) starting from its proximal segment. Right, Diffusion-weighted image (DWI) and pseudocolored apparent diffusion coefficient (ADC) map 24 hours after the thromboembolic stroke showing the typical cytotoxic edema usually observed in acute and subacute ischemic stroke encompassing a significant part of the right hemisphere. E, Representative T2-weighted MRI at 24 hours after thromboembolic stroke in saline and NAC+Anti-GpIIb/IIIa mice showing a smaller ischemic lesion in the NAC+Anti-GpIIb/IIIa–treated animal. F, Corresponding quantification (n=16–19/group). G, Mean neuroscore at 24 hours after thromboembolic stroke showing a significantly better neurological outcome (lower neuroscore) in NAC+Anti-GpIIb/IIIa–treated mice (n=20/group). H, Mean ΔNeuroscore corresponding to the difference between the neuroscore evaluated at +24 hours and the neuroscore evaluated at +1 hour after stroke onset, confirming the beneficial effect of NAC+Anti-GpIIb/IIIa treatment (n=20/group). * means significant versus saline. DAPI indicates 4′,6-diamidino-2-phenylindole; GR, GR144053 trihydrochloride; and NAC, N-acetylcysteine.

Using this model, we performed a controlled, randomized, and blinded preclinical trial of NAC+Anti-GpIIb/IIIa (the most efficient therapeutic strategy identified in our previous experiments) versus vehicle (saline) using 20 animals per group. The onset-to-treatment delay was chosen based on the median onset-to-treatment observed in the National Institute of Neurological Disorders and Stroke trials that demonstrated the beneficial effect of tPA in patients with stroke: 90 minutes. The primary end point was neurological outcome at 24 hours as assessed by a previously described neuroscore14 (higher score meaning more severe neurological deficits). Secondary outcomes were mortality, lesion size, and hemorrhagic transformation rate as assessed by T2-weighted and T2*-weighted imaging at 24 hours, respectively. It is important to note that to include only the mice with large-vessel occlusion resulting from thrombus embolization, only animals presenting significant neurological deficits (Neuroscore >1 and <4 (death) 60 minutes after thrombus embolization were randomly assigned (so that 36% of the performed mice were excluded before randomization because of the failure of the surgery to induce significant neurological deficit).

NAC+Anti-GpIIb/IIIa significantly improved neurological outcome and ischemic lesion size in comparison with control saline-treated mice when administered intravenously 90 minutes after stroke onset (Figure 6E through 6H). No significant difference was observed for mortality (20% in the saline group versus 5% in the NAC+Anti-GpIIb/IIIa group, P=0.1567 using χ2 test) and hemorrhagic transformation rates (0% in both groups). Thus, these data demonstrate that this thrombolytic strategy improves neurological functional status after stroke.

NAC Displays a Favorable Safety Profile in a Hemorrhagic Stroke Model

To further evidence of the safety of NAC regarding the risk of hemorrhagic transformations, we determined whether NAC treatment worsened the outcome of mice subjected to intracranial hemorrhage induced by intrastriatal administration of 0.1 UI type VII collagenase (Figure 7A).14 Unlike intravenous heparin administration (200 UI/kg in bolus, 75 minutes after collagenase administration) that led to hematoma expansion and a high mortality rate, NAC treatment (400 mg/kg in bolus, 75 minutes after collagenase administration) failed to promote hematoma expansion, worsen clinical score, or increase lesion sizes at any time points in comparison with saline-treated animals (Figure 7B through 7E).

Figure 7. NAC does not aggravate collagenase-induced hemorrhage.A, Schematic representation of the performed experiments. Mice received intravenous NAC (400 mg/kg), heparin (200 IU/kg), or an equivalent volume of saline 75 minutes after intrastriatal administration of collagenase (0.1 U). B, Hemorrhage growth between 1 hour and 4 hours after collagenase injection as assessed by T2* imaging (n=5–7 per group). C, Clinical score 4 hours, 24 hours, and 72 hours post–collagenase administration. D, Time course of hematoma size as assessed by T2*-weighted imaging (deoxyhemoglobin) in saline- and NAC-treated mice. The black arrow indicates time of treatment injection. E, Right, Mean final hematoma volume as assessed by T2-weighted imaging at 72-hour post–collagenase injection. Left, Representative T2-weighted images of saline- and NAC-treated animals (n=6–7 per group). * means significant versus saline. NAC, N-acetylcysteine; and NS, nonsignificant.

Discussion

In the present study, we have shown that NAC has a potent thrombolytic activity in the presence of arterial thrombi (Figure 8). This thrombolytic effect is essentially mediated by the cleavage of VWF that cross-links platelets. Moreover, using 3 different ischemic stroke models, we demonstrated that NAC alleviates ischemic lesions and improves neurological outcome when injected intravenously in combination with a GpIIb/IIIa antagonist. It is important to note that NAC did not worsen outcome or induce hematoma expansion in a model of intracranial hemorrhage, suggesting that it displays a favorable benefit-risk ratio.

Figure 8. Schematic representation of the main findings.A, Schematic representation. B, N-acetylcysteine reduces the disulfide bonds of the von Willebrand Factor multimers that maintain platelets linked in arterial thrombi, thereby inducing thrombus dissolution. VWF indicates von Willebrand Factor.

The dose injected in the present study is only slightly higher (but more rapidly injected) than the dose recommended to treat acetaminophen poisoning in human (300 mg/kg), supporting the potential for future clinical translation of our findings. It is important to note that GpIIb/IIIa inhibitors have been widely used in patients with acute coronary syndrome, and tirofiban, a nonpeptidic GpIIb/IIIa inhibitor similar to GR144053, has recently been shown to be safe in the acute phase of stroke,20 thereby opening the possibility to coadministrate NAC and a GpIIb/IIIa inhibitor to further improve its thrombolytic effect. The clinical relevance of our results is also supported by the fact that NAC has been successfully used as a VWF-degrading agent in a small series of patients presenting thrombotic thrombocytopenic purpura,21,22 a condition characterized by abnormally large circulating VWF multimers. Moreover, NAC has been shown to improve outcome in patients with acute myocardial infarction treated with streptokinase and nitroglycerine.23 In this study, the authors detected a trend toward faster coronary reperfusion in NAC-treated patients (–39%), albeit the reason for this effect remained elusive. More recently, the NACIAM trial (N-Acetylcysteine in Acute Myocardial Infarction) demonstrated that early use of N-acetylcysteine in patients with ST-segment–elevation myocardial infarction resulted in accelerated tissue reperfusion, more rapid chest pain resolution, and myocardial infarct size reduction by 33% on day 5 in comparison with placebo-treated controls.24 Our results raise the intriguing possibility that these beneficial effects of NAC are mediated by its thrombolytic effects.

Our data support that the observed thrombolytic effect of NAC administration is mainly mediated by cleavage of the VWF that cross-links platelets inside arterial thrombi. Indeed, we demonstrated that NAC is able to disaggregate platelets cross-linked by VWF. Moreover, we demonstrated that NAC does not act solely through platelet inhibition, coagulation inhibition, or fibrinolysis, because antiplatelet agents, anticoagulants, and fibrinolytic agents all failed to restore vessel patency in the FeCl 3 model. Moreover, NAC did not improve outcome in a permanent model of cerebral ischemia, suggesting that its other described neuroprotective properties are not sufficient to significantly improve stroke outcome in the present experimental conditions.25 It should be mentioned, however, that NAC can influence hemostasis by other pathways in vivo, including anticoagulation and antiplatelet effects,26 which may have participated in the observed thrombolytic effect. Whether NAC acts exclusively through direct VWF multimer cleavage or also through thiol-containing intermediates remains to be investigated. It raises the intriguing possibility that naturally occurring thiol-containing compound (such as glutathione) could actively participate in the endogenous thrombolytic process.27 Overall, our results suggest that disulfide bond–reducing agents constitute a new large family of thrombolytics. Accordingly, further studies may aim at developing an ideal thrombolytic agent with maximal VWF cleaving abilities and minimal side effects. It is noteworthy that our results are consistent with the multistep model of occlusive thrombus formation that we described previously.4 According to this model, in situ formed occlusive thrombi are made of a base, which constitutes, at low shear rates, an inner core, which constitutes at medium to high shear rates, and an outer part, which constitutes at very high shear rates. Our present results suggest that NAC destabilizes the outer part of the thrombus because it is predominantly made of platelet cross-linked by VWF, thereby exposing the thrombus core that is mainly made of platelet cross-linked by GpIIb/IIIa-fibrinogen interactions. Consequently, coadministration of a GpIIb/IIIa inhibitor with NAC destabilizes this inner part of the thrombus by competing with endogenous GpIIb/IIIa ligands and leads to complete recanalization.

This study has some limitations, however. First, human thrombi may have a different inner structure in comparison with FeCl 3 -induced thrombi in mice, and thrombus composition may change according to the occlusion site and the delay between occlusion and start of thrombolytic treatment. It is interesting to note that a recent study showed that thrombi retrieved from intracranial arteries in patients with acute ischemic stroke contain on average 20.3±10.1% VWF.7 Therefore, the target of NAC is present in a significant proportion in thrombi from human patients with stroke. Besides, even if we demonstrated that NAC+anti-GpIIb/IIIa treatment improved stroke outcome when injected up to 90 minutes after stroke onset in the large-vessel thromboembolic model, the influence of thrombus age on NAC efficacy deserves further investigations. Similarly, whether NAC induces downstream embolization of microthrombi because of destabilization of the thrombus structure after VWF multimer reduction remains unknown and could explain the observed cyclic flow reduction pattern in the FeCl 3 model (thrombus embolization followed by regrowth and reocclusion). Second, because there is no spontaneous hemorrhagic transformation in the ischemic stroke models performed in the present study, we investigated the prohemorrhagic effect of NAC in a dedicated intracranial hemorrhage model that may be less relevant to the human condition. Therefore, our safety data should be interpreted with caution. Regarding anti-GpIIb/IIIa treatments that enhance the thrombolytic efficacy of NAC, tirofiban has been shown to be safe in patients with ischemic stroke regarding the risk of hemorrhagic transformation in phase IIb studies.20,28 The fact that tirofiban was safe in human stroke and that we did not observe hemorrhagic transformation in 2 ischemic stroke models in mice after treatment with NAC and a tirofiban analog is reassuring for further investigation of this drug combination for human use. Third, NAC may influence the poststroke immune system. Indeed, low doses of NAC have been shown to reduce inflammatory biomarkers in postmenoposal women,29 to regulate the function of macrophages in endotoxic shock,30 and to have a weakly strengthening effect on the innate immune system.31 Regarding higher doses of NAC, data from clinical trials, in which NAC was administered to prevent the toxicity of acetaminophen poisoning (at 300 mg/kg versus 400 mg/kg in the present study), show that only 1% to 2% of the patients experienced infectious complications.32 However, because the immune system is already weakened in the first days following ischemic stroke,33 the effect of NAC on immune parameters should be monitored to ensure safe translational development of this treatment.

Overall, the superiority of NAC in comparison with clinically available antithrombotic treatments, combined with its favorable safety profile in a hemorrhagic stroke model, makes it a promising drug for the treatment of acute arterial thrombosis such as stroke, myocardial infarction, or acute limb ischemia. Intravenous NAC administration could provide an effective, cheap, and safe thrombolytic treatment, especially in low-income areas with limited access to expensive recombinant proteins and in the presence of platelet-rich thrombi. Clinical trials are now needed to provide definitive proof of NAC thrombolytic efficacy, for instance, by comparing NAC with a placebo for facilitated percutaneous coronary intervention in patients with acute coronary syndrome or to tPA in patients with ischemic stroke.

Acknowledgments

Drs Martinez de Lizarrondo and Gauberti designed the study, analyzed the data, and wrote the article; Drs Martinez de Lizarrondo, Herbig, Gakuba, Repessé, Diamond, and Gauberti performed the experiments; Drs Denis and Lenting provided critical reagents and performed in vitro VWF experiments; and Drs Ali, Touzé, and Vivien analyzed the data, secured the funding, and critically revised the article.

Sources of Funding This work was supported by the Institut National de la Santé Et de la Recherche Médicale, the French Ministry of Research and Technology, the Conseil Régional de Basse-Normandie, the Eurostroke-Arise Program (FP7/2007-2013-201024), and the Fondation pour la Recherche sur les AVC (FR-AVC-002).

Disclosures None.

Footnotes