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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., (© 2017 American Heart Association, Inc.)

Background and Purpose: Despite its fibrinolytic effect, tissue-type plasminogen activator (tPA) displays deleterious effects in the brain, including proexcitotoxicity, that can reduce the overall benefit from thrombolysis during stroke. We have proposed that tPA potentiates excitotoxicity by interacting with and cleaving the aminoterminal end of the NR1 subunit of N-methyl-d-aspartate receptors, leading to an increased calcium influx, Erk1/2 activation, and neurotoxicity. Because this mechanism is debated, our aim was to demonstrate its in vivo occurrence and relevance. Because tPA is released under ischemic conditions, we hypothesized that if it indeed processes NR1, then the released fragment should reactivate the immune system in animals that had been immunized long before with recombinant aminoterminal end of the NR1. This effect should be exacerbated in ischemic animals thrombolysed with recombinant tPA., Methods: At a time when specific antibodies could not be detected any longer, mice previously vaccinated with recombinant aminoterminal end of the NR1 were subjected to thromboembolic stroke induced by injecting thrombin in the middle cerebral artery alone or with intravenous thrombolysis., Results: Stroke performed 1 year after active immunization induced the reappearance of antibodies against the aminoterminal end of the NR1 in the plasma, an effect significantly increased when ischemia was followed by recombinant tPA-induced reperfusion. Moreover, immunization preventing the interaction of tPA with aminoterminal end of the NR1 reduced ischemic brain damages and extended the therapeutic window of tPA-induced thrombolysis., Conclusions: We demonstrate that the tPA-dependent interaction and cleavage of the NR1 subunit of N-methyl-d-aspartate receptors occurs in vivo after stroke and that this interaction is a relevant therapeutic target for stroke treatment.

Background: Accumulating evidence demonstrates a critical involvement of tissue-type plasminogen activator (tPA) in pathological and physiological brain conditions. Determining whether and how vascular tPA can cross the blood-brain barrier (BBB) to enter the brain is thus important, not only during stroke but also in physiological conditions., Methods and Results: In the present work, we provide evidence in vivo that intravenous injection of tPA increases NMDA-induced striatal lesion in the absence of BBB leakage. Accordingly, we show that tPA crosses the BBB both after excitotoxic lesion and in control conditions. Indeed, vascular injected tPA can be detected within the brain parenchyma and in the cerebrospinal fluid. By using an in vitro model of BBB, we have confirmed that tPA can cross the intact BBB. Its passage was blocked at 4 degrees C, was saturable, and was independent of its proteolytic activity. We have shown that tPA crosses the BBB by transcytosis, mediated by a member of the LDL receptor-related protein family., Conclusions: We demonstrate that blood-derived tPA can reach the brain parenchyma without alteration of the BBB. The molecular mechanism of the passage of tPA from blood to brain described here could represent an interesting target to improve thrombolysis in stroke.