Potent Thrombolytic Effect of N-Acetylcysteine on Arterial Thrombi

Abstract

Background: 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.

Methods: 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.

Results: 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.

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

Keywords: plasminogen activators; platelet aggregation; stroke; thrombolytic therapy; von Willebrand Factor.

Figure 1. Thrombi induced by FeCl₃ 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 FeCl3-induced MAC 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 FeCl3 induced platelet-rich thrombi. (B) Representative images of thionin-stained brain sections, 24 hours post-MCA occlusion (MCAo) induced by either thrombin or FeCl₃ (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 FeCl₃-induced stroke models, with or without tPA administration (n=10 per group). Although in the thrombin model, early and late tPA were respectively beneficial and deleterious (due to the deleterious effect of late tPA mediated reperfusion), tPA had no significant effect in the FeCl₃ model. (D) Mean angiographic score (see methods section) of longitudinally studied mice after thrombin- or FeCl₃-induced MCAo (n=5 per group) showing spontaneous and tPA-induced recanalization in the thrombin model. No recanalization occurred in the FeCl₃ model, with or without tPA treatment. (E) Representative MRI ΔR2* maps of mice immediately (20 minutes) or 24 hours after MCAo in thrombin- and FeCl₃-induced stroke models. Black arrows indicate areas of perfusion defect, confirming the lack of reperfusion in the FeCl3 model. (F) Quantitative assessment of perfusion index (see methods section) in the two models (n=4–6 per group). (*=p<0.05; ns = non-significant).

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 FeCl₃ injury on the MCA (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). 24 hours after MCAo in (E) thrombin-, (D) FeCl₃- and (E) electrocoagulation (permanent)-induced stroke models as assessed by T2-weighted imaging. Top: representative T2-weighted images of saline and NAC-treated animals. (* means significant versus saline).

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 mM) 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 due 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 three 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 purpose. (* means significant versus saline).

Figure 4. Adjunctive treatment with GpIIb/IIIa inhibitors further improves NAC-induced reperfusion

(A) Schematic representation of the performed experiments. (B) Representative Doppler flowmetry after FeCl₃ injury on the MCA (monitoring during 30 minutes) of saline-, NAC− (400 mg/kg) or NAC+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 MCAo. (E) Quantification of the lesion size (n=8 per group). (* means significant versus saline or otherwise indicated).

Figure 5. NAC restores vessel patency after common carotid artery thrombosis

(A) Schematic representation of the performed experiments. (B) Representative Doppler flowmetry after FeCl₃-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 FeCl₃-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 under 30% of baseline) and the monitoring lasted 1 hour after FeCl₃ treatment. (F) Mean values of the time to first occlusion. (G) Mean blood flow in the last 10 minutes of monitoring. (n=5 per group). (G) Representative Doppler flowmetry after FeCl₃-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).

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 randomized. The other mice were excluded. (B) Pictures from the operator view (upper panels) and schematic representation (lower panels) of the different steps of the thromboembolic model. First, the right common carotid artery (CCA) is isolated. Then, FeCl₃ (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 (3). (4) Lastly, 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 MCA (green) but the absence of flow in the right MCA (red) starting from its proximal segment. Right: Diffusion weighted image (DWI) and pseudo-colored 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).

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 min after intrastriatal administration of collagenase (0.1 U). (B) Hemorrhage growth between 1h and 4h 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).

Figure 8. Schematic representation of the main findings

(A) Schematic representation (B) NAC reduces the disulfide bonds of the VWF multimers that maintain platelets linked in arterial thrombi, thereby inducing thrombus dissolution.